Articles

Physical limits of flow sensing in the left-right organizer
- Ferreira Rita R.
- Vilfan Andrej
- Julicher Frank
- Supatto Willy
- Vermot Julien
eLife, eLife Sciences Publication , 2017, 25, pp.556 - 556 . Fluid flows generated by motile cilia are guiding the establishment of the left-right asymmetry of the body in the vertebrate left-right organizer. Competing hypotheses have been proposed: the direction of flow is sensed either through mechanosensation, or via the detection of chemical signals transported in the flow. We investigated the physical limits of flow detection to clarify which mechanisms could be reliably used for symmetry breaking. We integrated parameters describing cilia distribution and orientation obtained in vivo in zebrafish into a multiscale physical study of flow generation and detection. Our results show that the number of immotile cilia is too small to ensure robust left and right determination by mechanosensing, given the large spatial variability of the flow. However, motile cilia could sense their own motion by a yet unknown mechanism. Finally, transport of chemical signals by the flow can provide a simple and reliable mechanism of asymmetry establishment. (10.7554/eLife.25078)
DOI : 10.7554/eLife.25078
Stabilization of Collagen Fibrils by Gelatin Addition: A Study of Collagen/Gelatin Dense Phases
- Portier François
- Teulon Claire
- Nowacka-Perrin Agnieszka
- Guenneau Flavien
- Schanne-Klein Marie-Claire
- Mosser Gervaise
Langmuir, American Chemical Society , 2017, 33 (45), pp.12916-12925 . Collagen and its denatured form, gelatin, are biopolymers of fundamental interest in numerous fields ranging from living tissues to biomaterials, food, and cosmetics. This study aims at characterizing mixtures of those biopolymers at high concentrations (up to 100 mg·mL–1) at which collagen has mesogenic properties. We use a structural approach combining polarization-resolved multiphoton microscopy, polarized light microscopy, magnetic resonance imaging, and transmission electron microscopy to analyze gelatin and collagen/gelatin dense phases in their sol and gel states from the macroscopic to the microscopic scale. We first report the formation of a lyotropic crystal phase of gelatin A and show that gelatin must structure itself in particles to become mesogenic. We demonstrate that mixtures of collagen and gelatin phase segregate, preserving the setting of the pure collagen mesophase at a gelatin ratio of up to 20% and generating a biphasic fractal sample at all tested ratios. Moreover, differential scanning calorimetric analysis shows that each protein separates into two populations. Both populations of gelatins are stabilized by the presence of collagen, whereas only one population of collagen molecules is stabilized by the presence of gelatin, most probably those at the interface of the fibrillated microdomains and of the gelatin phase. Although further studies are needed to fully understand the involved mechanism, these new data should have a direct impact on the bioengineering of those two biopolymers. (10.1021/acs.langmuir.7b02142)
DOI : 10.1021/acs.langmuir.7b02142
Structural changes and picosecond to second dynamics of cytochrome c in interaction with nitric oxide in ferrous and ferric redox states
- Kruglik Sergei
- Yoo Byung-Kuk
- Lambry Jean-Christophe
- Martin Jean-Louis
- Negrerie Michel
Physical Chemistry Chemical Physics, Royal Society of Chemistry , 2017, 19 (32), pp.21317-21334 . Apart from its role in electron transfer, mitochondrial cytochrome c also plays a role in apoptosis and is subject to nitrosylation. The cleavage of the Fe–Met80 bond plays a role in several processes including the release of Cyt c from mitochondria or increase of its peroxidase activity. Nitrosylation of Cyt c precludes the reformation of the disrupted Fe–Met80 bond and was shown to occur during apoptosis. These physiological properties are associated with a conformational change of the heme center of Cyt c. Here, we demonstrate that NO binding induces pronounced heme conformational changes in the six-coordinate Cyt c–NO complex. Equilibrium and time-resolved Raman data reveal that the heme structural conformation depends both on the nature of the distal iron ligand (NO or Met80) and on the Fe2+ or Fe3+ heme redox state. Upon nitrosylation, the heme ruffling distortion is greatly enhanced for ferrous Cyt c. Contrastingly, the initial strong heme distortion in native ferric Cyt c almost disappears after NO binding. We measured the heme coordination dynamics in the picosecond to second time range and identified Met80 and NO rebinding phases using time-resolved Raman and absorption spectroscopies. Dissociation of NO instantly produces 5-coordinate heme with a domed structure which continues to rearrange within 15 ps, while the initial ruffling distortion disappears. The rates of Cyt c–NO complex formation measured by transient absorption are kon = 1.81 × 106 M−1 s−1 for ferric Cyt c and 83 M−1 s−1 for ferrous Cyt c. After NO dissociation and exit from the heme pocket, the rebinding of Met80 to the heme iron takes place 6 orders of magnitude more slowly (3–5 μs) than Met80 rebinding in the absence of NO (5 ps). Altogether, these data reveal the structural and dynamic properties of Cyt c in interaction with nitric oxide relevant for the molecular mechanism of apoptosis. (10.1039/C7CP02634J)
DOI : 10.1039/C7CP02634J
Multiscale control and rapid scanning of time delays ranging from picosecond to millisecond
- Solinas Xavier
- Antonucci Laura
- Bonvalet A.
