Articles

Large-scale live imaging of adult neural stem cells in their endogenous niche
- Dray Nicolas
- Bedu Sébastien
- Vuillemin Nelly
- Alunni Alessandro
- Coolen Marion
- Krecsmarik Monika
- Supatto Willy
- Beaurepaire Emmanuel
- Bally-Cuif Laure
Development (Cambridge, England), Company of Biologists , 2015, 142 (20), pp.3592-3600 . Live imaging of adult neural stem cells (aNSCs) in vivo is a technical challenge in the vertebrate brain. Here, we achieve long-term imaging of the adult zebrafish telencephalic neurogenic niche and track a population of >1000 aNSCs over weeks, by taking advantage of fish transparency at near-infrared wavelengths and of intrinsic multiphoton landmarks. This methodology enables us to describe the frequency, distribution and modes of aNSCs divisions across the entire germinal zone of the adult pallium, and to highlight regional differences in these parameters. (10.1242/dev.123018)
DOI : 10.1242/dev.123018
High-power, widely tunable, room-temperature picosecond optical parametric oscillator based on cylindrical 5%MgO:PPLN
- Chaitanya Kumar S.
- Wei Junxiong
- Debray J.
- Kemlin Vincent
- Boulanger B.
- Ishizuki Hideki
- Taira T.
- Ebrahim-Zadeh M.
Optics Letters, Optical Society of America - OSA Publishing , 2015, 40 (16), pp.3897-3900 . We report a high-power picosecond optical parametric oscillator (OPO) based on cylindrical MgO:PPLN synchronously pumped by an Yb-fiber laser. The singly resonant OPO is tunable in the near-infrared signal across 1413–1900 nm and mid-infrared idler over 2418–4307 nm by angle tuning of the crystal at room temperature. With non-optimized output coupling of ∼10%, the OPO simultaneously delivers 2.4 W of signal at 1664 nm and 1.7 W of idler at 2950 nm at an overall extraction efficiency of ∼45% with high beam-pointing stability <30 μrad and <14 μrad for the signal and idler, respectively. The generated signal and idler exhibit passive power stability better than 1% rms and 0.8% rms over 15 h, respectively, in high beam quality with TEM00 profile. The extracted signal pulses from the OPO have duration of 15.2 ps with a spectral bandwidth of 0.7 nm, corresponding to a time-bandwidth product of ΔvΔtau∼1.2. (10.1364/OL.40.003897)
DOI : 10.1364/OL.40.003897
Optimization of Picrosirius red staining protocol to determine collagen fiber orientations in vaginal and uterine cervical tissues by Mueller polarized microscopy
- Nazac André
- Bancelin Stéphane
- Teig Benjamin
- Haj Ibrahim Bicher
- Fernandez Hervé
- Schanne-Klein Marie-Claire
- de Martino Antonello
Microscopy Research and Technique, Wiley , 2015, 78 (8), pp.723-730 . Polarized microscopy provides unique information on anisotropic samples. In its most complete implementation, namely Mueller microscopy, this technique is well suited for the visualization of fibrillar proteins orientations, with collagen in the first place. However, the intrinsic optical anisotropy of unstained tissues has to be enhanced by Picrosirius Red (PR) staining to enable Mueller measurements. In this work, we compared the orientation mapping provided by Mueller and second harmonic generation (SHG) microscopies on PR stained samples of vaginal and uterine cervix tissues. SHG is a multiphoton technique that is highly specific to fibrillar collagen, and was taken as the gold standard for its visualization. We showed that Mueller microscopy can be safely used to determine collagen orientation in PR stained cervical tissue. In contrast, in vaginal samples, Mueller microscopy revealed orientations not only of collagen but also of other anisotropic structures. Thus PR is not fully specific to collagen, which necessitates comparison to SHG microscopy in every type of tissue. In addition to this study of PR specificity, we determined the optimal values of the staining parameters. We found that staining times of 5min, and sample thicknesses of 5 mu m were sufficient in cervical and vaginal tissues. Microsc. Res. Tech. 78:723-730, 2015. (c) 2015 Wiley Periodicals, Inc. (10.1002/jemt.22530)
DOI : 10.1002/jemt.22530
Kinetic Analysis of a Globin-Coupled Histidine Kinase, AfGcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity
- Fojtikova Veronika
- Stranava Martin
- Vos Marten H.
