Articles

Regulation of the ROS Response Dynamics and Organization to PDGF Motile Stimuli Revealed by Single Nanoparticle Imaging.
- Bouzigues Cedric I.
- Nguyên Thanh-Liêm
- Ramodiharilafy Rivo
- Claeson Amy
- Tharaux Pierre-Louis
- Alexandrou Antigoni
Chemistry and Biology, Elsevier , 2014, 21 (5), pp.647-56 . Although reactive oxygen species (ROS) are better known for their harmful effects, more recently, H2O2, one of the ROS, was also found to act as a secondary messenger. However, details of spatiotemporal organization of specific signaling pathways that H2O2 is involved in are currently missing. Here, we use single nanoparticle imaging to measure the local H2O2 concentration and reveal regulation of the ROS response dynamics and organization to platelet-derived growth factor (PDGF) signaling. We demonstrate that H2O2 production is controlled by PDGFR kinase activity and EGFR transactivation, requires a persistent stimulation, and is regulated by membrane receptor diffusion. This temporal filtering is impaired in cancer cells, which may determine their pathological migration. H2O2 subcellular mapping reveals that an external PDGF gradient induces an amplification-free asymmetric H2O2 concentration profile. These results support a general model for the control of signal transduction based only on membrane receptor diffusion and second messenger degradation. (10.1016/j.chembiol.2014.02.020)
DOI : 10.1016/j.chembiol.2014.02.020
Comparative Study of the Folding/Unfolding Dynamics of Poly(glutamic acid) in Light and Heavy Water
- Mendonça Lucille
- Steinbacher Andreas
- Bouganne Raphaël
- Hache François
Journal of Physical Chemistry B, American Chemical Society , 2014, 118 (20), pp.5350-5356 . The folding/unfolding equilibrium is investigated in poly(glutamic acid) (PGA) by two complementary sets of experiments: temperature-dependent steady-state circular dichroism spectra on the one hand and time-resolved circular dichroism measurements coupled with a T-jump experiment on the other hand. The experiments are performed for PGA dissolved in water for various pH values, as well as in heavy water. The kinetic and thermodynamic parameters extracted from these measurements are shown to be markedly different between light and heavy water, which is assigned to the difference in hydrogen bond energies in both solvents. (10.1021/jp501282z)
DOI : 10.1021/jp501282z
T-cell therapy using a bank of EBV-specific cytotoxic T cells: lessons from a phase I/II feasibility and safety study.
- Gallot Géraldine
- Vollant Solène
- Saïagh Soraya
- Clémenceau Béatrice
- Vivien Régine
- Cerato Evelyne
- Bignon Jean-D
- Ferrand Christophe
- Jaccard Arnaud
- Vigouroux Stéphane
- Choquet Sylvain
- Dalle Jean-Hugues
- Frachon Irène
- Bruno Bénédicte
- Mothy Mohamad
- Mechinaud Françoise
- Leblond Véronique
- Milpied Noël
- Vié Henri
Journal of Immunotherapy, Lippincott, Williams & Wilkins , 2014, 37 (3), pp.170-9 . We report herein the results we obtained and the limitations we experienced during the production and use of a bank of Epstein-Barr virus (EBV)-transformed human cytotoxic T lymphocytes (EBV-CTLs). To assess the feasibility and toxicity of this strategy, we selected and stored, in liquid nitrogen, 4 billion EBV-CTLs from each of the 13 selected donors. Subsequently, in a multicenter phase I/II study, 11 patients with EBV-associated lymphoma resistant to conventional treatments received 1-3 doses of 5 million EBV-CTLs/kg with 1-3 and 0-4 compatibilities for human leukocyte antigen (HLA)-I and HLA-II, respectively. Except for one event of fever after injection, no immediate or delayed toxicity, no graft versus host disease, and no graft rejection attributable to CTL infusion were observed. Three patients presented complete remission and 1 partial remission after treatment. Considering the clinical options currently available, and the constrains associated with CTL preparation and implementation, we conclude that CTL banks should consist of a reasonably small number of cell lines with documented specificities. This objective could be more easily achieved if the few homozygous donors for the most frequent HLA alleles of the targeted population could be made available for such a project. (10.1097/CJI.0000000000000031)
DOI : 10.1097/CJI.0000000000000031
Multiplex Cell and Lineage Tracking with Combinatorial Labels
- Loulier Karine
- Barry Raphaëlle
- Mahou Pierre
- Le franc Yann
- Supatto Willy
- Matho Katherine s.
