Articles

Télomères et Télomérase : des cibles toujours pertinentes en oncologie ?
- Mergny Jean‐louis
- Guittat Lionel
- Ségal-Bendirdjian Évelyne
Bulletin du Cancer, Elsevier , 2020, 108, pp.30-54 . (10.1016/j.bulcan.2020.10.007)
DOI : 10.1016/j.bulcan.2020.10.007
Going Platinum to the Tune of a Remarkable Guanine Quadruplex Binder: Solution‐ and Solid‐State Investigations.
- Miron Caitlin
- van Staalduinen Laura
- Rangaswamy Alana
- Chen Mickey
- Liang Yushi
- Jia Zongchao
- Mergny Jean‐louis
- Petitjean Anne
Angewandte Chemie International Edition, Wiley-VCH Verlag , 2020, pp.Online ahead of print . Guanine quadruplex recognition has gained increasing attention, inspired by the growing awareness of the key roles played by these non-canonical nucleic acid architectures in cellular regulatory processes. We report here the solution and solid-state studies of a novel planar platinum(II) complex that is easily assembled from a simple ligand, and exhibits notable binding affinity for guanine quadruplex structures, while maintaining good selectivity for guanine quadruplex over duplex structures. A crystal structure of this ligand complexed with a telomeric quadruplex confirms double end-capping, with dimerization at the 5' interface. (10.1002/anie.202012520)
DOI : 10.1002/anie.202012520
Circular dichroism second-harmonic generation microscopy probes the polarity distribution of collagen fibrils
- Schmeltz Margaux
- Teulon Claire
- Pinsard Maxime
- Hansen Uwe
- Alnawaiseh Maged
- Ghoubay Djida
- Borderie Vincent
- Mosser Gervaise
- Aimé Carole
- Légaré François
- Latour Gael
- Schanne-Klein Marie-Claire
Optica, Optical Society of America - OSA Publishing , 2020, 7 . Second-harmonic generation (SHG) microscopy is currently the preferred technique for visualizing collagen in intact tissues, but the usual implementations struggle to reveal collagen fibrils oriented out of the imaging plane. Recently, an advanced SHG modality, circular dichroism SHG (CD-SHG), has been proposed to specifically highlight out-of-plane fibrils. In this study, we present a theoretical analysis of CD-SHG signals that goes beyond the electric dipolar approximation to account for collagen chirality. We demonstrate that magnetic dipolar contributions are necessary to analyze CD-SHG images of human cornea sections and other collagen-rich samples. We show that the sign of CD-SHG signals does not reveal whether collagen fibrils point upwards or downwards as tentatively proposed previously. CD-SHG instead probes the polarity distribution of out-of-plane fibril assemblies at submicrometer scale, namely homogeneous polarity versus a mix of antiparallel fibrils. This makes CD-SHG a powerful tool for characterizing collagen organization in tissues, specifically the degree of disorder, which is affected during pathological remodeling. CD-SHG may thus serve to discriminate healthy and diseased collagen-rich tissues. (10.1364/optica.399246)
DOI : 10.1364/optica.399246
Human papillomavirus G-rich regions as potential antiviral drug 1 targets
- Carvalho Josué
- Lopes-Nunes Jéssica
- Campello Maria P C
- Paulo António
- Milici Janice
- Meyers Craig
- Mergny Jean‐louis
- Salgado Gilmar F
- Queiroz João A
- Cruz Carla
Nucleic Acid Therapeutics, Mary Ann Liebert, Inc. publishers , 2020 . Herein we report for the first time the screening of several ligands in terms of their ability to bind and stabilize G-quadruplexes found in seven human Papillomavirus (HPV) genomes. Using a variety of biophysical assays, HPV G-quadruplexes were shown to possess a high degree of structural polymorphism upon ligand binding which may have an impact on transcription, replication and viral protein production. A sequence found in high-risk HPV16 genotype folds into multiple non-canonical DNA structures; it was converted into a major G-quadruplex conformation upon interaction with a well-characterized highly selective G4-ligand, PhenDC3, which may have animpact on the viral infection. Likewise, HPV57 and 58, which fold into multiple G-quadruplex structures, were found to form single stable complexes in the presence of two other G4-ligands, C8and Pyridostatin, respectively. Additionally, one of the selected compounds, the acridine derivative C8, demonstrated a significant antiviral effect in HPV18-infected organotypic raft cultures. Altogether, these results indicate that targeting HPV G-quadruplexes may be an alternative route for the development of novel antiviral therapies.