- Liebl Ursula
- Hranicek Jakub
- Kitanishi Kenichi
- Shimizu Toru
- Martinkova Marketa
Biochemistry, American Chemical Society , 2015, 54 (32), pp.5017–5029 . The globin-coupled histidine kinase, AfGcHK, is a part of the two-component signal transduction system from the soil bacterium Anaeromyxobacter sp. Fw109-5. Activation of its sensor domain significantly increases its autophosphorylation activity, which targets the His183 residue of its functional domain. The phosphate group of phosphorylated AfGcHK is then transferred to the cognate response regulator. We investigated the effects of selected variables on the autophosphorylation reaction’s kinetics. The kcat values of the heme Fe(III)-OH–, Fe(III)-cyanide, Fe(III)-imidazole, and Fe(II)-O2 bound active AfGcHK forms were 1.1–1.2 min–1, and their KmATP values were 18.9–35.4 μM. However, the active form bearing a CO-bound Fe(II) heme had a kcat of 1.0 min–1 but a very high KmATP value of 357 μM, suggesting that its active site structure differs strongly from the other active forms. The Fe(II) heme-bound inactive form had kcat and KmATP values of 0.4 min–1 and 78 μM, respectively, suggesting that its low activity reflects a low affinity for ATP relative to that of the Fe(III) form. The heme-free form exhibited low activity, with kcat and KmATP values of 0.3 min–1 and 33.6 μM, respectively, suggesting that the heme iron complex is essential for high catalytic activity. Overall, our results indicate that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme’s catalytic activity because they modulate its ATP binding affinity and thus change its kcat/KmATP value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discussed (10.1021/acs.biochem.5b00517)
DOI : 10.1021/acs.biochem.5b00517
Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease
- Camus S. M.
- de Moraes J. A.
- Bonnin P.
- Abbyad P.
- Le Jeune S.
- Lionnet F.
- Loufrani L.
- Grimaud L.
- Lambry J.-C.
- Charue D.
- Kiger L.
- Renard J.-M.
- Larroque C.
- Le Clesiau H.
- Tedgui A.
- Bruneval P.
- Barja-Fidalgo C.
- Alexandrou A.
- Tharaux P.-L.
- Boulanger C. M.
- Blanc-Brude O. P.
Blood, American Society of Hematology , 2015, 125 (24), pp.3805-3814 . Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that hemeladen MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of hemeladen MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD. (10.1182/blood-2014-07-589283)
DOI : 10.1182/blood-2014-07-589283
Ultrafast Dynamics of Carboxy-Hemoglobin: Two-Dimensional Infrared Spectroscopy Experiments and Simulations
- Falvo Cyril
- Daniault Louis
- Vieille Thibault
- Kemlin Vincent
- Lambry Jean-Christophe
- Meier Christoph
- Vos Marten H.
- Bonvalet Adeline
- Joffre Manuel
Journal of Physical Chemistry Letters, American Chemical Society , 2015, 6 (12), pp.2216–2222 . This Letter presents a comparison between experimental and simulated 2D mid-infrared spectra of carboxy-hemoglobin in the spectral region of the carbon monoxide stretching mode. The simulations rely on a fluctuating potential energy surface that includes both the effect of heme and the protein surroundings computed from molecular dynamics simulations. A very good agreement between theory and experiment is obtained with no adjustable parameters. The simulations show that the effect of the distal histidine through the hydrogen bond is strong and is directly responsible for the slow decay of the frequency–frequency correlation function on a 10 ps time scale. This study confirms that fluctuations in carboxy-hemoglobin are more inhomogeneous than those in the more frequently studied carboxy-myoglobin. The comparison between simulations and experiments brings valuable information on the complex relation between protein structure and spectral diffusion. (10.1021/acs.jpclett.5b00811)
DOI : 10.1021/acs.jpclett.5b00811
Targeting of Helicobacter pylori thymidylate synthase ThyX by non-mitotoxic hydroxy-naphthoquinones
- Skouloubris S.
- Djaout K.
- Lamarre I.
- Lambry J.-C.
- Anger K.