- Joffre Manuel
Optics Express, Optical Society of America - OSA Publishing , 2017, 25 (15), pp.17811-17819 . Femtosecond amplifiers seeded by two independent femtosecond oscillators normally produce amplified pulse pairs with a timing jitter equal to the oscillator period, which is typically around 12 ns for Titanium:Sapphire lasers. Combining Arbitrary-Detuning Asynchronous Optical Sampling (AD-ASOPS) with an appropriate selection of amplified pulses, we demonstrate that the time-delay distribution can be narrowed down to a 25-ps time window, allowing to produce spectral interference fringes for each amplified pulse pair. Subsequent AD-ASOPS determination of the actual time delay with subpicosecond accuracy allows to tailor the delay distribution with an electronic control all the way to the repetition period of the amplifiers. We thus demonstrate rapid scanning of the time delays up to nearly 1 ms with a sub-picosecond accuracy, which makes this method an ideal tool for multiscale pump-probe spectroscopy. (10.1364/oe.25.017811)
DOI : 10.1364/oe.25.017811
Life-Long Neurogenic Activity of Individual Neural Stem Cells and Continuous Growth Establish an Outside-In Architecture in the Teleost Pallium
- Furlan Giacomo
- Cuccioli Valentina
- Vuillemin Nelly
- Dirian Lara
- Muntasell Anna Janue
- Coolen Marion
- Dray Nicolas
- Bedu Sébastien
- Houart Corinne
- Beaurepaire Emmanuel
- Foucher Isabelle
- Bally-Cuif Laure
Current Biology, Elsevier , 2017, 27 (21), pp.3288-3301.e3 . Spatiotemporal variations of neurogenesis are thought to account for the evolution of brain shape. In the dorsal telencephalon (pallium) of vertebrates, it remains unresolved which ancestral neurogenesis mode prefigures the highly divergent cytoarchitectures that are seen in extant species. To gain insight into this question, we developed genetic tools to generate here the first 4-dimensional (3D + birthdating time) map of pallium construction in the adult teleost zebrafish. Using a Tet-On-based genetic birthdating strategy, we identify a “sequential stacking” construction mode where neurons derived from the zebrafish pallial germinal zone arrange in outside-in, age-related layers from a central core generated during embryogenesis. We obtained no evidence for overt radial or tangential neuronal migrations. Cre-lox-mediated tracing, which included following Brainbow clones, further demonstrates that this process is sustained by the persistent neurogenic activity of individual pallial neural stem cells (NSCs) from embryo to adult. Together, these data demonstrate that the spatiotemporal control of NSC activity is an important driver of the macroarchitecture of the zebrafish adult pallium. This simple mode of pallium construction shares distinct traits with pallial genesis in mammals and non-mammalian amniotes such as birds or reptiles, suggesting that it may exemplify the basal layout from which vertebrate pallial architectures were elaborated. (10.1016/j.cub.2017.09.052)
DOI : 10.1016/j.cub.2017.09.052
A novel microstructural interpretation for the biomechanics of mouse skin derived from multiscale characterization
- Lynch Barbara
- Bancelin Stéphane
- Bonod-Bidaud Christelle
- Gueusquin Jean-Baptiste
- Ruggiero Florence
- Schanne-Klein Marie-Claire
- Allain Jean-Marc
Acta Biomaterialia, Elsevier , 2017, 50, pp.302-311 . Skin is a complex, multi-layered organ, with important functions in the protection of the body. The der-mis provides structural support to the epidermal barrier, and thus has attracted a large number of mechanical studies. As the dermis is made of a mixture of stiff fibres embedded in a soft non-fibrillar matrix, it is classically considered that its mechanical response is based on an initial alignment of the fibres, followed by the stretching of the aligned fibres. Using a recently developed setup combining mul-tiphoton microscopy with mechanical assay, we imaged the fibres network evolution during dermis stretching. These observations, combined with a wide set of mechanical tests, allowed us to challenge the classical microstructural interpretation of the mechanical properties of the dermis: we observed a continuous alignment of the collagen fibres along the stretching. All our results can be explained if each fibre contributes by a given stress to the global response. This plastic response is likely due to inner sliding inside each fibre. The non-linear mechanical response is due to structural effects of the fibres network in interaction with the surrounding non-linear matrix. This multiscale interpretation explains our results on genetically-modified mice with a simple alteration of the dermis microstructure. Statement of Significance: Soft tissues, as skin, tendon or aorta, are made of