- Ieng Siohoi
- Fouquet Stéphane
- Dupin Elisabeth
- Benosman Ryad
- Chédotal Alain
- Beaurepaire Emmanuel
- Morin Xavier
- Livet Jean
Neuron, Elsevier , 2014, 81 (3), pp.505–520 . (10.1016/j.neuron.2013.12.016)
DOI : 10.1016/j.neuron.2013.12.016
Substrate interaction dynamics and oxygen control in the active site of thymidylate synthase ThyX
- Becker Hubert f.
- Djaout Kamel
- Lamarre Isabelle
- Ulmer Jonathan e.
- Schaming Delphine
- Balland Véronique
- Liebl Ursula
- Myllykallio Hannu
- Vos Marten h.
Biochemical Journal, Portland Press , 2014, 459 (1), pp.37-45 . Thymidylate synthase ThyX, required for DNA synthesis in many pathogenic bacteria, is considered a promising antimicrobial target. It binds FAD and three substrates, producing dTMP (2′-deoxythymidine-5′-monophosphate) from dUMP (2′-deoxyuridine-5′-monophosphate). However, ThyX proteins also act as NADPH oxidase by reacting directly with O2. In the present study we investigated the dynamic interplay between the substrates and their role in competing with this wasteful and potentially harmful oxidase reaction in catalytically efficient ThyX from Paramecium bursaria Chlorella virus-1. dUMP binding accelerates the O2-insensitive half-reaction between NADPH and FAD by over four orders of magnitude to ~30 s−1. Thus, although dUMP does not have a direct role in FAD reduction, any turnover with molecular O2 requires its presence. Inversely, NADPH accommodation accelerates dUMP binding ~3-fold and apparently precedes dUMP binding under physiological conditions. In the oxidative half-reaction, excess CH2H4folate (N5,N10-methylene-5,6,7,8-tetrahydrofolate) was found to re-oxidize FADH2 within 1 ms, thus very efficiently competing with FADH2 oxidation by O2 (1.5 s−1 under aerobic conditions). The resulting reaction scheme points out how the interplay between the fast reactions with the native substrates, although not rate-limiting for overall catalysis, avoids NADPH oxidase activity in aerobic micro-organisms, including many pathogens. These observations also explain why ThyX proteins are also present in aerobic micro-organisms (10.1042/BJ20131567)
DOI : 10.1042/BJ20131567
Single YVO4:Eu nanoparticle emission spectra using direct Eu3+ ion excitation with a sum-frequency 465-nm solid-state laser
- Nguyên Thanh-Liêm
- Castaing Marc
- Gacoin Thierry
- Boilot Jean-Pierre
- Balembois François
- Georges Patrick
- Alexandrou Antigoni
Optics Express, Optical Society of America - OSA Publishing , 2014, 22 (17), pp.20542 . We report emission spectrum measurements on single YxEu1-xVO4 nanoparticles. The inhomogeneous widths of the emission peaks are identical for single nanoparticles and for ensembles of nanoparticles, while being broader than those of the bulk material. This indicates that individual nanoparticles are identical in terms of the distribution of different local Eu3+ sites due to crystalline defects and confirms their usability as identical, single-particle oxidant biosensors. Moreover, we report a 465 nm solid-state laser based on sum-frequency mixing that provides a compact, efficient solution for direct Eu3+ excitation of these nanoparticles. Both these two aspects should broaden the scope of Eu-doped nanoparticle applications. (10.1364/OE.22.020542)
DOI : 10.1364/OE.22.020542
Differential Interaction Kinetics of a Bipolar Structure- Specific Endonuclease with DNA Flaps Revealed by Single-Molecule Imaging Differential Interaction Kinetics of a Bipolar Structure-Specific Endonuclease with DNA Flaps
- Rezgui Rachid
- Lestini Roxane
- Künh Joëlle
- Fave Xenia
- Mcleod Lauren
- Myllykallio Hannu
- Alexandrou Antigoni
- Bouzigues Cedric
PLoS ONE, Public Library of Science , 2014 (november 20), pp.0113493 . As DNA repair enzymes are essential for preserving genome integrity, understanding their substrate interaction dynamics and the regulation of their catalytic mechanisms is crucial. Using single-molecule imaging, we investigated the association and dissociation kinetics of the bipolar endonuclease NucS from Pyrococcus abyssi (Pab) on 59 and 39-flap structures under various experimental conditions. We show that association of the PabNucS with ssDNA flaps is largely controlled by diffusion in the NucS-DNA energy landscape and does not require a free 59 or 39 extremity. On the other hand, NucS dissociation is independent of the flap length and thus independent of sliding on the single-stranded portion of the flapped DNA substrates. Our kinetic measurements have revealed previously unnoticed asymmetry in dissociation kinetics from these substrates that is markedly modulated by the replication clamp PCNA. We propose that the replication clamp PCNA enhances the cleavage specificity of NucS proteins by accelerating NucS loading at the ssDNA/dsDNA junctions and by minimizing the nuclease interaction time with its DNA substrate. Our data are also consistent with marked reorganization of ssDNA and nuclease domains occurring during NucS catalysis, and indicate that NucS binds its substrate directly at the ssDNA-dsDNA junction and then threads the ssDNA extremity into the catalytic site. The powerful techniques used here for probing the dynamics of DNA-enzyme binding at the single-molecule have provided new insight regarding substrate specificity of NucS nucleases. (10.1371/journal.pone.0113493)
DOI : 10.1371/journal.pone.0113493
Investigating the Cell Membrane via Single Particle Tracking, Bayesian Inference and Hydrodynamic Force Application
- Richly Maximilian
- Türkcan Silvan
- Bouzigues Cédric
- Popoff Michel R.
