Articles

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
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
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
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
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
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
An Overview of data science uses in bioimage informatics
- Chessel Anatole
Methods, Elsevier , 2017, 115, pp.110 - 118 . (10.1016/j.ymeth.2016.12.014)
DOI : 10.1016/j.ymeth.2016.12.014
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
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
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
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
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
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
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
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
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
Ultra-wide range field-dependent measurements of the relaxivity of Gd1−xEuxVO4 nanoparticle contrast agents using a mechanical sample-shuttling relaxometer
- Chou Ching-Yu
- Abdesselem Mouna
- Bouzigues Cedric
- Chu Minglee
- Guiga Angelo
- Huang Tai-Huang
- Ferrage Fabien
- Gacoin Thierry
- Alexandrou Antigoni
- Sakellariou Dimitris
Scientific Reports, Nature Publishing Group , 2017, 7, pp.44770 . The current trend for Magnetic Resonance Imaging points towards higher magnetic fields. Even though sensitivity and resolution are increased in stronger fields, T1 contrast is often reduced, and this represents a challenge for contrast agent design. Field-dependent measurements of relaxivity are thus important to characterize contrast agents. At present, the field-dependent curves of relaxivity are usually carried out in the field range of 0 T to 2 T, using fast field cycling relaxometers. Here, we employ a high-speed sample shuttling device to switch the magnetic fields experienced by the nuclei between virtually zero field, and the center of any commercial spectrometer. We apply this approach on rare-earth (mixed Gadolinium-Europium) vanadate nanoparticles, and obtain the dispersion curves from very low magnetic field up to 11.7 T. In contrast to the relaxivity profiles of Gd chelates, commonly used for clinical applications, which display a plateau and then a decrease for increasing magnetic fields, these nanoparticles provide maximum contrast enhancement for magnetic fields around 1–1.5 T. These field-dependent curves are fitted using the so-called Magnetic Particle (MP) model and the extracted parameters discussed as a function of particle size and composition. We finally comment on the new possibilities offered by this approach. (10.1038/srep44770)
DOI : 10.1038/srep44770
Correction: Ultrafast photochemistry of the bc 1 complex
- Vos Marten H.
- Reeder Brandon J.
- Daldal Fevzi
- Liebl Ursula
Physical Chemistry Chemical Physics, Royal Society of Chemistry , 2017, 19 (13), pp.9320 - 9320 . (10.1039/C7CP90057K)
DOI : 10.1039/C7CP90057K
Ultrafast photochemistry of the bc 1 complex
- Vos Marten H.
- Reeder Brandon J.
- Daldal Fevzi
- Liebl Ursula
Physical Chemistry Chemical Physics, Royal Society of Chemistry , 2017, 19 (9), pp.6807-6813 . We present a full investigation of ultrafast light-induced events in the membraneous cytochrome bc1 complex by transient absorption spectroscopy. This energy-transducing complex harbors four redox-active components per monomer: heme c1, two 6-coordinate b-hemes and a [2Fe-2S] cluster. Using excitation of these components in different ratios under various excitation conditions, probing in the full visible range and under three well-defined redox conditions, we demonstrate that for all ferrous hemes of the complex photodissociation of axial ligands takes place and that they rebind in 5-7 ps, as in other 6-coordinate heme proteins, including cytoglobin, which is included as a reference in this study. By contrast, the signals are not consistent with photooxidation of the b hemes. This conclusion contrasts with a recent assessment based on a more limited data set. The binding kinetics of internal and external ligands are indicative of a rigid heme environment, consistent with the electron transfer function. We also report, for the first time, photoactivity of the very weakly absorbing iron-sulfur center. This yields the unexpected perspective of studying photochemistry, initiated by excitation of iron-sulfur clusters, in a range of protein complexes. (10.1039/C7CP00193B)
DOI : 10.1039/C7CP00193B