Fast in vivo multiphoton light-sheet microscopy with optimal pulse frequency
- Maioli Vincent
- Boniface Antoine
- Mahou Pierre
- Ortas Júlia Ferrer
- Abdeladim Lamiae
- Beaurepaire Emmanuel
- Supatto Willy
Biomedical optics express, Optical Society of America - OSA Publishing , 2020, 11 (10), pp.6012-6026 . Improving the imaging speed of multiphoton microscopy is an active research field. Among recent strategies, light-sheet illumination holds distinctive advantages for achieving fast imaging in vivo. However, photoperturbation in multiphoton light-sheet microscopy remains poorly investigated. We show here that the heart beat rate of zebrafish embryos is a sensitive probe of linear and nonlinear photoperturbations. By analyzing its behavior with respect to laser power, pulse frequency and wavelength, we derive guidelines to find the best balance between signal and photoperturbation. We then demonstrate one order-of-magnitude signal enhancement over previous implementations by optimizing the laser pulse frequency. These results open new opportunities for fast live tissue imaging. (10.1364/BOE.400113)
DOI : 10.1364/BOE.400113
Rôle pathogénique de l’expression anormale de la tétraspanine CD9 par les cellules épithéliales pariétales dans les glomérulopathies extracapillaires
- Lazareth Hélène
- Lenoir Olivia
- Hénique Carole
- Bouzigues Cédric
- Boucheix Claude
- Tharaux Pierre-Louis
Médecine/Sciences, EDP Sciences , 2020, 36 (10), pp.852-855 . No abstract available (10.1051/medsci/2020154)
DOI : 10.1051/medsci/2020154
G-Quadruplexes in the Archaea Domain
- Brázda Václav
- Luo Yu
- Bartas Martin
- Kaura Patrik
- Porubiaková Otilia
- Šťastný Jiří
- Pečinka Petr
- Verga Daniela
- da Cunha Violette
- Takahashi Tomio S
- Forterre Patrick
- Myllykallio Hannu
- Fojta Miroslav
- Mergny Jean‐louis
Biomolecules, MDPI , 2020, 10 (9), pp.E1349 . The importance of unusual DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, G-quadruplexes (G4s) have gained in popularity during the last decade, and their presence and functional relevance at the DNA and RNA level has been demonstrated in a number of viral, bacterial, and eukaryotic genomes, including humans. Here, we performed the first systematic search of G4-forming sequences in all archaeal genomes available in the NCBI database. In this article, we investigate the presence and locations of G-quadruplex forming sequences using the G4Hunter algorithm. G-quadruplex-prone sequences were identified in all archaeal species, with highly significant differences in frequency, from 0.037 to 15.31 potential quadruplex sequences per kb. While G4 forming sequences were extremely abundant in Hadesarchaea archeon (strikingly, more than 50% of the Hadesarchaea archaeon isolate WYZ-LMO6 genome is a potential part of a G4-motif), they were very rare in the Parvarchaeota phylum. The presence of G-quadruplex forming sequences does not follow a random distribution with an over-representation in non-coding RNA, suggesting possible roles for ncRNA regulation. These data illustrate the unique and non-random localization of G-quadruplexes in Archaea. (10.3390/biom10091349)
DOI : 10.3390/biom10091349
Development, structure, and bioengineering of the human corneal stroma: A review
- Tidu Aurélien
- Schanne-Klein Marie-Claire
- Borderie Vincent
Experimental Eye Research, Elsevier , 2020, 200, pp.108256 . (10.1016/j.exer.2020.108256)
DOI : 10.1016/j.exer.2020.108256
In-place molecular preservation of cellulose in 5,000-year-old archaeological textiles
- Reynaud Corentin
- Thoury Mathieu
- Dazzi Alexandre
- Latour Gael
- Scheel Mario
- Li Jiayi
- Thomas Ariane
- Moulherat Christophe
- Didier Aurore
- Bertrand Loïc
Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2020, 117 (33), pp.19670-19676 . The understanding of fossilization mechanisms at the nanoscale remains extremely challenging despite its fundamental interest and its implications for paleontology, archaeology, geoscience, and environmental and material sciences. The mineralization mechanism by which cellulosic, keratinous, and silk tissues fossilize in the vicinity of archaeological metal artifacts offers the most exquisite preservation through a mechanism unexplored on the nanoscale. It is at the center of the vast majority of ancient textiles preserved under nonextreme conditions, known through extremely valuable fragments. Here we show the reconstruction of the nanoscale mechanism leading to the preservation of an exceptional collection of ancient cellulosic textiles recovered in the ancient Near East (4,000 to 5,000 years ago). We demonstrate that even the most mineralized fibers, which contain inorganic compounds throughout their histology, enclose preserved cel-lulosic remains in place. We evidence a process that combines the three steps of water transport of biocidal metal cations and soil solutes, degradation and loss of crystallinity of cellulosic polysaccharides, and silicification. cultural heritage | fossilization | cellulosic textiles | nanoimaging | synchrotron (10.1073/pnas.2004139117)
DOI : 10.1073/pnas.2004139117
Dynamics of Cell Membrane Permeabilization by Saponins Using Terahertz Attenuated Total Reflection
- Zheng Xiujun
- Gallot Guilhem
Biophysical Journal, Biophysical Society , 2020, 119 (4), pp.749-755 . Understanding the relevant parameters of the formation of pores during permeabilization is very challenging for medical applications. Several components are involved: the arrival of the permeabilizing molecules to the membrane, the efficiency of formation of the pores and their specific dynamics, and the flux of molecules through the plasma membrane. Using attenuated total reflection in the terahertz domain, we studied the dynamics of Madine-Darby canine kidney cells after permeabilization by saponin molecules. We developed an analytical model taking into account saponin molecule diffusion, cell geometry, cytosol molecule diffusion, and pore dynamics. We also studied the effect of possible pore overlapping on the cell membrane, introducing a dimensionless quantity that is the ratio between overlapping and diffusive effects. Pores are found to be static within 1 h after their creation, hinting that the diffusion of the saponin molecules to the membrane is the limiting factor in our experiments. (10.1016/j.bpj.2020.05.040)
DOI : 10.1016/j.bpj.2020.05.040
Genome wide distribution of G-quadruplexes and their impact on gene expression in malaria parasites
- Gazanion Elodie
- Lacroix Laurent
- Alberti Patrizia
- Gurung Pratima
- Wein Sharon
- Cheng Mingpan
- Mergny Jean‐louis
- Gomes Ana Rita
- Lopez‑rubio José‑juan
PLoS Genetics, Public Library of Science , 2020, 16 (7), pp.e1008917 . Mechanisms of transcriptional control in malaria parasites are still not fully understood. The positioning patterns of G-quadruplex (G4) DNA motifs in the parasite's AT-rich genome, especially within the var gene family which encodes virulence factors, and in the vicinity of recombination hotspots, points towards a possible regulatory role of G4 in gene expression and genome stability. Here, we carried out the most comprehensive genome-wide survey, to date, of G4s in the Plasmodium falciparum genome using G4Hunter, which identifies G4 forming sequences (G4FS) considering their G-richness and G-skewness. We show an enrichment of G4FS in nucleosome-depleted regions and in the first exon of var genes, a pattern that is conserved within the closely related Laverania Plasmodium parasites. Under G4-stabilizing conditions, i.e., following treatment with pyridostatin (a