Articles

Dual-color deep-tissue three-photon microscopy with a multiband infrared laser
- Guesmi Khmaies
- Abdeladim Lamiae
- Tozer Samuel
- Mahou Pierre
- Kumamoto Takuma
- Jurkus Karolis
- Rigaud Philippe
- Loulier Karine
- Dray Nicolas
- Georges Patrick
- Hanna Marc
- Livet Jean
- Supatto Willy
- Beaurepaire Emmanuel
- Druon Frédéric
Light: Science and Applications, Nature Publishing Group , 2018, 7 (1), pp.12 . Multiphoton microscopy combined with genetically encoded fluorescent indicators is a central tool in biology. Three-photon (3P) microscopy with excitation in the short-wavelength infrared (SWIR) water transparency bands at 1.3 and 1.7 µm opens up new opportunities for deep-tissue imaging. However, novel strategies are needed to enable in-depth multicolor fluorescence imaging and fully develop such an imaging approach. Here, we report on a novel multiband SWIR source that simultaneously emits ultrashort pulses at 1.3 and 1.7 µm that has characteristics optimized for 3P microscopy: sub-70 fs duration, 1.25 MHz repetition rate, and µJ-range pulse energy. In turn, we achieve simultaneous 3P excitation of green fluorescent protein (GFP) and red fluorescent proteins (mRFP, mCherry, tdTomato) along with third-harmonic generation. We demonstrate in-depth dual-color 3P imaging in a fixed mouse brain, chick embryo spinal cord, and live adult zebrafish brain, with an improved signal-to-background ratio compared to multicolor two-photon imaging. This development opens the way towards multiparametric imaging deep within scattering tissues. (10.1038/s41377-018-0012-2)
DOI : 10.1038/s41377-018-0012-2
A mechanism for CO regulation of ion channels
- Kapetanaki Sofia M.
- Burton Mark J.
- Basran Jaswir
- Uragami Chiasa
- Moody Peter C. E.
- Mitcheson John S.
- Schmid Ralf
- Davies Noel W.
- Dorlet Pierre
- Vos Marten H.
- Storey Nina M.
- Raven Emma
Nature Communications, Nature Publishing Group , 2018, 9, pp.907 . Despite being highly toxic, carbon monoxide (CO) is also an essential intracellular signalling molecule. The mechanisms of CO-dependent cell signalling are poorly defined, but are likely to involve interactions with heme proteins. One such role for CO is in ion channel regulation. Here, we examine the interaction of CO with K$_{ATP}$ channels. We find that CO activates K$_{ATP}$ channels and that heme binding to a CXXHX$_{16}$H motif on the SUR2A receptor is required for the CO-dependent increase in channel activity. Spectroscopic and kinetic data were used to quantify the interaction of CO with the ferrous heme-SUR2A complex. The results are significant because they directly connect CO-dependent regulation to a heme-binding event on the channel. We use this information to present molecular-level insight into the dynamic processes that control the interactions of CO with a heme-regulated channel protein, and we present a structural framework for understanding the complex interplay between heme and CO in ion channel regulation. (10.1038/s41467-018-03291-z)
DOI : 10.1038/s41467-018-03291-z
Discovery of a Novel and Selective Indoleamine 2,3-Dioxygenase (IDO-1) Inhibitor 3-(5-Fluoro-1 H -indol-3-yl)pyrrolidine-2,5-dione (EOS200271/PF-06840003) and Its Characterization as a Potential Clinical Candidate
- Crosignani Stefano
- Bingham Patrick
- Bottemanne Pauline
- Cannelle Hélène
- Cauwenberghs Sandra
- Cordonnier Marie
- Dalvie Deepak
- Deroose Frederik
- Feng Jun Li
- Gomes Bruno
- Greasley Samantha
- Kaiser Stephen
- Kraus Manfred
- Negrerie Michel
- Maegley Karen
- Miller Nichol
- Murray Brion
- Schneider Manfred
- Soloweij James
- Stewart Albert
- Tumang Joseph
- Torti Vince
- van den Eynde Benoit
- Wythes Martin
Journal of Medicinal Chemistry, American Chemical Society , 2017, 60 (23), pp.9617 - 9629 . (10.1021/acs.jmedchem.7b00974)
DOI : 10.1021/acs.jmedchem.7b00974
How aging impacts skin biomechanics: a multiscale study in mice
- Lynch Barbara
- Bonod-Bidaud Christelle
- Ducourthial Guillaume
- Affagard Jean-Sébastien
- Bancelin Stéphane
- Psilodimitrakopoulos Sotiris
- Ruggiero Florence
- Allain Jean-Marc
- Schanne-Klein Marie-Claire
Scientific Reports, Nature Publishing Group , 2017, 7 (1) . Skin aging is a complex process that strongly affects the mechanical behavior of skin. This study aims at deciphering the relationship between age-related changes in dermis mechanical behavior and the underlying changes in dermis microstructure. To that end, we use multiphoton microscopy to monitor the reorganization of dermal collagen during mechanical traction assays in ex vivo skin from young and old mice. The simultaneous variations of a full set of mechanical and microstructural parameters are analyzed in the framework of a multiscale mechanical interpretation. They show consistent results for wild-type mice as well as for genetically-modified mice with modified collagen V synthesis. We mainly observe an increase of the tangent modulus and a lengthening of the heel region in old murine skin from all strains, which is attributed to two different origins that may act together: (i) increased cross-linking of collagen fibers and (ii) loss of water due to proteoglycans deterioration, which impedes inner sliding within these fibers. In contrast, the microstructure reorganization upon stretching shows no age-related difference, which can be attributed to opposite effects of the decrease of collagen content and of the increase of collagen cross-linking in old mice. Aging is a complex