- Masson Jean-Baptiste
- Allain Jean-Marc
- Alexandrou Antigoni
Biophysical Journal, Biophysical Society , 2014, 106 (2), pp.p633a . We investigate the potential felt by membrane receptors inside membrane microdomains via tracking of single receptors labelled with rare-earth doped luminescent nanoparticles and Bayesian inference analysis of the recorded trajectories. We demonstrated that the potential felt by peptidic toxin receptors confined in lipid rafts is well described by a second-order polynomial potential, possibly due to an inhomogeneous lipid and protein distribution [Türkcan et al., Biophys. J. 2012] In contrast, the potential experienced by transferrin receptors in cytoskeleton-delimited microdomains is localized at the border of the confinement domain. (10.1016/j.bpj.2013.11.3504)
DOI : 10.1016/j.bpj.2013.11.3504
Advances in whole-embryo imaging: a quantitative transition is underway
- Pantazis Periklis
- Supatto Willy
Nature Reviews Molecular Cell Biology, Nature Publishing Group , 2014, 15, pp.327-339 . With the advent of imaging probes and live microscopy, developmental biologists have markedly extended our understanding of the molecular and cellular details of embryonic development. To fully comprehend the complex mechanistic framework that forms the developing organism, quantitative studies with high fidelity in space and time are now required. We discuss how integrating established, newly introduced and future imaging tools with quantitative analysis will ensure that imaging can fulfil its promise to elucidate how new life begins. (10.1038/nrm3786)
DOI : 10.1038/nrm3786
Calibrating optical tweezers with Bayesian inference
- Richly Maximilian U.
- Türkcan Silvan
- Le Gall Antoine
- Fiszman Nicolas
- Masson Jean-Baptiste
- Westbrook Nathalie
- Perronet Karen
- Alexandrou Antigoni
Optics Express, Optical Society of America - OSA Publishing , 2013, 21 (25), pp.31578 . We present a new method for calibrating an optical-tweezer setup that does not depend on input parameters and is less affected by systematic errors like drift of the setup. It is based on an inference approach that uses Bayesian probability to infer the diffusion coefficient and the potential felt by a bead trapped in an optical or magnetic trap. It exploits a much larger amount of the information stored in the recorded bead trajectory than standard calibration approaches. We demonstrate that this method outperforms the equipartition method and the power-spectrum method in input information required (bead radius and trajectory length) and in output accuracy. (10.1364/OE.21.031578)
DOI : 10.1364/OE.21.031578
Intracellular dynamics of archaeal FANCM homologue Hef in response to halted DNA replication.
- Lestini Roxane
- Laptenok Sergey P.