- Briffotaux J.
- Liebl U.
- de Reuse H.
- Myllykallio H.
Open Microbiology Journal, Bentham Open , 2015, 5 (6), pp.150015 . ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the functionally analogous human enzyme, thus providing means for selective inhibition of bacterial growth. To identify novel compounds with anti-bacterial activity against the human pathogenic bacterium Helicobacter pylori, based on our earlier biochemical and structural analyses, we designed a series of eighteen 2-hydroxy-1,4-naphthoquinones (2-OH-1,4-NQs) that target HpThyX. Our lead-like molecules markedly inhibited the NADPH oxidation and 2′-deoxythymidine-5′-monophosphate-forming activities of HpThyX enzyme in vitro, with inhibitory constants in the low nanomolar range. The identification of non-cytotoxic and non-mitotoxic 2-OH-1,4-NQ inhibitors permitted testing their in vivo efficacy in a mouse model for H. pylori infections. Despite the widely assumed toxicity of naphthoquinones (NQs), we identified tight-binding ThyX inhibitors that were tolerated in mice and can be associated with a modest effect in reducing the number of colonizing bacteria. Our results thus provide proof-of-concept that targeting ThyX enzymes is a highly feasible strategy for the development of therapies against H. pylori and a high number of other ThyX-dependent pathogenic bacteria. We also demonstrate that chemical reactivity of NQs does not prevent their exploitation as anti-microbial compounds, particularly when mitotoxicity screening is used to prioritize these compounds for further experimentation. (10.1098/rsob.150015)
DOI : 10.1098/rsob.150015
Hotspots in an obligate homodimeric anticancer target. Structural and functional effects of interfacial mutations in human thymidylate synthase.
- Salo-Ahen Outi M H
- Tochowicz Anna
- Pozzi Cecilia
- Cardinale Daniela
- Ferrari Stefania
- Boum Yap
- Mangani Stefano
- Stroud Robert M
- Saxena Puneet
- Myllykallio Hannu
- Costi Maria Paola
- Ponterini Glauco
- Wade Rebecca C
Journal of Medicinal Chemistry, American Chemical Society , 2015, 58 (8), pp.3572-81 . Human thymidylate synthase (hTS), a target for antiproliferative drugs, is an obligate homodimer. Single-point mutations to alanine at the monomer-monomer interface may enable the identification of specific residues that delineate sites for drugs aimed at perturbing the protein-protein interactions critical for activity. We computationally identified putative hotspot residues at the interface and designed mutants to perturb the intersubunit interaction. Dimer dissociation constants measured by a FRET-based assay range from 60 nM for wild-type hTS up to about 1 mM for single-point mutants and agree with computational predictions of the effects of these mutations. Mutations that are remote from the active site retain full or partial activity, although the substrate KM values were generally higher and the dimer was less stable. The lower dimer stability of the mutants can facilitate access to the dimer interface by small molecules and thereby aid the design of inhibitors that bind at the dimer interface. (10.1021/acs.jmedchem.5b00137)
DOI : 10.1021/acs.jmedchem.5b00137
Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase
- Yoo Byung-Kuk
- Lamarre Isabelle
- Martin Jean-Louis
- Rappaport Fabrice
- Negrerie Michel
Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2015, 112 (14), pp.E1697-704 . We investigated the changes of heme coordination in purified soluble guanylate cyclase (sGC) by time-resolved spectroscopy in a time range encompassing 11 orders of magnitude (from 1 ps to 0.2 s). After dissociation, NO either recombines geminately to the 4-coordinate (4c) heme (τG1 = 7.5 ps; 97 ± 1% of the population) or exits the heme pocket (3 ± 1%). The proximal His rebinds to the 4c heme with a 70-ps time constant. Then, NO is distributed in two approximately equal populations (1.5%). One geminately rebinds to the 5c heme (τG2 = 6.5 ns), whereas the other diffuses out to the solution, from where it rebinds bimolecularly (τ = 50 μs with [NO] = 200 μM) forming a 6c heme with a diffusion-limited rate constant of 2 × 10(8) M(-1)⋅s(-1). In both cases, the rebinding of NO induces the cleavage of the Fe-His bond that can be observed as an individual reaction step. Saliently, the time constant of bond cleavage differs depending on whether NO binds geminately or from solution (τ5C1 = 0.66 μs and τ5C2 = 10 ms, respectively). Because the same event occurs with rates separated by four orders of magnitude, this measurement implies that sGC is in different structural states in both cases, having different strain exerted on the Fe-His bond. We show here that this structural allosteric transition takes place in the range 1-50 μs. In this context, the detection of NO binding to the proximal side of sGC heme is discussed. (10.1073/pnas.1423098112)
DOI : 10.1073/pnas.1423098112
Theoretical, numerical and experimental study of geometrical parameters that affect anisotropy measurements in polarization-resolved SHG microscopy.
