Articles

A Molecular Mechanics Model for Flavins
- Aleksandrov Alexey
Journal of Computational Chemistry, Wiley , 2019, 40 (32), pp.2834-2842 . Flavin containing molecules form a group of important cofactors that assist a wide range of enzymatic reactions. Flavins use the redox‐active isoalloxazine system, which is capable of one‐ and two‐electron transfer reactions and can exist in several protonation states. In this work, molecular mechanics force field parameters compatible with the CHARMM36 all‐atom additive force field were derived for biologically important flavins, including riboflavin, flavin mononucleotide, and flavin adenine dinucleotide. The model was developed for important protonation and redox states of the isoalloxazine group. The partial charges were derived using the CHARMM force field parametrization strategy, where quantum mechanics water–solute interactions are used to target optimization. In addition to monohydrate energies and geometries, electrostatic potential around the compound was used to provide additional restraints during the charge optimization. Taking into account the importance of flavin‐containing molecules special attention was given to the quality of bonded terms. All bonded terms, including stiff terms and torsion angle parameters, were parametrized using exhaustive potential energy surface scans. In particular, the model reproduces well the butterfly motion of isoalloxazine in the oxidized and reduced forms as predicted by quantum mechanics in gas phase. The model quality is illustrated by simulations of four flavoproteins. Overall, the presented molecular mechanics model will be of utility to model flavin cofactors in different redox states. (10.1002/jcc.26061)
DOI : 10.1002/jcc.26061
Structural and functional insight into serine hydroxymethyltransferase from Helicobacter pylori
- Sodolescu Andreea
- Dian Cyril
- Terradot Laurent
- Bouzhir-Sima Latifa
- Lestini Roxane
- Myllykallio Hannu
- Skouloubris Stéphane
- Liebl Ursula
PLoS ONE, Public Library of Science , 2018, 13 (12), pp.e0208850 . Serine hydroxymethyltransferase (SHMT), encoded by the glyA gene, is a ubiquitous pyridoxal 5’-phosphate (PLP)-dependent enzyme that catalyzes the formation of glycine from serine. The thereby generated 5,10-methylene tetrahydrofolate (MTHF) is a major source of cellular one-carbon units and a key intermediate in thymidylate biosynthesis. While in virtually all eukaryotic and many bacterial systems thymidylate synthase ThyA, SHMT and dihydrofolate reductase (DHFR) are part of the thymidylate/folate cycle, the situation is different in organisms using flavin-dependent thymidylate synthase ThyX. Here the distinct catalytic reaction directly produces tetrahydrofolate (THF) and consequently in most ThyX-containing organisms, DHFR is absent. While the resulting influence on the folate metabolism of ThyX-containing bacteria is not fully understood, the presence of ThyX may provide growth benefits under conditions where the level of reduced folate derivatives is compromised. Interestingly, the third key enzyme implicated in generation of MTHF, serine hydroxymethyltransferase (SHMT), has a universal phylogenetic distribution, but remains understudied in ThyX-containg bacteria. To obtain functional insight into these ThyX-dependent thymidylate/folate cycles, we characterized the predicted SHMT from the ThyX-containing bacterium Helicobacter pylori. Serine hydroxymethyltransferase activity was confirmed by functional genetic complementation of a glyA-inactivated E. coli strain. A H. pylori ΔglyA strain was obtained, but exhibited markedly slowed growth and had lost the virulence factor CagA. Biochemical and spectroscopic evidence indicated formation of a characteristic enzyme-PLP-glycine-folate complex and revealed unexpectedly weak binding affinity of PLP. The three-dimensional structure of the H. pylori SHMT apoprotein was determined at 2.8Ǻ resolution, suggesting a structural basis for the low affinity of the enzyme for its cofactor. Stabilization of the proposed inactive configuration using small molecules has potential to provide a specific way for inhibiting HpSHMT. (10.1371/journal.pone.0208850)
DOI : 10.1371/journal.pone.0208850
Color Imaging with Multimodal Three-Photon Microscopy
- Abdeladim Lamiae
- Mahou Pierre
- Supatto Willy
- Beaurepaire Emmanuel
- Guesmi Khmaies
- Jurkus Karolis
- Rigaud Philippe
- Georges Patrick
- Hanna Marc
- Druon Frédéric
- Tozer Samuel
- Kumamoto Takuma
- Loulier Karine
- Livet Jean
- Dray Nicolas
Optics and photonics news, Optical Society of America - OSA Publishing , 2018 .