- Kühn Joëlle
- Hink Mark A
- Schanne-Klein Marie-Claire
- Liebl Ursula
- Myllykallio Hannu
Nucleic Acids Research, Oxford University Press , 2013, 41 (22), pp.10358-10370 . Hef is an archaeal member of the DNA repair endonuclease XPF (XPF)/Crossover junction endonuclease MUS81 (MUS81)/Fanconi anemia, complementation group M (FANCM) protein family that in eukaryotes participates in the restart of stalled DNA replication forks. To investigate the physiological roles of Hef in maintaining genome stability in living archaeal cells, we studied the localization of Hef-green fluorescent protein fusions by fluorescence microscopy. Our studies revealed that Haloferax volcanii Hef proteins formed specific localization foci under regular growth conditions, the number of which specifically increased in response to replication arrest. Purification of the full-length Hef protein from its native host revealed that it forms a stable homodimer in solution, with a peculiar elongated configuration. Altogether our data indicate that the shape of Hef, significant physicochemical constraints and/or interactions with DNA limit the apparent cytosolic diffusion of halophilic DNA replication/repair complexes, and demonstrate that Hef proteins are dynamically recruited to archaeal eukaryotic-like chromatin to counteract DNA replication stress. We suggest that the evolutionary conserved function of Hef/FANCM proteins is to enhance replication fork stability by directly interacting with collapsed replication forks. (10.1093/nar/gkt816)
DOI : 10.1093/nar/gkt816
From single cells to tissues: interactions between the matrix and human breast cells in real time.
- Barnes Clifford
- Speroni Lucia
- Quinn Kyle P
- Montévil Maël
- Saetzler Kurt
- Bode-Animashaun Gbemisola
- Mckerr George
- Georgakoudi Irene
- Downes C Stephen
- Sonnenschein Carlos
- Howard C Vyvyan
- Soto Ana
PLoS ONE, Public Library of Science , 2013, 9 (4), pp.e93325 . Mammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma--epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis. The formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology. Localized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs. (10.1371/journal.pone.0093325)
DOI : 10.1371/journal.pone.0093325
Ultrafast carbonyl motion of the Photoactive Yellow Protein chromophore probed by femtosecond circular dichroism
- Mendonça Lucille
- Hache François
- Changenet-Barret Pascale
- Plaza Pascal
- Chosrowjan Haik
- Taniguchi Seiji
- Imamoto Yasushi
Journal of the American Chemical Society, American Chemical Society , 2013, 135, pp.14637 . Motions of the trans-p-coumaric acid carbonyl group following the photoexcitation of the R52Q mutant of Photoactive Yellow Protein (PYP) are investigated, for the first time, by ultrafast time-resolved circular dichroism (TRCD) spectroscopy. TRCD is monitored in the near ultraviolet, over a timescale of 10 ps. Immediately after excitation, TRCD is found to exhibit a large negative peak, which decays within a few picoseconds. A quantitative analysis of the signals shows that, upon excitation, the carbonyl group undergoes a fast (< (10.1021/ja404503q)
DOI : 10.1021/ja404503q
A Bottom-Up Approach to Build the Hyperpolarizability of Peptides and Proteins from their Amino-Acids
- Duboisset Julien
- Deniset-Besseau Ariane
- Benichou Emmanuel
- Russier-Antoine Isabelle
- Lascoux Noëlle
- Jonin Christian
- Hache François
- Schanne-Klein Marie-Claire
- Brevet Pierre-Francois
Journal of Physical Chemistry B, American Chemical Society , 2013, 117 (34), pp.9877-9881 . We experimentally demonstrate that some peptides and proteins lend themselves to an elementary analysis where their first hyperpolarizability can be decomposed into the coherent superposition of the first hyperpolarizability of their elementary units. We then show that those elementary units can be associated with the amino acids themselves in the case of nonaromatic amino acids and nonresonant second harmonic generation. As a case study, this work investigates the experimentally determined first hyperpolarizability of rat tail Type I collagen and compares it to that of the shorter peptide [(PPG)10]3, where P and G are the one-letter code for Proline and Glycine, respectively, and that of the triamino acid peptides PPG and GGG. An absolute value of (0.16 ± 0.01) × 10-30 esu for the first hyperpolarizability of nonaromatic amino acids is then obtained by using the newly defined 0.087 × 10-30 esu reference value for water. By using a collagen like model, the microscopic hyperpolarizability along the peptide bond can be evaluated at (0.7 ± 0.1) × 10-30 esu. (10.1021/jp312574q)
DOI : 10.1021/jp312574q
Receptor displacement in the cell membrane by hydrodynamic force amplification through nanoparticles.
- Türkcan Silvan
- Richly Maximilian
- Bouzigues Cedric I.