process that affects the function of all organs and tissues and most often has an irreversible impact on their mechanical behavior. The most visible effects of aging are observed in skin and have been extensively studied for medical and cosmetic purposes. The three skin layers are affected both structurally and functionally. However, aging primary impacts the mechanical integrity of the dermis. At macroscopic scale, the mechanical behavior of aged dermis shows an increased stiffness and a decreased ability to recoil 1–3. At lower scales, a complex multi-parameters process eventually results in a decrease of collagen and elastin contents due to an imbalance between matrix proteins synthesis and degradation by matrix metalloproteinases, an increase of collagen cross-linking, a deterioration of proteoglycans and a subsequent loss of water 4–9. However, the link between these microstructural modifications and the mechanical changes has so far been inferred rather than experimentally demonstrated due to the technical issues encountered when trying to obtain multiscale data. Collagens are the main component of the dermis and other connective tissues 7,10. Fibril-forming collagens assemble into striated fibrils, the diameter and three-dimensional organization of which are tissue-specific. They form multiprotein networks with other matrix proteins such as the elastin fibers and non-fibrillar matrix (pro-teoglycans, glycoaminoglycans…) that determine the mechanical behavior of dermis and other collagen-rich tissues 11–18. Collagen fibers are usually heterotypic structures. In dermis, they are made of type I, III and V col-lagens. Type V collagen is a minor component that acts as a regulatory fibril-forming collagen 19,20. As such, it plays an important role in the pathogenesis of the classical Ehlers-Danlos (EDS) syndrome. This rare connective tissue disease illustrates the close link between collagen microstructure and tissue mechanics since it is caused by mutations in collagen V genes, while being primary characterized by skin hyperextensibility 19–21. Moreover, EDS patients show a prematurely aged skin, which illustrates the close link between collagen microstructure and skin aging. (10.1038/s41598-017-13150-4)
DOI : 10.1038/s41598-017-13150-4
Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing
- Stringari Chiara
- Abdeladim Lamiae
- Malkinson Guy
- Mahou Pierre
- Solinas Xavier
- Lamarre Isabelle
- Brizion Sébastien
- Galey Jean-Baptiste
- Supatto Willy
- Legouis Renaud
- Pena Ana-Maria
- Beaurepaire Emmanuel
Scientific Reports, Nature Publishing Group , 2017, 7 (1), pp.3792 - 3792 . Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750–1040 nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041 nm, an additional equivalent two-photon excitation wavelength at 879 nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image co-registration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development. Multiphoton microscopy is a powerful tool for label-free and non-invasive functional imaging in small organisms and tissues 1, 2. Pulsed near infrared excitation light allows in-depth imaging based on contrasts such as endog-enous fluorescence 2 , second harmonic generation (SHG) 3 and third harmonic generation (THG) 4. Endogenous fluorescence in living tissues arises from several intrinsic biomarkers that play important roles in physiological processes 2. The primary intracellular sources are NAD(P)H and FAD, the two major cofactors of redox reactions in the cell and central regulators of energy production and metabolism 5, 6. Their fluorescence reports on the metabolic activity of cells allowing tissue physiology and processes such as stem cell differentiation, cancer development and neurodegenerative diseases to being non-invasively monitored 7–12. The fluorescence lifetimes of NADH and FAD are different upon binding to the protein during the electron transport chain. FLIM provides sensitive measurements of the free and protein-bound NAD(P)H ratio and of the redox states (NADH/NAD +) of cells, and can be used to distinguish glycolytic and oxidative phosphorylation metabolic states 13–17. Monitoring lifetime of free and protein-bound FAD has also been exploited to quantify redox ratio FAD/FADH 2 , and used as a biomarker of precancerous epithelial cells 12. It is well established that retinoids play a crucial role in stem cell differentiation and embryo development 18, 19 and their concentration and gradients have been detected in vivo during zebrafish development 9, 20. Other intrinsic fluorophores such as porphyrin, collagen, elastin, keratin, (10.1038/s41598-017-03359-8)
DOI : 10.1038/s41598-017-03359-8
Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea
- Ghoubay-Benallaoua Djida
- de Sousa Céline
- Martos Raphaël
- Latour Gaël
- Schanne-Klein Marie-Claire
- Dupin Elisabeth
- Borderie Vincent
PLoS ONE, Public Library of Science , 2017, 12 (11), pp.e0188398 . Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC) and epithelial limbal (LSC) stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8), E8 supplemented with EGF (E8+) or Green’s medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green’s media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes. (10.1371/journal.pone.0188398)
DOI : 10.1371/journal.pone.0188398
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
Physical limits of flow sensing in the left-right organizer
- Ferreira Rita R.