- Teulon Claire
- Gusachenko Ivan
- Latour Gaël
- Schanne-Klein Marie-Claire
Optics Express, Optical Society of America - OSA Publishing , 2015, 23 (7), pp.9313-28 . Polarization-resolved second harmonic generation (P-SHG) microscopy is an efficient imaging modality for in situ observation of biopolymers structure in tissues, providing information about their mean in-plane orientation and their molecular structure and 3D distribution. Nevertheless, P-SHG signal build-up in a strongly focused regime is not throroughly understood yet, preventing reliable and reproducible measurements. In this study, theoretical analysis, vectorial numerical simulations and experiments are performed to understand how geometrical parameters, such as excitation and collection numerical apertures and detection direction, affect P-SHG imaging in homogeneous collagen tissues. A good agreement is obtained in tendon and cornea, showing that detection geometry significantly affects the SHG anisotropy measurements, but not the measurements of collagen in-plane orientation. (10.1364/OE.23.009313)
DOI : 10.1364/OE.23.009313
Whole-brain functional imaging with two-photon light-sheet microscopy
- Wolf Sébastien
- Supatto Willy
- Debrégeas Georges
- Mahou Pierre
- Kruglik Sergei G
- Sintes Jean-Marc
- Beaurepaire Emmanuel
- Candelier Raphaël
Nature Methods, Nature Publishing Group , 2015, 12 (5), pp.379-380 . (10.1038/nmeth.3371)
DOI : 10.1038/nmeth.3371
Long-term consequences of invasive deer on songbird communities: Going from bad to worse?
- Chollet Simon
- Bergman Carita
- Gaston Anthony
- Martin Jean-Louis
Biological Invasions, Springer Verlag , 2015, 17 (2), pp.777-790 . Although we understand many initial effects of invasive deer on songbirds, we do not yet understand how their longer-term effects unfold. We looked for such potential long-term effects on songbirds in the context of an archipelago where deer were introduced over a century ago. Initial data consist of vegetation plots and songbird point-counts on 57 islands in Haida Gwaii (British Columbia, Canada) taken in 1989 when the impacts of introduced deer lacking predators had already developed for [50 years. Twenty years later, we surveyed these islands using the same methods. To isolate the effects of deer, we compare results to nearby islands never colonized by deer and assess how canopy birds have fared relative to understory birds. We also compare responses between islands of ‘‘moderate deer impact’’ where the understory vegetation was only moderately depleted by deer in 1989 and those with ‘‘severe deer impact’’ where understory vegetation was strongly depleted even in 1989. In 1989 all islands with deer were impoverished in songbirds that depend on understory vegetation, but the moderate impact islandsstill had richer and more abundant understory forestbird communities than found on severe impact islands.Islands with the fewest deer impacts in 1989 were small and isolated from sources of deer colonization.By 2009, severe deer impacts extended to islands that were initially less affected by deer. The severity ofimpacts also increased even on islands that had been dramatically affected by 1989. Declines in bird abundance occurred before declines in bird diversity. These results support the need for actions by wildlife managers to curtail deer impacts as soon as these become evident, especially in reserves and protected areas that lack hunters and other deer predators. (10.1007/s10530-014-0768-0)
DOI : 10.1007/s10530-014-0768-0
Time-resolved circular dichroism : what can we learn on conformational changes ?
- Hache François
Display and Imaging , 2015, 1 (3-4), pp.197-209 . Time-resolved circular dichroism (CD) measurements can yield relevant information on the dynamics of conformational changes in molecules on the condition that some a priori knowledge is obtained. This can be the use of simple models such as the excitonic coupling or the octant rule or the phenomenological relationship between far-UV CD and secondary structures in proteins for example. This article describes such experiments where CD has brought relevant conformational information.