extra-cellular matrix, with very few cells embedded inside. The matrix is a mixture of water and biomolecules, which include the collagen fibre network. The role of the collagen is fundamental since the network is supposed to control the tissue mechanical properties and remodeling: the cells attach to the collagen fibres and feel the network deformations. This paper challenges the classical link between fibres organization and mechanical properties. To do so, it uses multiscale observations combined to a large set of mechanical loading. It thus appears that the behaviour at low stretches is mostly controlled by the network structural response, while, at large stretches, the fibre inner-sliding dominate. (10.1016/j.actbio.2016.12.051)
DOI : 10.1016/j.actbio.2016.12.051
Fast quantitative ROS detection based on dual-color single rare-earth nanoparticle imaging reveals signaling pathway kinetics in living cells
- Abdesselem M.
- Ramodiharilafy R.
- Devys L.
- Gacoin T.
- Alexandrou A.
- Bouzigues C. I.
Nanoscale, Royal Society of Chemistry , 2017, 9 (2), pp.656 - 665 . Reactive oxygen species (ROS), and notably hydrogen peroxide H2O2, are cellular second messengers that are known to control a variety of signaling processes. They can finely regulate the dynamics of signal transduction, cell response and ultimately tissue function. However, there are very few local, quantitative and time-resolved descriptions of their cellular organization at the scale of molecular reactions, due to the lack of efficient sensors. We thus developed a novel nanoprobe-based ROS detection system using the simultaneous imaging of single lanthanide nanoparticles (YAG:Ce and chemically reduced Gd0.6Eu0.4VO4). We reveal that both particle luminescence signals are controlled by their H2O2 local environment. By simultaneously tracking their luminescence, we devised a new approach providing a quantitative (0.5 μM accuracy in the 1–10 μM range) H2O2 measurement with a 500 ms time resolution, surpassing all existing methods by two orders of magnitude, and revealing previously inaccessible molecular events controlling ROS concentration. We used this nanoprobe in living cells to track fast signaling pathways, by measuring the dynamics of H2O2 intracellular concentrations, induced by endothelin-1 (ET-1) stimulation. We thus revealed the mechanisms controlling ROS production, notably the activity modulation of the ROS-producing enzyme NADPH oxidase by fast (<10 s) EGFR transactivation, and measured quantitatively their kinetic parameters through a minimal analytical model. Altogether, these results illustrate how lanthanide nanoparticle-based sensors are a powerful tool to dynamically probe molecular mechanisms shaping the oxidative cell response. (10.1039/C6NR07413H)
DOI : 10.1039/C6NR07413H
Affine kinematics in planar fibrous connective tissues: an experimental investigation
- Jayyosi Charles
- Affagard Jean-Sébastien
- Ducourthial Guillaume
- Bonod-Bidaud Christelle
- Lynch Barbara
- Bancelin Stéphane
- Ruggiero Florence
- Schanne-Klein Marie-Claire
- Allain Jean-Marc
- Bruyère-Garnier Karine
- Coret Michel
Biomechanics and Modeling in Mechanobiology, Springer Verlag , 2017, pp.1–15 . The affine transformation hypothesis is usually adopted in order to link the tissue scale with the fibers scale in structural constitutive models of fibrous tissues. Thanks to the recent advances in imaging techniques, such as multiphoton microscopy, the microstructural behavior and kinematics of fibrous tissues can now be monitored at different stretching within the same sample. Therefore, the validity of the affine hypothesis can be investigated. In this paper, the fiber reorientation predicted by the affine assumption is compared to experimental data obtained during mechanical tests on skin and liver capsule coupled with microstructural imaging using multiphoton microscopy. The values of local strains and the collagen fibers orientation measured at increasing loading levels are used to compute a theoretical estimation of the affine reorientation of collagen fibers. The experimentally measured reorientation of collagen fibers during loading could not be successfully reproduced with this simple affine model. It suggests that other phenomena occur in the stretching process of planar fibrous connective tissues, which should be included in structural constitutive modeling approaches. (10.1007/s10237-017-0899-1)
DOI : 10.1007/s10237-017-0899-1
Stromal striae: a new insight into corneal physiology and mechanics
- Grieve Kate
- Ghoubay Djida
- Georgeon Cristina
- Latour Gaël
- Nahas Amir
- Plamann Karsten
- Crotti Caroline
- Bocheux Romain
- Borderie Marie
- Nguyen Thu-Mai
- Andreiuolo Felipe
- Schanne-Klein Marie-Claire
- Borderie Vincent M.