Author Correction: A mechanism for CO regulation of ion channels
- Kapetanaki Sofia
- Burton Mark
- Basran Jaswir
- Uragami Chiasa
- Moody Peter
- Mitcheson John
- Schmid Ralf
- Davies Noel
- Dorlet Pierre
- Vos Marten H.
- Storey Nina
- Raven Emma
Nature Communications, Nature Publishing Group , 2018, 9, pp.3354 . The originally published version of this article contained an error in the subheading ‘Heme is required for CO-dependent channel activation’, which was incorrectly given as ‘Hame is required for CO-dependent channel activation’. This has now been corrected in both the PDF and HTML versions of the Article (10.1038/s41467-018-05622-6)
DOI : 10.1038/s41467-018-05622-6
Advances and challenges in drug design against tuberculosis: application of in silico approaches
- Aleksandrov Alexey
- Myllykallio Hannu
Expert Opinion on Drug Discovery, Informa Healthcare , 2018, pp.1 - 12 . (10.1080/17460441.2019.1550482)
DOI : 10.1080/17460441.2019.1550482
Chiral Cilia Orientation in the Left-Right Organizer
- Ferreira Rita
- Pakula Guillaume
- Klaeyle Lhéanna
- Fukui Hajime
- Vilfan Andrej
- Supatto Willy
- Vermot Julien
Cell Reports, Elsevier Inc , 2018, 25 (8), pp.2008-2016.e4 . (10.1016/j.celrep.2018.10.069)
DOI : 10.1016/j.celrep.2018.10.069
Snapshots of archaeal DNA replication and repair in living cells using super-resolution imaging
- Delpech Floriane
- Collien Yoann
- Mahou Pierre
- Beaurepaire Emmanuel
- Myllykallio Hannu
- Lestini Roxane
Nucleic Acids Research, Oxford University Press , 2018 . (10.1093/nar/gky829)
DOI : 10.1093/nar/gky829
Combining the polarizable Drude force field with a continuum electrostatic Poisson-Boltzmann implicit solvation model
- Aleksandrov Alexey
- Lin Fang-Yu
- Roux Benoît
- Mackerell Alexander
Journal of Computational Chemistry, Wiley , 2018, 39 (22), pp.1707-1719 . (10.1002/jcc.25345)
DOI : 10.1002/jcc.25345
Resonant broadband stimulated Raman scattering in myoglobin
- Ferrante C.
- Batignani G.
- Fumero G.
- Pontecorvo E.
- Virga A.
- Montemiglio L.
- Cerullo G.
- Vos Marten H.
- Scopigno T.