- Vilfan Andrej
- Julicher Frank
- Supatto Willy
- Vermot Julien
eLife, eLife Sciences Publication , 2017, 25, pp.556 - 556 . Fluid flows generated by motile cilia are guiding the establishment of the left-right asymmetry of the body in the vertebrate left-right organizer. Competing hypotheses have been proposed: the direction of flow is sensed either through mechanosensation, or via the detection of chemical signals transported in the flow. We investigated the physical limits of flow detection to clarify which mechanisms could be reliably used for symmetry breaking. We integrated parameters describing cilia distribution and orientation obtained in vivo in zebrafish into a multiscale physical study of flow generation and detection. Our results show that the number of immotile cilia is too small to ensure robust left and right determination by mechanosensing, given the large spatial variability of the flow. However, motile cilia could sense their own motion by a yet unknown mechanism. Finally, transport of chemical signals by the flow can provide a simple and reliable mechanism of asymmetry establishment. (10.7554/eLife.25078)
DOI : 10.7554/eLife.25078
Stabilization of Collagen Fibrils by Gelatin Addition: A Study of Collagen/Gelatin Dense Phases
- Portier François
- Teulon Claire
- Nowacka-Perrin Agnieszka
- Guenneau Flavien
- Schanne-Klein Marie-Claire
- Mosser Gervaise
Langmuir, American Chemical Society , 2017, 33 (45), pp.12916-12925 . Collagen and its denatured form, gelatin, are biopolymers of fundamental interest in numerous fields ranging from living tissues to biomaterials, food, and cosmetics. This study aims at characterizing mixtures of those biopolymers at high concentrations (up to 100 mg·mL–1) at which collagen has mesogenic properties. We use a structural approach combining polarization-resolved multiphoton microscopy, polarized light microscopy, magnetic resonance imaging, and transmission electron microscopy to analyze gelatin and collagen/gelatin dense phases in their sol and gel states from the macroscopic to the microscopic scale. We first report the formation of a lyotropic crystal phase of gelatin A and show that gelatin must structure itself in particles to become mesogenic. We demonstrate that mixtures of collagen and gelatin phase segregate, preserving the setting of the pure collagen mesophase at a gelatin ratio of up to 20% and generating a biphasic fractal sample at all tested ratios. Moreover, differential scanning calorimetric analysis shows that each protein separates into two populations. Both populations of gelatins are stabilized by the presence of collagen, whereas only one population of collagen molecules is stabilized by the presence of gelatin, most probably those at the interface of the fibrillated microdomains and of the gelatin phase. Although further studies are needed to fully understand the involved mechanism, these new data should have a direct impact on the bioengineering of those two biopolymers. (10.1021/acs.langmuir.7b02142)
DOI : 10.1021/acs.langmuir.7b02142
Structural changes and picosecond to second dynamics of cytochrome c in interaction with nitric oxide in ferrous and ferric redox states
- Kruglik Sergei
- Yoo Byung-Kuk
- Lambry Jean-Christophe
- Martin Jean-Louis
- Negrerie Michel
Physical Chemistry Chemical Physics, Royal Society of Chemistry , 2017, 19 (32), pp.21317-21334 . Apart from its role in electron transfer, mitochondrial cytochrome c also plays a role in apoptosis and is subject to nitrosylation. The cleavage of the Fe–Met80 bond plays a role in several processes including the release of Cyt c from mitochondria or increase of its peroxidase activity. Nitrosylation of Cyt c precludes the reformation of the disrupted Fe–Met80 bond and was shown to occur during apoptosis. These physiological properties are associated with a conformational change of the heme center of Cyt c. Here, we demonstrate that NO binding induces pronounced heme conformational changes in the six-coordinate Cyt c–NO complex. Equilibrium and time-resolved Raman data reveal that the heme structural conformation depends both on the nature of the distal iron ligand (NO or Met80) and on the Fe2+ or Fe3+ heme redox state. Upon nitrosylation, the heme ruffling distortion is greatly enhanced for ferrous Cyt c. Contrastingly, the initial strong heme distortion in native ferric Cyt c almost disappears after NO binding. We measured the heme coordination dynamics in the picosecond to second time range and identified Met80 and NO rebinding phases using time-resolved Raman and absorption spectroscopies. Dissociation of NO instantly produces 5-coordinate heme with a domed structure which continues to rearrange within 15 ps, while the initial ruffling distortion disappears. The rates of Cyt c–NO complex formation measured by transient absorption are kon = 1.81 × 106 M−1 s−1 for ferric Cyt c and 83 M−1 s−1 for ferrous Cyt c. After NO dissociation and exit from the heme pocket, the rebinding of Met80 to the heme iron takes place 6 orders of magnitude more slowly (3–5 μs) than Met80 rebinding in the absence of NO (5 ps). Altogether, these data reveal the structural and dynamic properties of Cyt c in interaction with nitric oxide relevant for the molecular mechanism of apoptosis. (10.1039/C7CP02634J)
DOI : 10.1039/C7CP02634J
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