Ex vivo multiscale quantitation of skin biomechanics in wild-type and genetically-modified mice using multiphoton microscopy
- Bancelin Stéphane
- Lynch Barbara
- Bonod-Bidaud Christelle
- Ducourthial Guillaume
- Psilodimitrakopoulos Sotiris
- Dokládal Petr
- Allain Jean-Marc
- Schanne-Klein Marie-Claire
- Ruggiero, Florence
Scientific Reports, Nature Publishing Group , 2015, 5, pp.17635 . Soft connective tissues such as skin, tendon or cornea are made of about 90% of extracellular matrix proteins, fibrillar collagens being the major components. Decreased or aberrant collagen synthesis generally results in defective tissue mechanical properties as the classic form of Elhers-Danlos syndrome (cEDS). This connective tissue disorder is caused by mutations in collagen V genes and is mainly characterized by skin hyperextensibility. To investigate the relationship between the microstructure of normal and diseased skins and their macroscopic mechanical properties, we imaged and quantified the microstructure of dermis of ex vivo murine skin biopsies during uniaxial mechanical assay using multiphoton microscopy. We used two genetically-modified mouse lines for collagen V: a mouse model for cEDS harboring a Col5a2 deletion (a.k.a. pN allele) and the transgenic K14-COL5A1 mice which overexpress the human COL5A1 gene in skin. We showed that in normal skin, the collagen fibers continuously align with stretch, generating the observed increase in mechanical stress. Moreover, dermis from both transgenic lines exhibited altered collagen reorganization upon traction, which could be linked to microstructural modifications. These findings show that our multiscale approach provides new crucial information on the biomechanics of dermis that can be extended to all collagen-rich soft tissues. (10.1038/srep17635)
DOI : 10.1038/srep17635
Quantitative measurement of permeabilization of living cells by terahertz attenuated total reflection
- Grognot Marianne
- Gallot Guilhem
Applied Physics Letters, American Institute of Physics , 2015, 107 (10), pp.103702 . Using Attenuated Total Reflection imaging technique in the terahertz domain, we demonstrate non-invasive, non-staining real time measurements of cytoplasm leakage during permeabilization of epithelial cells by saponin. The terahertz signal is mostly sensitive to the intracellular protein concentration in the cells, in a very good agreement with standard bicinchoninic acid protein measurements. It opens the way to in situ real time dynamics of protein content and permeabilization in live cells. (10.1063/1.4930168)
DOI : 10.1063/1.4930168
Time-resolved infrared spectroscopic studies of ligand dynamics in the active site from cytochrome c oxidase
- Vos Marten H.
- Liebl Ursula
Biochimica et Biophysica Acta (BBA) - Reviews on Bioenergetics, Elsevier , 2015, 1847 (1), pp.79–85 . The catalytic site of heme–copper oxidases encompasses two close-lying ligand binding sites: the heme, where oxygen is bound and reduced and the CuB atom, which acts as ligand entry and release port. Diatomic gaseous ligands with a dipole moment, such as the signaling molecules carbon monoxide (CO) and nitric oxide (NO), carry clear infrared spectroscopic signatures in the different states that allow characterization of the dynamics of ligand transfer within, into and out of the active site using time-resolved infrared spectroscopy. We review the nature and diversity of these processes that have in particular been characterized with CO as ligand and which take place on time scales ranging from femtoseconds to milliseconds. These studies have advanced our understanding of the functional ligand pathways and reactivity in enzymes and more globally represent intriguing model systems for mechanisms of ligand motion in a confined protein environment. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems. (10.1016/j.bbabio.2014.07.018)
DOI : 10.1016/j.bbabio.2014.07.018
In Vivo Single-Cell Detection of Metabolic Oscillations in Stem Cells
- Stringari Chiara
- Wang Hong
- Geyfman Mikhail
- Crosignani Viera
- Kumar Vivek
- Takahashi Joseph s.