Journal of Raman Spectroscopy, Wiley , 2018, 49 (6), pp.913-920 . (10.1002/jrs.5323)
DOI : 10.1002/jrs.5323
Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication
- Ishino Sonoko
- Skouloubris Stéphane
- Kudo Hanae
- L’hermitte-Stead Caroline
- Es-Sadik Asmae
- Lambry Jean-Christophe
- Ishino Yoshizumi
- Myllykallio Hannu
Nucleic Acids Research, Oxford University Press , 2018 . The mismatch repair (MMR) system, exemplified by the MutS/MutL proteins, is widespread in Bacteria and Eukarya. However, molecular mechanisms how numerous archaea and bacteria lacking the mutS/mutL genes maintain high replication fidelity and genome stability have remained elusive. EndoMS is a recently discovered hyperthermophilic mismatch-specific endonuclease encoded by nucS in Thermococcales. We deleted the nucS from the actinobacterium Corynebacterium glutamicum and demonstrated a drastic increase of spontaneous transition mutations in the nucS deletion strain. The observed spectra of these mutations were consistent with the enzymatic properties of EndoMS in vitro. The robust mismatch-specific endonuclease activity was detected with the purified C. glutamicum EndoMS protein but only in the presence of the β-clamp (DnaN). Our biochemical and genetic data suggest that the frequently occurring G/T mismatch is efficiently repaired by the bacterial EndoMS-β−clamp complex formed via a carboxy-terminal sequence motif of EndoMS proteins. Our study thus has great implications for understanding how the activity of the novel MMR system is coordinated with the replisome and provides new mechanistic insight into genetic diversity and mutational patterns in industrially and clinically (e.g. Mycobacteria) important archaeal and bacterial phyla previously thought to be devoid of the MMR system. (10.1093/nar/gky460)
DOI : 10.1093/nar/gky460
Unique Features and Anti-microbial Targeting of Folate- and Flavin-Dependent Methyltransferases Required for Accurate Maintenance of Genetic Information
- Myllykallio Hannu
- Sournia Pierre
- Héliou Alice
- Liebl Ursula
Frontiers in Microbiology, Frontiers Media , 2018, 9, pp.918 . Comparative genome analyses have led to the discovery and characterization of novel flavin- and folate-dependent methyltransferases that mainly function in DNA precursor synthesis and post-transcriptional RNA modification by forming (ribo) thymidylate and its derivatives. Here we discuss the recent literature on the novel mechanistic features of these enzymes sometimes referred to as “uracil methyltransferases,” albeit we prefer to refer to them as (ribo) thymidylate synthases. These enzyme families attest to the convergent evolution of nucleic acid methylation. Special focus is given to describing the unique characteristics of these flavin- and folate-dependent enzymes that have emerged as new models for studying the non-canonical roles of reduced flavin cofactors (FADH2) in relaying carbon atoms between enzyme substrates. This ancient enzymatic methylation mechanism with a very wide phylogenetic distribution may be more commonly used for biological methylation reactions than previously anticipated. This notion is exemplified by the recent discovery of additional substrates for these enzymes. Moreover, similar reaction mechanisms can be reversed by demethylases, which remove methyl groups e.g., from human histones. Future work is now required to address whether the use of different methyl donors facilitates the regulation of distinct methylation reactions in the cell. It will also be of great interest to address whether the low activity flavin-dependent thymidylate synthases ThyX represent ancestral enzymes that were eventually replaced by the more active thymidylate synthases of the ThyA family to facilitate the maintenance of larger genomes in fast-growing microbes. Moreover, we discuss the recent efforts from several laboratories to identify selective anti-microbial compounds that target flavin-dependent thymidylate synthase ThyX. Altogether we underline how the discovery of the alternative flavoproteins required for methylation of DNA and/or RNA nucleotides, in addition to providing novel targets for antibiotics, has provided new insight into microbial physiology and virulence. (10.3389/fmicb.2018.00918)