high affinity G4 ligand), we show that a bona fide G4 found in the non-coding strand of var promoters modulates reporter gene expression. Furthermore, transcriptional profiling of pyridostatin-treated parasites, shows large scale perturbations, with deregulation affecting for instance the ApiAP2 family of transcription factors and genes involved in ribosome biogenesis. Overall, our study highlights G4s as important DNA secondary structures with a role in Plasmodium gene expression regulation, sub-telomeric recombination and var gene biology. (10.1371/journal.pgen.1008917)
DOI : 10.1371/journal.pgen.1008917
Role of surface defects in colloidal cadmium selenide (CdSe) nanocrystals in the specificity of fluorescence quenching by metal cations
- Mrad Randa
- Poggi Mélanie
- Ben Chaâbane Rafik
- Negrerie Michel
Journal of Colloid and Interface Science, Elsevier , 2020, 571, pp.368 - 377 . This study aimed to answer the question as whether crystal defects at the surface of soluble capped CdSe nanocrystals (or quantum dots, QDs) in water colloidal suspension are involved in the mechanism of fluorescence quenching induced by metal cations. Nanocrystals of CdSe were synthesized by an aqueous protocol, varying the ratio between the CdSe precursors and the grafted ligand mercaptosuccinic acid (MSA). Changing the MSA/CdSe ratio during synthesis impacts the crystal nucleation growth, which plays an important role in surface construction of CdSe QDs and changes the surface state. In this way, we could modulate the crystal surface defects of CdSe, as verified by analysis of the individual bands which constitute the emission spectra and are associated with different relaxation processes. We found that the various tested metal cations, which interact in solution with the MSA ligand grafted on the QDs, quench their fluorescence differently, depending on the MSA/CdSe ratio used in synthesis. The crystal defects modulate the excitonic relaxation in CdSe and we demonstrated here that the surface defects intervene in the quenching of QDs induced by the binding of cations. (10.1016/j.jcis.2020.03.058)
DOI : 10.1016/j.jcis.2020.03.058
Monitoring the molecular composition of live cells exposed to electric pulses via label-free optical methods
- Azan Antoine
- Grognot Marianne
- García Sánchez Tomás
- Descamps Lucie
- Untereiner Valérie
- Piot Olivier
- Gallot Guilhem
- Mir Lluis M
Scientific Reports, Nature Publishing Group , 2020, 10 (1), pp.10471 . The permeabilization of the live cells membrane by the delivery of electric pulses has fundamental interest in medicine, in particular in tumors treatment by electrochemotherapy. Since underlying mechanisms are still not fully understood, we studied the impact of electric pulses on the biochemical composition of live cells thanks to label-free optical methods: confocal Raman microspectroscopy and terahertz microscopy. A dose effect was observed after cells exposure to different field intensities and a major impact on cell peptide/protein content was found. Raman measurements reveal that protein structure and/or environment are modified by the electric pulses while terahertz measurements suggest a leakage of proteins and other intracellular compounds. We show that Raman and terahertz modalities are a particularly attractive complement to fluorescence microscopy which is the reference optical technique in the case of electropermeabilization. Finally, we propose an analytical model for the influx and efflux of non-permeant molecules through transiently (electro)permeabilized cell membranes. (10.1038/s41598-020-67402-x)
DOI : 10.1038/s41598-020-67402-x
pKa Calculations with the Polarizable Drude Force Field and Poisson–Boltzmann Solvation Model
- Aleksandrov Alexey
- Roux Benoît
- Mackerell Alexander D.