- Allain Jean-Marc
- Alexandrou Antigoni
Biophysical Journal, Biophysical Society , 2013, 105 (1), pp.116-126 . We introduce an intrinsically multiplexed and easy to implement method to apply an external force to a biomolecule and thus probe its interaction with a second biomolecule or, more generally, its environment (for example, the cell membrane). We take advantage of the hydrodynamic interaction with a controlled fluid flow within a microfluidic channel to apply a force. By labeling the biomolecule with a nanoparticle that acts as a kite and increases the hydrodynamic interaction with the fluid, the drag induced by convection becomes important. We use this approach to track the motion of single membrane receptors, the Clostridium perfringens ε-toxin (CPεT) receptors that are confined in lipid raft platforms, and probe their interaction with the environment. Under external force, we observe displacements over distances up to 10 times the confining domain diameter due to elastic deformation of a barrier and return to the initial position after the flow is stopped. Receptors can also jump over such barriers. Analysis of the receptor motion characteristics before, during, and after a force is applied via the flow indicates that the receptors are displaced together with their confining raft platform. Experiments before and after incubation with latrunculin B reveal that the barriers are part of the actin cytoskeleton and have an average spring constant of 2.5 ± 0.6 pN/μm before vs. 0.6 ± 0.2 pN/μm after partial actin depolymerization. Our data, in combination with our previous work demonstrating that the ε-toxin receptor confinement is not influenced by the cytoskeleton, imply that it is the raft platform and its constituents rather than the receptor itself that encounters and deforms the barriers formed by the actin cytoskeleton. (10.1016/j.bpj.2013.05.045)
DOI : 10.1016/j.bpj.2013.05.045
Discovery of two new inhibitors of Botrytis cinerea chitin synthase by a chemical library screening.
- Magellan Hervé
- Boccara Martine
- Drujon Thierry
- Soulié Marie-Christine
- Guillou Catherine
- Dubois Joëlle
- Becker Hubert F.
Bioorganic and Medicinal Chemistry, Elsevier , 2013, 21 (17), pp.4997–5003 . : Chitin synthases polymerize UDP-GlcNAC to form chitin polymer, a key component of fungal cell wall biosynthesis. Furthermore, chitin synthases are desirable targets for fungicides since chitin is absent in plants and mammals. Two potent Botrytis cinerea chitin synthase inhibitors, 2,3,5-tri-O-benzyl-d-ribose (compound 1) and a 2,5-functionalized imidazole (compound 2) were identified by screening a chemical library. We adapted the wheat germ agglutinin (WGA) test for chitin synthase activity detection to allow miniaturization and robotization of the screen. Both identified compounds inhibited chitin synthases in vitro with IC50 values of 1.8 and 10μM, respectively. Compounds 1 and 2 were evaluated for their antifungal activity and were found to be active against B. cinerea BD90 strain with MIC values of 190 and 100μM, respectively. Finally, we discovered that both compounds confer resistance to plant leaves against the attack of the fungus by reducing the propagation of lesions by 37% and 23%, respectively. Based on the inhibitory properties found in different assays, compounds 1 and 2 can be considered as antifungal hit inhibitors of chitin synthase, allowing further optimization of their pharmacological profile to improve their antifungal properties. (10.1016/j.bmc.2013.06.058)
DOI : 10.1016/j.bmc.2013.06.058
Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX.
- Laptenok Sergey P.
- Bouzhir-Sima Latifa
- Lambry Jean-Christophe
- Myllykallio Hannu
- Liebl Ursula
- Vos Marten H.
Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2013, 110 (22), pp.8924-8929 . In many bacteria the flavoenzyme thymidylate synthase ThyX produces the DNA nucleotide deoxythymidine monophosphate from dUMP, using methylenetetrahydrofolate as carbon donor and NADPH as hydride donor. Because all three substrates bind in close proximity to the catalytic flavin adenine dinucleotide group, substantial flexibility of the ThyX active site has been hypothesized. Using femtosecond time-resolved fluorescence spectroscopy, we have studied the conformational heterogeneity and the conformational interconversion dynamics in real time in ThyX from the hyperthermophilic bacterium Thermotoga maritima. The dynamics of electron transfer to excited flavin adenine dinucleotide from a neighboring tyrosine residue are used as a sensitive probe of the functional dynamics of the active site. The fluorescence decay spanned a full three orders of magnitude, demonstrating a very wide range of conformations. In particular, at physiological temperatures, multiple angstrom cofactor-residue displacements occur on the picoseconds timescale. These experimental findings are supported by molecular dynamics simulations. Binding of the dUMP substrate abolishes this flexibility and stabilizes the active site in a configuration where dUMP closely interacts with the flavin cofactor and very efficiently quenches fluorescence itself. Our results indicate a dynamic selected-fit mechanism where binding of the first substrate dUMP at high temperature stabilizes the enzyme in a configuration favorable for interaction with the second substrate NADPH, and more generally have important implications for the role of active site flexibility in enzymes interacting with multiple poly-atom substrates and products. Moreover, our data provide the basis for exploring the effect of inhibitor molecules on the active site dynamics of ThyX and other multisubstrate flavoenzymes. (10.1073/pnas.1218729110)