DOI : 10.3389/fmicb.2018.00918
Use of MPA-capped CdS quantum dots for sensitive detection and quantification of Co2+ ions in aqueous solution
- Bel Haj Mohamed Naim
- Ben Brahim Nassim
- Mrad Randa
- Haouari Mohamed
- Ben Chaâbane Rafik
- Négrerie Michel
Analytica Chimica Acta, Elsevier Masson , 2018, 1028, pp.50 - 58 . Water soluble CdS quantum dots (QDs) were synthesized by a simple aqueous chemical route using mercaptopropionic acid (MPA) as a stabilizer. These QDs had a fluorescence emission band maximum at 540 nm with a FWHM ∼130 nm and a quantum yield of ∼12%. Transmission electronic microscopy images were used to determine the QD diameter of 8.9 ± 0.4 nm. From this value we calculated the molecular mass M(QD) = 1.17 × 106 g mol-1 and the extinction coefficient at the band edge (450 nm) ε450 = 4.7 × 106 cm-1 M-1, which allowed to determine the true molar concentration of 17 nM for spectroscopic measurements in solution. The fluorescence intensity of MPA-CdS QDs was quenched only in the presence of Co2+ ions, but not in the presence of thirteen other metal cations. The fluorescence quenching of MPA-CdS QDs appeared proportional to the Co2+ concentration in the range 0.04-2 μM. Based on a fluorescence peak position and a lifetime both independent from Co2+ concentration, the quenching mechanism of MPA-CdS QDs appeared static. Because the strong electronic absorption of Co2+ overlaps the emission of QDs, our results can be explained by Förster energy transfer from QD to the bound Co2+ cations. (10.1016/j.aca.2018.04.041)
DOI : 10.1016/j.aca.2018.04.041
Density of Grafted Chains in Thioglycerol-Capped CdS Quantum Dots Determines Their Interaction with Aluminum(III) in Water
- Ben Brahim Nassim
- Poggi Mélanie
- Lambry Jean-Christophe
- Bel Haj Mohamed Naim
- Ben Chaâbane Rafik
- Négrerie Michel
Inorganic Chemistry, American Chemical Society , 2018, 57, pp.4979 - 4988 . We aimed to quantify the interaction of watersoluble-functionalized CdS quantum dots (QDs) with metal cations from their composition and physical properties. From the diameter of thioglycerol-capped nanoparticles (TG-CdS QDs) measured by electronic microscopy (D = 12.3 ± 0.3 nm), we calculated the molecular mass of the individual particle MA QD = (3 ± 0.5) × 10 6 g•mol-1 and its molar absorption coefficient ε 450 = 21 × 10 6 M-1 •cm-1. We built a three-dimensional model of the TG-CdS QDs in agreement with the structural data, which allowed us to quantify the number of thioglycerol grafted chains to ∼2000 per QD. This value fully matches the saturation binding curve of Al 3+ cations interacting with TG-CdS QDs. The reaction occurred with a slow association rate (k on = 2.1 × 10 3 M-1 •s-1), as expected for heavy QDs. The photophysical properties of the functionalized QDs were studied using an absolute QD concentration of 7 nM, which allowed us to investigate the interaction with 14 metallic cations in water. The fluorescence intensity of TG-CdS QDs could be quenched only in the presence of Al 3+ ions in the range 0.2-10 μM but not with other cations and was not observed with other kinds of grafting chains. (10.1021/acs.inorgchem.7b03254)
DOI : 10.1021/acs.inorgchem.7b03254
Isolation and identification of two extremely halophilic archaea from sebkhas in the Algerian Sahara
- Khallef Sakina
- Lestini Roxane
- Myllykallio Hannu
- Houali Karim
Cellular and Molecular Biology, CMB Association , 2018, 64 (4), pp.83–91 . (10.14715/cmb/2018.64.4.14)
DOI : 10.14715/cmb/2018.64.4.14
Analyse d’une carte du monde du VIIIe siècle : la Mappa mundi d’Albi
- Robinet Laurianne
- Deschaux Jocelyne
- Tournié Aurélie
- Stéphane Vaidelich
- Latour Gael
- Michelin Anne
- Thao Sylvie
- Andraud Christine
- Schanne-Klein Marie-Claire
- Lavédrine Bertrand
Support Tracé, Association pour la Recherche Scientifique sur les Arts Graphiques (ARSAG) , 2018, 17, pp.5-13 .