Journal of Chemical Theory and Computation, American Chemical Society , 2020, 16 (7), pp.4655–4668 . Electronic polarization effects have been suggested to play an important role in proton binding to titratable residues in proteins. In this work, we describe a new computational method for pKa calculations, using Monte Carlo (MC) simulations to sample protein protonation states with the Drude polarizable force field and Poisson–Boltzmann (PB) continuum electrostatic solvent model. While the most populated protonation states at the selected pH, corresponding to residues that are half-protonated at that pH, are sampled using the exact relative free energies computed with Drude particles optimized in the field of the PB implicit solvation model, we introduce an approximation for the protein polarization of low-populated protonation states to reduce the computational cost. The highly populated protonation states used to compute the polarization and pKa’s are then iteratively improved until convergence. It is shown that for lysozyme, when considering 9 of the 18 titratable residues, the new method converged within two iterations with computed pKa’s differing only by 0.02 pH units from pKa’s estimated with the exact approach. Application of the method to predict pKa’s of 94 titratable side chains in 8 proteins shows the Drude-PB model to produce physically more correct results as compared to the additive CHARMM36 (C36) force field (FF). With a dielectric constant of two assigned to the protein interior the Root Mean Square (RMS) deviation between computed and experimental pKa’s is 2.07 and 3.19 pH units with the Drude and C36 models, respectively, and the RMS deviation using the Drude-PB model is relatively insensitive to the choice of the internal dielectric constant in contrast to the additive C36 model. At the higher internal dielectric constant of 20, pKa’s computed with the additive C36 model converge to the results obtained with the Drude polarizable force field, indicating the need to artificially overestimate electrostatic screening in a nonphysical way with the additive FF. In addition, inclusion of both syn and anti orientations of the proton in the neutral state of acidic groups is shown to yield improved agreement with experiment. The present work, which is the first example of the use of a polarizable model for the prediction of pKa’s in proteins, shows that the use of a polarizable model represents a more physically correct model for the treatment of electrostatic contributions to pKa shifts in proteins. (10.1021/acs.jctc.0c00111)
DOI : 10.1021/acs.jctc.0c00111
Electronic measurement of femtosecond time delays for arbitrary-detuning asynchronous optical sampling
- Antonucci Laura
- Solinas Xavier
- Bonvalet Adeline
- Joffre Manuel
Optics Express, Optical Society of America - OSA Publishing , 2020, 28 (12), pp.18251 . (10.1364/OE.393887)
DOI : 10.1364/OE.393887
Enabling large-scale genome editing at repetitive elements by reducing DNA nicking
- Smith Cory J
- Castanon Oscar
- Said Khaled
- Volf Verena
- Khoshakhlagh Parastoo
- Hornick Amanda
- Ferreira Raphael
- Wu Chun-Ting
- Güell Marc
- Garg Shilpa
- Ng Alex H M
- Myllykallio Hannu
- Church George M
Nucleic Acids Research, Oxford University Press , 2020, 48 (9), pp.5183-5195 . To extend the frontier of genome editing and enable editing of repetitive elements of mammalian genomes, we made use of a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks and single-strand breaks. We used a set of gRNAs targeting repetitive elements-ranging in target copy number from about 32 to 161 000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ∼13 200 and ∼12 200 loci in 293T and human induced pluripotent stem cells (hiP-SCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering. (10.1093/nar/gkaa239)
DOI : 10.1093/nar/gkaa239
Corneal stromal stem cells restore transparency after N 2 injury in mice
- Ghoubay Djida
- Borderie Marie
- Grieve Kate
- Martos Raphaël
- Bocheux Romain
- Nguyen Thu-Mai
- Callard Patrice
- Chédotal Alain
- Borderie Vincent M.
Stem Cells Translational Medicine, Wiley , 2020, 9 (8), pp.917-935 . Corneal scarring associated with various corneal conditions is a leading cause of blindness worldwide. The present study aimed to test the hypothesis that corneal stromal stem cells have a therapeutic effect and are able to restore the extracellular matrix organization and corneal transparency in vivo. We first developed a mouse model of corneal stromal scar induced by liquid nitrogen (N2 ) application. We then reversed stromal scarring by injecting mouse or human corneal stromal stem cells in injured cornea. To characterize the mouse model developed in this study and the therapeutic effect of corneal stromal stem cells, we used a combination of in vivo (slit lamp, optical coherence