- Andersen Bogi
- Gratton Enrico
Cell Reports, Elsevier Inc , 2015, 10 (1), pp.1-7 . Through the use of bulk measurements in metabolic organs, the circadian clock was shown to play roles in organismal energy homeostasis. However, the relationship between metabolic and circadian oscillations has not been studied in vivo at a single-cell level. Also, it is unknown whether the circadian clock controls metabolism in stem cells. We used a sensitive, noninvasive method to detect metabolic oscillations and circadian phase within epidermal stem cells in live mice at the single-cell level. We observe a higher NADH/NAD+ ratio, reflecting an increased glycolysis/oxidative phosphorylation ratio during the night compared to the day. Furthermore, we demonstrate that single-cell metabolic heterogeneity within the basal cell layer correlates with the circadian clock and that diurnal fluctuations in NADH/NAD+ ratio are Bmal1 dependent. Our data show that, in proliferating stem cells, the circadian clock coordinates activities of oxidative phosphorylation and glycolysis with DNA synthesis, perhaps as a protective mechanism against genotoxicity. (10.1016/j.celrep.2014.12.007)
DOI : 10.1016/j.celrep.2014.12.007
Multifunctional rare-Earth vanadate nanoparticles: luminescent labels, oxidant sensors, and MRI contrast agents.
- Abdesselem Mouna
- Schoeffel Markus
- Maurin Isabelle
- Ramodiharilafy Rivo
- Autret Gwennhael
- Clément Olivier
- Tharaux Pierre-Louis
- Boilot Jean-Pierre
- Gacoin Thierry
- Bouzigues Cedric
- Alexandrou Antigoni
ACS Nano, American Chemical Society , 2014, 8 (11), pp.11126-37 . Collecting information on multiple pathophysiological parameters is essential for understanding complex pathologies, especially given the large interindividual variability. We report here multifunctional nanoparticles which are luminescent probes, oxidant sensors, and contrast agents in magnetic resonance imaging (MRI). Eu(3+) ions in an yttrium vanadate matrix have been demonstrated to emit strong, nonblinking, and stable luminescence. Time- and space-resolved optical oxidant detection is feasible after reversible photoreduction of Eu(3+) to Eu(2+) and reoxidation by oxidants, such as H2O2, leading to a modulation of the luminescence emission. The incorporation of paramagnetic Gd(3+) confers in addition proton relaxation enhancing properties to the system. We synthesized and characterized nanoparticles of either 5 or 30 nm diameter with compositions of GdVO4 and Gd0.6Eu0.4VO4. These particles retain the luminescence and oxidant detection properties of YVO4:Eu. Moreover, the proton relaxivity of GdVO4 and Gd0.6Eu0.4VO4 nanoparticles of 5 nm diameter is higher than that of the commercial Gd(3+) chelate compound Dotarem at 20 MHz. Nuclear magnetic resonance dispersion spectroscopy showed a relaxivity increase above 10 MHz. Complexometric titration indicated that rare-earth leaching is negligible. The 5 nm nanoparticles injected in mice were observed with MRI to concentrate in the liver and the bladder after 30 min. Thus, these multifunctional rare-earth vanadate nanoparticles pave the way for simultaneous optical and magnetic resonance detection, in particular, for in vivo localization evolution and reactive oxygen species detection in a broad range of physiological and pathophysiological conditions.
Map the intracellular concentration of reactive oxygen species
- Bouzigues Cédric
- Alexandrou Antigoni
Médecine/Sciences, EDP Sciences , 2014, 30 (10), pp.848 - 850 . L’organisation dans le temps et l’espace des voies de signalisation est un élément essentiel dans le façonnage de la réponse cellulaire. Ceci est vrai notamment dans les processus chimiotactiques, où l’apparition d’une organisation intracellulaire asymétrique est physiologiquement indispensable. Dans ce contexte, la signalisation par les espèces oxygénées réactives, ou ROS, est singulière. En effet, ces molécules, comme le peroxyde d’hydrogène H2O2, sont connues principalement pour leur activité bactéricide et les dommages induits par le stress oxydant. Elles jouent cependant en parallèle un rôle essentiel dans plusieurs voies de signalisation physiologiques contrôlant des réponses variées – contraction, prolifération, migration – dans de nombreux tissus (systèmes nerveux, hépatique, vasculaire, etc.). L’homéostasie locale des ROS est donc un processus vital dans la vie cellulaire pour préserver l’action physiologique sans effet nuisible. (10.1051/medsci/20143010010)
DOI : 10.1051/medsci/20143010010
Determination of collagen fiber orientation in histological slides using Mueller microscopy and validation by second harmonic generation imaging.