DOI : 10.1073/pnas.1218729110
Structure-Based Discovery of the Novel Antiviral Properties of Naproxen against the Nucleoprotein of Influenza A Virus
- Lejal Nathalie
- Tarus Bogdan
- Bouguyon Edwige
- Chenavas Sylvie
- Bertho Nicolas
- Delmas Bernard
- Ruigrok Rob W H
- Di Primo Carmelo
- Slama-Schwok Anny
Antimicrobial Agents and Chemotherapy, American Society for Microbiology , 2013, 57 (5), pp.2231 - 2242 . The nucleoprotein (NP) binds the viral RNA genome and associates with the polymerase in a ribonucleoprotein complex (RNP) required for transcription and replication of influenza A virus. NP has no cellular counterpart, and the NP sequence is highly conserved, which led to considering NP a hot target in the search for antivirals. We report here that monomeric nucleoprotein can be inhibited by a small molecule binding in its RNA binding groove, resulting in a novel antiviral against influenza A virus. We identified naproxen, an anti-inflammatory drug that targeted the nucleoprotein to inhibit NP-RNA association required for NP function, by virtual screening. Further docking and molecular dynamics (MD) simulations identified in the RNA groove two NP-naproxen complexes of similar levels of interaction energy. The predicted naproxen binding sites were tested using the Y148A, R152A, R355A, and R361A proteins carrying single-point mutations. Surface plasmon resonance, fluorescence, and other in vitro experiments supported the notion that naproxen binds at a site identified by MD simulations and showed that naproxen competed with RNA binding to wild-type (WT) NP and protected active monomers of the nucleoprotein against proteolytic cleavage. Naproxen protected Madin-Darby canine kidney (MDCK) cells against viral challenges with the H1N1 and H3N2 viral strains and was much more effective than other cyclooxygenase inhibitors in decreasing viral titers of MDCK cells. In a mouse model of intranasal infection, naproxen treatment decreased the viral titers in mice lungs. In conclusion, naproxen is a promising lead compound for novel antivirals against influenza A virus that targets the nucleoprotein in its RNA binding groove. (10.1128/AAC.02335-12)
DOI : 10.1128/AAC.02335-12
Monomeric Nucleoprotein of Influenza A Virus
- Chenavas Sylvie
- Estrozi Leandro F.
- Slama-Schwok Anny
- Delmas Bernard
- Di Primo Carmelo
- Baudin Florence
- Li Xinping
- Crépin Thibaut
- Ruigrok Rob W. H.
PLoS Pathogens, Public Library of Science , 2013, 9 (3) . Isolated influenza A virus nucleoprotein exists in an equilibrium between monomers and trimers. Samples containing only monomers or only trimers can be stabilized by respectively low and high salt. The trimers bind RNA with high affinity but remain trimmers, whereas the monomers polymerise onto RNA forming nucleoprotein-RNA complexes. When wild type (wt) nucleoprotein is crystallized, it forms trimers, whether one starts with monomers or trimers. We therefore crystallized the obligate monomeric R416A mutant nucleoprotein and observed how the domain exchange loop that leads over to a neighbouring protomer in the trimer structure interacts with equivalent sites on the mutant monomer surface, avoiding polymerisation. The C-terminus of the monomer is bound to the side of the RNA binding surface, lowering its positive charge. Biophysical characterization of the mutant and wild type monomeric proteins gives the same results, suggesting that the exchange domain is folded in the same way for the wild type protein. In a search for how monomeric wt nucleoprotein may be stabilized in the infected cell we determined the phosphorylation sites on nucleoprotein isolated from virus particles. We found that serine 165 was phosphorylated and conserved in all influenza A and B viruses. The S165D mutant that mimics phosphorylation is monomeric and displays a lowered affinity for RNA compared with wt monomeric NP. This suggests that phosphorylation may regulate the polymerisation state and RNA binding of nucleoprotein in the infected cell. The monomer structure could be used for finding new anti influenza drugs because compounds that stabilize the monomer may slow down viral infection. (10.1371/journal.ppat.1003275)
DOI : 10.1371/journal.ppat.1003275
Multimodal Highlighting of Structural Abnormalities in Diabetic Rat and Human Corneas.