tomography, in vivo confocal microscopy, optical coherence tomography shear wave elastography, and optokinetic tracking response) and ex vivo (full field optical coherence microscopy, flow cytometry, transmission electron microscopy, and histology) techniques. The mouse model obtained features early inflammation, keratocyte apoptosis, keratocyte transformation into myofibroblasts, collagen type III synthesis, impaired stromal ultrastructure, corneal stromal haze formation, increased corneal rigidity, and impaired visual acuity. Injection of stromal stem cells in N2 -injured cornea resulted in improved corneal transparency associated with corneal stromal stem cell migration and growth in the recipient stroma, absence of inflammatory response, recipient corneal epithelial cell growth, decreased collagen type III stromal content, restored stromal ultrastructure, decreased stromal haze, decreased corneal rigidity, and improved vision. Our study demonstrates the ability of corneal stromal stem cells to promote regeneration of transparent stromal tissue after corneal scarring induced by liquid nitrogen. (10.1002/sctm.19-0306)
DOI : 10.1002/sctm.19-0306
Fast In Vivo Imaging of SHG Nanoprobes with Multiphoton Light-Sheet Microscopy
- Malkinson Guy
- Mahou Pierre
- Chaudan Élodie
- Gacoin Thierry
- Sonay Ali
- Pantazis Periklis
- Beaurepaire Emmanuel
- Supatto Willy
ACS photonics, American Chemical Society , 2020, 7 (4), pp.1036-1049 . (10.1021/acsphotonics.9b01749)
DOI : 10.1021/acsphotonics.9b01749
Fast In Vivo Imaging of SHG Nanoprobes with Multiphoton Light-Sheet Microscopy
- Malkinson Guy
- Mahou Pierre
- Chaudan Élodie
- Gacoin Thierry
- Sonay Ali
- Pantazis Periklis
- Beaurepaire Emmanuel
- Supatto Willy
ACS photonics, American Chemical Society , 2020, 7 (4), pp.1036-1049 . Two-photon light-sheet microscopy (2P-SPIM) provides a unique combination of advantages for fast and deep fluorescence imaging in live tissues. Detecting coherent signals such as second-harmonic generation (SHG) in 2P-SPIM in addition to fluorescence would open further imaging opportunities. However, light-sheet microscopy involves an orthogonal configuration of illumination and detection that questions the ability to detect coherent signals. Indeed, coherent scattering from micron-sized structures occurs predominantly along the illumination beam. By contrast, point-like sources such as SHG nanocrystals can efficiently scatter light in multiple directions and be detected using the orthogonal geometry of a light-sheet microscope. This study investigates the suitability of SHG light-sheet microscopy (SHG-SPIM) for fast imaging of SHG nanoprobes. Parameters that govern the detection efficiency of KTiOPO 4 and BaTiO 3 nanocrystals using SHG-SPIM are investigated theoretically and experimentally. The effects of incident polarization, detection numerical aperture, nanocrystal rotational motion, and second-order susceptibility tensor symmetries on the detectability of SHG nanoprobes in this specific geometry are clarified. Guidelines for optimizing SHG-SPIM imaging are established, enabling fast in vivo light-sheet imaging combining SHG and two-photon excited fluorescence. Finally, microangiography was achieved in live zebrafish embryos by SHG imaging at up to 180 frames per second and single-particle tracking of SHG nanoprobes in the blood flow. (10.1021/acsphotonics.9b01749)
DOI : 10.1021/acsphotonics.9b01749
Native Collagen: Electrospinning of Pure, Cross-Linker-Free, Self-Supported Membrane
- Dems Dounia
- Rodrigues da Silva Julien
- Hélary Christophe
- Wien Frank
- Marchand Marion
- Debons Nicolas
- Muller Laurent
- Chen Yong
- Schanne-Klein Marie-Claire
- Laberty-Robert Christel
- Krins Natacha
- Aimé Carole
ACS Applied Bio Materials, ACS Publications , 2020 . Rebuilding biological environments is crucial when facing the challenges of fundamental and biomedical research. Thus, preserving the native state of biomolecules is essential. We use electrospinning (ES), which is an extremely promising method for the preparation of fibrillar membranes to mimic the ECM of native tissues. Here, we report for the first time (1) the ES of pure and native collagen into a self-supported membrane in absence of crosslinker and polymer support, (2) the preservation of the membrane integrity in hydrated media in absence of crosslinker and (3) the preservation of the native molecular structure and recovery of the hierarchical assembly of collagen. We use a multiscale approach to characterize collagen native structure at the molecular level using circular dichroism, and to investigate collagen hierarchical organization within the self-supported membrane using a combination of multiphoton and electron microscopies. Finally, we show that the membranes are perfectly suited for cell adhesion and spreading, making them very promising candidates for the development of biomaterials and finding applications in biomedical research. (10.1021/acsabm.0c00006)