- Bancelin Stéphane
- Nazac André
- Ibrahim Bicher Haj
- Dokládal Petr
- Decencière Etienne
- Teig Benjamin
- Haddad Huda
- Fernandez Hervé
- Schanne-Klein Marie-Claire
- de Martino Antonello
Optics Express, Optical Society of America - OSA Publishing , 2014, 22 (19), pp.22561-22574 . We studied the azimuthal orientations of collagen fibers in histological slides of uterine cervical tissue by two different microscopy techniques, namely Mueller polarimetry (MP) and Second Harmonic Generation (SHG). SHG provides direct visualization of the fibers with high specificity, which orientations is then obtained by suitable image processing. MP provides images of retardation (among other polarimetric parameters) due to the optical anisotropy of the fibers, which is enhanced by Picrosirius Red staining. The fiber orientations are then assumed to be those of the retardation slow axes. The two methods, though fully different from each other, provide quite similar maps of average fiber orientations. Overall, our results confirm that MP microscopy provides reliable images of dominant fiber orientations at a much lower cost that SHG, which remains the "gold standard" for specific imaging of collagen fibers using optical microscopy. (10.1364/OE.22.022561)
DOI : 10.1364/OE.22.022561
Dynamics of the Heme-binding Bacterial Gas-sensing Dissimilative Nitrate Respiration Regulator (DNR) and Activation Barriers for Ligand Binding and Escape.
- Lobato Laura
- Bouzhir-Sima Latifa
- Yamashita Taku
- Wilson Michael T
- Vos Marten H
- Liebl Ursula
Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology , 2014, 289 (38), pp.26514-24 . DNR (dissimilative nitrate respiration regulator) is a heme-binding transcription factor that is involved in the regulation of denitrification in Pseudomonas aeruginosa. In the ferrous deoxy state, the heme is 6-coordinate; external NO and CO can replace an internal ligand. Using fluorescence anisotropy, we show that high-affinity sequence-specific DNA binding occurs only when the heme is nitrosylated, consistent with the proposed function of DNR as NO sensor and transcriptional activator. This role is moreover supported by the NO "trapping" properties revealed by ultrafast spectroscopy that are similar to those of other heme-based NO sensor proteins. Dissociated CO-heme pairs rebind in an essentially barrierless way. This process competes with migration out of the heme pocket. The latter process is thermally activated (Ea ∼7 kJ/mol). This result is compared with other heme proteins, including the homologous CO sensor/transcription factor CooA, variants of the 5-coordinate mycobacterial sensor DosT and the electron transfer protein cytochrome c. This comparison indicates that thermal activation of ligand escape from the heme pocket is specific for systems where an external ligand replaces an internal one. The origin of this finding and possible implications are discussed. (10.1074/jbc.M114.571398)
DOI : 10.1074/jbc.M114.571398
Determination of collagen fibril size via absolute measurements of second-harmonic generation signals.