The Future of Multiplexed Eukaryotic Genome Engineering
- Thompson David
- Aboulhouda Soufiane
- Hysolli Eriona
- Smith Cory
- Wang Stan
- Castanon Oscar
- Church George
ACS Chemical Biology, American Chemical Society , 2018, 13 (2), pp.313 - 325 . Multiplex genome editing is the simultaneous introduction of multiple distinct modifications to a given genome. Though in its infancy, maturation of this field will facilitate powerful new biomedical research approaches and will enable a host of far-reaching biological engineering applications, including new therapeutic modalities and industrial applications, as well as “genome writing” and de-extinction efforts. In this Perspective, we focus on multiplex editing of large eukaryotic genomes. We describe the current state of multiplexed genome editing, the current limits of our ability to multiplex edits, and provide perspective on the many applications that fully realized multiplex editing technologies would enable in higher eukaryotic genomes. We offer a broad look at future directions, covering emergent CRISPR-based technologies, advances in intracellular delivery, and new DNA assembly approaches that may enable future genome editing on a massively multiplexed scale. (10.1021/acschembio.7b00842)
DOI : 10.1021/acschembio.7b00842
Evolutionary insights into Trm112-methyltransferase holoenzymes involved in translation between archaea and eukaryotes
- Van tran Nhan
- Muller Leslie
- Ross Robert L
- Lestini Roxane
- Létoquart Juliette
- Ulryck Nathalie
- Limbach Patrick A
- De crécy-Lagard Valérie
- Cianférani Sarah
- Graille Marc
Nucleic Acids Research, Oxford University Press , 2018, 46 (16), pp.8483 - 8499 . Protein synthesis is a complex and highly coordinated process requiring many different protein factors as well as various types of nucleic acids. All translation machinery components require multiple maturation events to be functional. These include post-transcriptional and post-translational modification steps and methylations are the most frequent among these events. In eukaryotes, Trm112, a small protein (COG2835) conserved in all three domains of life, interacts and activates four methyltransferases (Bud23, Trm9, Trm11 and Mtq2) that target different components of the translation machinery (rRNA, tRNAs, release factors). To clarify the function of Trm112 in archaea, we have characterized functionally and structurally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies. Interestingly, among the identified enzymes, some are functionally orthologous to eukaryotic Trm112 partners, emphasizing again the similarity between eukaryotic and archaeal translation machineries. Other partners display some similarities with bacterial methyltransferases, suggesting that Trm112 is a general partner for methyltransferases in all living organisms. (10.1093/nar/gky638)
DOI : 10.1093/nar/gky638
Highly concentrated collagen solutions leading to transparent scaffolds of controlled three-dimensional organizations for corneal epithelial cell colonization
- Tidu Aurelien
- Ghoubay-Benallaoua Djida
- Teulon Claire
- Asnacios Sophie
- Grieve Kate
- Portier François
- Schanne-Klein Marie-Claire
- Borderie Vincent M.
- Mosser Gervaise
Biomaterials Science, Royal Society of Chemistry (RSC) , 2018, 6 (6), pp.1492-1502 . This study aimed at controlling both the organization and the transparency of dense collagen scaffolds making use of the lyotropic mesogen properties of collagen. Cholesteric or plywood-like liquid crystal phases were achieved using mixtures of acetic and hydrochloric acids as solvents. The critical pH at which the switch between the two phases occurred was around pH = 3. The use of the two acids led to fibril-lated collagen I scaffolds, whose visual aspect ranged from opaque to transparent. Rheological investigations showed that viscoelastic properties of the plywood-like solutions were optimized for molding due to faster recovery. They also confirmed the correlation between the elastic modulus and the diameter of collagen fibrils obtained after fibrillogenesis under ammonia vapor. Human corneal epithelial cells, grown from donor limbal explants, were cultured both on transparent plywood-like matrices and on human amniotic membranes for 14 days. The development of corneal epithelium and the preservation of epi-thelial stem cells were checked by optical microscopy, colony formation assay, immuno-fluorescence and quantitative polymerase chain reaction. A higher level of amplification of limbal stem cells was obtained with collagen matrices compared with amniotic membranes, showing the high biocompatibility of our scaffolds. We therefore suggest that collagen solutions presenting both plywood-like organization and transparency might be of interest for biomedical applications in ophthalmology. (10.1039/c7bm01163f)
DOI : 10.1039/c7bm01163f
Multiscale conformational dynamics probed by time-resolved circular dichroism.
- Schmid Marco
- Changenet Pascale
- Hache François
Proceedings of SPIE, the International Society for Optical Engineering, SPIE, The International Society for Optical Engineering , 2018 .
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