- Kowalczuk Laura
- Latour Gaël
- Bourges Jean-Louis
- Savoldelli Michèle
- Jeanny Jean-Claude
- Plamann Karsten
- Schanne-Klein Marie-Claire
- Behar-Cohen Françine
Translational vision science & technology, Association for Research in Vision and Ophthalmology (ARVO) , 2013, 2 (2), pp.3 . PURPOSE: This study aimed to highlight structural corneal changes in a model of type 2 diabetes, using in vivo corneal confocal microscopy (CCM). The abnormalities were also characterized by transmission electron microscopy (TEM) and second harmonic generation (SHG) microscopy in rat and human corneas. METHODS: Goto-Kakizaki (GK) rats were observed at age 12 weeks (n = 3) and 1 year (n = 6), and compared to age-matched controls. After in vivo CCM examination, TEM and SHG microscopy were used to characterize the ultrastructure and the three-dimensional organization of the abnormalities. Human corneas from diabetic (n = 3) and nondiabetic (n = 3) patients were also included in the study. RESULTS: In the basal epithelium of GK rats, CCM revealed focal hyper-reflective areas, and histology showed proliferative cells with irregular basement membrane. In the anterior stroma, extracellular matrix modifications were detected by CCM and confirmed in histology. In the Descemet's membrane periphery of all the diabetic corneas, hyper-reflective deposits were highlighted using CCM and characterized as long-spacing collagen fibrils by TEM. SHG microscopy revealed these deposits with high contrast, allowing specific detection in diabetic human and rat corneas without preparation and characterization of their three-dimensional organization. CONCLUSION: Pathologic findings were observed early in the development of diabetes in GK rats. Similar abnormalities have been found in corneas from diabetic patients. TRANSLATIONAL RELEVANCE: This multidisciplinary study highlights diabetes-induced corneal abnormalities in an animal model, but also in diabetic donors. This could constitute a potential early marker for diagnosis of hyperglycemia-induced tissue changes. (10.1167/tvst.2.2.3)
DOI : 10.1167/tvst.2.2.3
Probing ordered lipid assemblies with polarized third-harmonic-generation microscopy
- Zimmerley Maxwell
- Mahou Pierre
- Débarre Delphine
- Schanne-Klein Marie-Claire
- Beaurepaire Emmanuel
Physical Review X, American Physical Society , 2013, 3 (1), pp.11002 . Ordered lipid assemblies are responsible for important physiological functions including skin barrier and axon conductivity. However, techniques commonly used to probe molecular order such as X-ray scattering and nuclear magnetic resonance are not suited for in-situ tissue studies. Here, we identify and characterize a novel contrast mechanism in nonlinear optical microscopy which is sensitive to molecular ordering in multilamellar lipid vesicles (MLVs) and in samples obtained from human skin biopsy: polarized third-harmonic generation (P-THG). We develop a multiscale theoretical framework to calculate the anisotropic, nonlinear optical response of lipid arrays as a function of molecular order. This analysis reveals that conserved carbon-carbon bond and aliphatic tail directionality are the atomic- and molecular-scale sources of the observed P-THG response, respectively. Agreement between calculations and experiments on lipid droplets and MLVs validates the use of P-THG as a probe of lipid ordering. Finally, we show that P-THG can be used to map molecular ordering in the multilamellar, intercorneocyte lipid matrix of the stratum corneum of human skin. These results provide the foundation for the use of P-THG in probing molecular order and highlight a novel biomedical application of multiphoton microscopy in an optically accessible tissue relevant to monitoring lipid-related disorder. (10.1103/PhysRevX.3.011002)
DOI : 10.1103/PhysRevX.3.011002
Non-invasive monitoring of cell metabolism and lipid production in 3D engineered human adipose tissues using label-free multiphoton microscopy
- Chang Tyler
- Zimmerley Maxwell
- Quinn Kyle P.