- Bancelin Stéphane
- Aimé Carole
- Gusachenko Ivan
- Kowalczuk Laura
- Latour Gaël
- Coradin Thibaud
- Schanne-Klein Marie-Claire
Nature Communications, Nature Publishing Group , 2014, septembre (5), pp.4920 . The quantification of collagen fibril size is a major issue for the investigation of pathological disorders associated with structural defects of the extracellular matrix. Second-harmonic generation microscopy is a powerful technique to characterize the macromolecular organization of collagen in unstained biological tissues. Nevertheless, due to the complex coherent building of this nonlinear optical signal, it has never been used to measure fibril diameter so far. Here we report absolute measurements of second-harmonic signals from isolated fibrils down to 30 nm diameter, via implementation of correlative second-harmonic-electron microscopy. Moreover, using analytical and numerical calculations, we demonstrate that the high sensitivity of this technique originates from the parallel alignment of collagen triple helices within fibrils and the subsequent constructive interferences of second-harmonic radiations. Finally, we use these absolute measurements as a calibration for ex vivo quantification of fibril diameter in the Descemet's membrane of a diabetic rat cornea. (10.1038/ncomms5920)
DOI : 10.1038/ncomms5920
Mitigating Phototoxicity during Multiphoton Microscopy of Live Drosophila Embryos in the 1.0–1.2 mm Wavelength Range
- Débarre Delphine
- Olivier Nicolas
- Supatto Willy
- Beaurepaire Emmanuel
PLoS ONE, Public Library of Science , 2014, pp.0104250 . Light-induced toxicity is a fundamental bottleneck in microscopic imaging of live embryos. In this article, after a review of photodamage mechanisms in cells and tissues, we assess photo-perturbation under illumination conditions relevant for point-scanning multiphoton imaging of live Drosophila embryos. We use third-harmonic generation (THG) imaging of developmental processes in embryos excited by pulsed near-infrared light in the 1.0–1.2 mm range. We study the influence of imaging rate, wavelength, and pulse duration on the short-term and long-term perturbation of development and define criteria for safe imaging. We show that under illumination conditions typical for multiphoton imaging, photodamage in this system arises through 2-and/or 3-photon absorption processes and in a cumulative manner. Based on this analysis, we derive general guidelines for improving the signal-to-damage ratio in two-photon (2PEF/SHG) or THG imaging by adjusting the pulse duration and/or the imaging rate. Finally, we report label-free time-lapse 3D THG imaging of gastrulating Drosophila embryos with sampling appropriate for the visualisation of morphogenetic movements in wild-type and mutant embryos, and long-term multiharmonic (THG-SHG) imaging of development until hatching. Citation: Débarre D, Olivier N, Supatto W, Beaurepaire E (2014) Mitigating Phototoxicity during Multiphoton Microscopy of Live Drosophila Embryos in the 1.0– 1.2 mm Wavelength Range. PLoS ONE 9(8): e104250. (10.1371/journal.pone.0104250)
DOI : 10.1371/journal.pone.0104250
Fibrillogenesis from nanosurfaces: multiphoton imaging and stereological analysis of collagen 3D self-assembly dynamics
- Bancelin Stéphane
- Decencière Etienne
- Machairas Vaïa
- Albert Claire
- Coradin Thibaud
- Schanne-Klein Marie-Claire
- Aimé Carole
Soft Matter, Royal Society of Chemistry , 2014, pp.6651-6657 . The assembly of proteins into fibrillar structures is an important process that concerns different biological contexts, including molecular medicine and functional biomaterials. Engineering of hybrid biomaterials can advantageously provide synergetic interactions of the biopolymers with an inorganic component to ensure specific supramolecular organization and dynamics. To this aim, we designed hybrid systems associating collagen and surface-functionalized silica particles and we built a new strategy to investigate fibrillogenesis processes in such multicomponents systems, working at the crossroads of chemistry, physics and mathematics. The self-assembly process was investigated by bimodal multiphoton imaging coupling second harmonic generation (SHG) and 2 photon excited fluorescence (2PEF). The in-depth spatial characterization of the system was further achieved using the three-dimensional analysis of the SHG/2PEF data via mathematical morphology processing. Quantitation of collagen distribution around particles offers strong evidence that the chemically induced confinement of the protein on the silica nanosurfaces has a key influence on the spatial extension of fibrillogenesis. This new approach is unique in the information it can provide on 3D dynamic hybrid systems and may be extended to other associations of fibrillar molecules with optically responsive nano-objects. (10.1039/c4sm00819g)
DOI : 10.1039/c4sm00819g
Multicolor two-photon light-sheet microscopy
- Mahou Pierre
- Vermot Julien
- Beaurepaire Emmanuel
- Supatto Willy
Nature Methods, Nature Publishing Group , 2014, 11, pp.600-601 . Two-photon microscopy is the most effective approach for deep-tissue fluorescence cellular imaging; however, its application to high-throughput or high-content imaging is often hampered by low pixel rates, challenging multicolor excitation and potential cumulative photodamage. To overcome these limitations, we extended our prior work and combined two-photon scanned light-sheet... (10.1038/nmeth.2963)
DOI : 10.1038/nmeth.2963