Scientific Reports, Nature Publishing Group , 2017, 7, pp.13584 . We uncover the significance of a previously unappreciated structural feature in corneal stroma, important to its biomechanics. Vogt striae are a known clinical indicator of keratoconus, and consist of dark, vertical lines crossing the corneal depth. However we detected stromal striae in most corneas, not only keratoconus. We observed striae with multiple imaging modalities in 82% of 118 human corneas, with pathology-specific differences. Striae generally depart from anchor points at Descemet’s membrane in the posterior stroma obliquely in a V-shape, whereas in keratoconus, striae depart vertically from posterior toward anterior stroma. Optical coherence tomography shear wave elastography showed discontinuity of rigidity, and second harmonic generation and scanning electron microscopies showed undulation of lamellae at striae locations. Striae visibility decreased beyond physiological pressure and increased beyond physiological hydration. Immunohistology revealed striae to predominantly contain collagen VI, lumican and keratocan. The role of these regions of collagen VI linking sets of lamellae may be to absorb increases in intraocular pressure and external shocks. (10.1038/s41598-017-13194-6)
DOI : 10.1038/s41598-017-13194-6
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
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
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
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
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
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
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
Ultrafast Spectroscopy Evidence for Picosecond Ligand Exchange at the Binding Site of a Heme Protein: Heme-Based Sensor YddV
- Lambry Jean-Christophe
- Stranava Martin
- Lobato Laura
- Martinkova Marketa
- Shimizu Toru
- Liebl Ursula
- Vos Marten H.
Journal of Physical Chemistry Letters, American Chemical Society , 2016, 7 (1), pp.69-74 . An important question for the functioning of heme proteins is whether different ligands present within the protein moiety can readily exchange with heme-bound ligands. Studying the dynamics of the heme domain of the Escherichia coli sensor protein YddV upon dissociation of NO from the ferric heme by ultrafast spectroscopy, we demonstrate that when the hydrophobic leucine residue in the distal heme pocket is mutated to glycine, in a substantial fraction of the protein water replaces NO as an internal ligand in as fast as ∼4 ps. This process, which is near-barrierless and occurs orders of magnitude faster than the corresponding process in myoglobin, corresponds to a ligand swap of NO with a water molecule present in the heme pocket, as corroborated by molecular dynamics simulations. Our findings provide important new insight into ligand exchange in heme proteins that functionally interact with different external ligands. (10.1021/acs.jpclett.5b02517)
DOI : 10.1021/acs.jpclett.5b02517
Probing the 3D structure of cornea-like collagen liquid crystals with polarization-resolved SHG microscopy
- Teulon Claire
- Tidu Aurélien
- Portier François
- Mosser Gervaise
- Schanne-Klein Marie-Claire
Optics Express, Optical Society of America - OSA Publishing , 2016, 24, pp.16084 - 10 . This work aims at characterizing the three-dimensional organization of liquid crystals composed of collagen, in order to determine the physico-chemical conditions leading to highly organized structures found in biological tissues such as cornea. To that end, we use second-harmonic generation (SHG) microscopy, since aligned collagen structures have been shown to exhibit intrinsic SHG signals. We combine polarization-resolved SHG experiments (P-SHG) with the theoretical derivation of the SHG signal of collagen molecules tilted with respect to the focal plane. Our P-SHG images exhibit striated patterns with variable contrast, as expected from our analytical and numerical calculations for plywood-like nematic structures similar to the ones found in the cornea. This study demonstrates the benefits of P-SHG microscopy for in situ characterization of highly organized biopolymers at micrometer scale, and the unique sensitivity of this nonlinear optical technique to the orientation of collagen molecules. (10.1364/OE.24.016084.v001)
DOI : 10.1364/OE.24.016084.v001
Predictive modeling targets thymidylate synthase ThyX in Mycobacterium tuberculosis
- Djaout Kamel
- Singh Vinayak
- Boum Yap
- Katawera Victoria
- Becker Hubert F.