- Lamarre-Jouenne Isabelle
- Kaplan David
- Beaurepaire Emmanuel
- Georgakoudi Irene
Biomaterials, Elsevier , 2013, 34 (34), pp.8607-8616 . Non-linear optical microscopy methods can characterize over time multiple functional properties of engineered tissues during development. Here, we demonstrate how the combined use of third-harmonic generation (THG) and two-photon excited fluorescence (2PEF) imaging can provide direct quantitative biomarkers of adipogenic stem cell differentiation and metabolic state, respectively. Specifically, we imaged over nine weeks silk scaffolds embedded with human mesenchymal stem cells and exposed to either propagation (PM) or adipogenic differentiation media (AM). THG was employed to visualize the formation of lipid droplets. 2PEF was used to assess the metabolic state of the cells through the redox ratio defined based on the endogenous FAD and NADH fluorescence. The redox ratio of cells in the AM scaffold was significantly lower than that in the PM scaffold during week 5 and 9, and correlated with significant increases in lipid-to-cell volume ratio, and number and size of lipid droplets in the AM scaffold. These findings indicate that the decrease in redox ratio during adipogenic differentiation is associated with fatty acid synthesis and lipid accumulation. Our methods therefore enabled us to identify and measure dynamic correlations between lipid droplet formation and cell metabolic state, while providing insight on the spatial heterogeneity of the observed signals. (10.1016/j.biomaterials.2013.07.066)
DOI : 10.1016/j.biomaterials.2013.07.066
Probing membrane protein interactions with their lipid raft environment using single-molecule tracking and Bayesian inference analysis.
- Türkcan Silvan
- Richly Maximilian U.
- Alexandrou Antigoni
- Masson Jean-Baptiste
PLoS ONE, Public Library of Science , 2013, 8 (1), pp.e53073 . The statistical properties of membrane protein random walks reveal information on the interactions between the proteins and their environments. These interactions can be included in an overdamped Langevin equation framework where they are injected in either or both the friction field and the potential field. Using a Bayesian inference scheme, both the friction and potential fields acting on the ε-toxin receptor in its lipid raft have been measured. Two types of events were used to probe these interactions. First, active events, the removal of cholesterol and sphingolipid molecules, were used to measure the time evolution of confining potentials and diffusion fields. Second, passive rare events, de-confinement of the receptors from one raft and transition to an adjacent one, were used to measure hopping energies. Lipid interactions with the ε-toxin receptor are found to be an essential source of confinement. ε-toxin receptor confinement is due to both the friction and potential field induced by cholesterol and sphingolipids. Finally, the statistics of hopping energies reveal sub-structures of potentials in the rafts, characterized by small hopping energies, and the difference of solubilization energy between the inner and outer raft area, characterized by higher hopping energies. (10.1371/journal.pone.0053073)
DOI : 10.1371/journal.pone.0053073
Picosecond binding of the his ligand to four-coordinate heme in cytochrome c ': A one-way gate for releasing proximal NO
- Yoo Byung-Kuk
- Lamarre Isabelle
- Martin Jean-Louis
- Andrew Colin R.
- Negrerie Michel
Journal of the American Chemical Society, American Chemical Society , 2013, 135 (8), pp.3248-3254 . We provide a direct demonstration of a "kinetic trap" mechanism in the proximal 5-coordinate heme-nitrosyl complex (5c-NO) of cytochrome c' from Alcaligenes xylosoxidans (AXCP) in which picosecond rebinding of the endogenous His ligand following heme-NO dissociation acts as a one-way gate for the release of proximal NO into solution. This demonstration is based upon picosecond transient absorption changes following NO photodissociation of the proximal 5c-NO AXCP complex. We have determined the absolute transient absorption spectrum of 4-coordinate ferrous heme to which NO rebinds with a time constant tNO = 7 ps (kNO = 1.4 × 1011 s-1) and shown that rebinding of the proximal histidine to the 4-coordinate heme takes place with a time constant tHis = 100 ± 10 ps (kHis = 1010 s-1) after the release of NO from the proximal heme pocket. This rapid His reattachment acts as a one-way gate for releasing proximal NO by precluding direct proximal NO rebinding once it has left the proximal heme pocket and requiring NO rebinding from solution to proceed via the distal heme face. Cop. 2013 American Chemical Society. (10.1021/ja312140f)
DOI : 10.1021/ja312140f
Arbitrary-detuning asynchronous optical sampling pump-probe spectroscopy of bacterial reaction centers
- Antonucci Laura
- Bonvalet Adeline
- Solinas Xavier
- Jones Mickael R.
- Vos Marten H.
- Joffre Manuel
Optics Letters, Optical Society of America - OSA Publishing , 2013, 38 (17), pp.3322-3324 . A recently reported variant of asynchronous optical sampling compatible with arbitrary unstabilized laser repetition rates is applied to pump-probe spectroscopy. This makes possible the use of a 5.1 MHz chirped pulse oscillator as the pump laser, thus extending the available time window to almost 200 ns with a time resolution as good as about 320 fs. The method is illustrated with the measurement in a single experiment of the complete charge transfer dynamics of the reaction center from Rhodobacter sphaeroides. © 2013 Optical Society of America (10.1364/OL.38.003322)
DOI : 10.1364/OL.38.003322