- Bush Natassja G.
- Hearnshaw Stephen J.
- Pritchard Jennifer E.
- Bourbon Pauline
- Madrid Peter B.
- Maxwell Anthony
- Mizrahi Valerie
- Myllykallio Hannu
- Ekins Sean
Scientific Reports, Nature Publishing Group , 2016, 6, pp.27792 . There is an urgent need to identify new treatments for tuberculosis (TB), a major infectious disease caused by Mycobacterium tuberculosis (Mtb), which results in 1.5 million deaths each year. We have targeted two essential enzymes in this organism that are promising for antibacterial therapy and reported to be inhibited by naphthoquinones. ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the human enzyme. DNA gyrase is a DNA topoisomerase present in bacteria and plants but not animals. The current study set out to understand the structure-activity relationships of these targets in Mtb using a combination of cheminformatics and in vitro screening. Here, we report the identification of new Mtb ThyX inhibitors, 2-chloro-3-(4-methanesulfonylpiperazin-1-yl)-1,4-dihydronaphthalene-1,4-dione) and idebenone, which show modest whole-cell activity and appear to act, at least in part, by targeting ThyX in Mtb. (10.1038/srep27792)
DOI : 10.1038/srep27792
Direct observation of subpicosecond vibrational dynamics in photoexcited myoglobin
- Ferrante C.
- Pontecorvo E.
- Cerullo Giulio
- Vos Marten H.
- Scopigno T.
Nature Chemistry-, Springer Nature , 2016 (8), pp.1137–1143 . Determining the initial pathway for ultrafast energy redistribution within biomolecules is a challenge, and haem proteins, for which energy can be deposited locally in the haem moiety using short light pulses, are suitable model systems to address this issue. However, data acquired using existing experimental techniques that fail to combine sufficient structural sensitivity with adequate time resolution have resulted in alternative hypotheses concerning the interplay between energy flow among highly excited vibrational levels and potential concomitant electronic processes. By developing a femtosecond-stimulated Raman set-up, endowed with the necessary tunability to take advantage of different resonance conditions, here we visualize the temporal evolution of energy redistribution over different vibrational modes in myoglobin. We establish that the vibrational energy initially stored in the highly excited Franck–Condon manifold is transferred with different timescales into low- and high-frequency modes, prior to slow dissipation through the protein. These findings demonstrate that a newly proposed mechanism involving the population dynamics of specific vibrational modes settles the controversy on the existence of transient electronic intermediates. (10.1038/nchem.2569)
DOI : 10.1038/nchem.2569
Thioglycerol-functionalized CdSe quantum dots detecting cadmium ions
- Brahim Nassim Ben
- Mohamed Naim Bel Haj
- Echabaane Mosaab
- Haouari Mohamed
- Chaâbane Rafik Ben
- Negrerie Michel
- Ouada Hafedh Ben
Sensors and Actuators B: Chemical, Elsevier , 2015, 220, pp.1346-1353 . Water-soluble CdSe quantum dots (QDs) were synthesized using thioglycerol (TG) as the surface capping agent through a one-step process at low temperature T (100 degrees C). The CdSe quantum dots were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, UV-visible absorption and fluorescence spectroscopies. These measurements revealed that the TG-capped CdSe QDs possess a high crystalline quality with an average diameter in the range 2.5-2.8 nm and exhibit particular optical properties. The UV-visible absorption of CdSe QDs is enhanced by the addition of cadmium ions, with a simultaneous shift of the edge band (400 nm), while seventeen other tested metal cations have no effect on the absorption of QDs. Moreover, the binding of Cd2+ ions induces a quenching of the fluorescence emission of TG-CdSe QDs. At particular absorption wavelengths, the response is linearly proportional to the cadmium ions concentration ranging from 1.0 to 22 mu M with a detection limit of 0.32 mu M (37 mu g L-1). Based on these optical properties, the TG-CdSe QDs could be used as a highly selective probe for the detection of Cd2+ ions in aqueous solutions, a species highly toxic for cells. (C) 2015 Elsevier B.V. All rights reserved. (10.1016/j.snb.2015.07.049)
DOI : 10.1016/j.snb.2015.07.049