DOI : 10.1021/acsabm.0c00006
In vitro and intracellular activities of frog skin temporins against Legionella pneumophila and its eukaryotic hosts
- Crépin Alexandre
- Jégou Jean-Francois
- André Sonia
- Ecale Florine
- Croitoru Anastasia
- Cantereau Anne
- Berjeaud Jean-Marc
- Ladram Ali
- Verdon Julien
Scientific Reports, Nature Publishing Group , 2020, 10 (1), pp.3978 . Temporin-SHa (SHa) is a small cationic host defence peptide (HDP) produced in skin secretions of the Sahara frog Pelophylax saharicus. This peptide has a broad-spectrum activity, efficiently targeting bacteria, parasites and viruses. Noticeably, SHa has demonstrated an ability to kill Leishmania infantum parasites (amastigotes) within macrophages. Recently, an analog of SHa with an increased net positive charge, named [K3]SHa, has been designed to improve those activities. SHa and [K3]SHa were both shown to exhibit leishmanicidal activity mainly by permeabilization of cell membranes but could also induce apoptotis-like death. Temporins are usually poorly active against Gram-negative bacteria whereas many of these species are of public health interest. Among them, Legionella pneumophila, the etiological agent of Legionnaire's disease, is of major concern. Indeed, this bacterium adopts an intracellular lifestyle and replicate inside alveolar macrophages likewise inside its numerous protozoan hosts. Despite several authors have studied the antimicrobial activity of many compounds on L. pneumophila released from host cells, nothing is known about activity on intracellular L. pneumophila within their hosts, and subsequently mechanisms of action that could be involved. Here, we showed for the first time that SHa and [K3]SHa were active towards several species of Legionella. Both peptides displayed bactericidal activity and caused a loss of the bacterial envelope integrity leading to a rapid drop in cell viability. Regarding amoebae and THP-1-derived macrophages, SHa was less toxic than [K3]SHa and exhibited low half maximal lethal concentrations (LC50). When used at non-toxic concentration (6.25 µM), SHa killed more than 90% L. pneumophila within amoebae and around 50% within macrophages. Using SHa labeled with the fluorescent dye Cy5, we showed an evenly diffusion within cells except in vacuoles. Moreover, SHa was able to enter the nucleus of amoebae and accumulate in the nucleolus. This subcellular localization seemed specific as macrophages nucleoli remained unlabeled. Finally, no modifications in the expression of cytokines and HDPs were recorded when macrophages were treated with 6.25 µM SHa. By combining all data, we showed that temporin-SHa decreases the intracellular L. pneumophila load within amoebae and macrophages without being toxic for eukaryotic cells. This peptide was also able to reach the nucleolus of amoebae but was not capable to penetrate inside vacuoles. These data are in favor of an indirect action of SHa towards intracellular Legionella and make this peptide a promising template for further developments. (10.1038/s41598-020-60829-2)
DOI : 10.1038/s41598-020-60829-2
Terahertz sensing in biology and medicine
- Gallot Guilhem
Photoniques, EDP Sciences , 2020 (101), pp.53-58 . Terahertz radiation offers new contrasts with biological systems, without markers or staining, at the molecular, cellular or tissue level. Thanks to technological advances, it is increasingly emerging as a solution of choice for directly probing the interaction with molecules and biological solutions. Applications range from dynamics of biological molecules to imaging of cancerous tissues, including ion, protein and membrane sensors. (10.1051/photon/202010153)
DOI : 10.1051/photon/202010153
Ultrafast Dynamics and Vibrational Relaxation in Six-Coordinate Heme Proteins Revealed by Femtosecond Stimulated Raman Spectroscopy
- Ferrante Carino
- Batignani Giovanni
- Pontecorvo Emanuele
- Montemiglio Linda
- Vos Marten H.
- Scopigno Tullio
Journal of the American Chemical Society, American Chemical Society , 2020, 142 (5), pp.2285-2292 . Identifying the structural rearrangement and the active sites in photo-induced reactions is a fundamental challenge to understand from a microscopic perspective the underlying dynamics ruling the functional mechanisms of heme proteins. Here, femtosecond stimulated Raman spectroscopy is used to follow the ultrafast evolution of two 1 six-coordinate heme proteins. By exploiting the sensitivity of Raman spectra to the structural conguration, we investigate the eects of photolysis and binding of amino acid residues in cytochrome c and neuroglobin. Comparing the system response for different time delays and Raman pump resonances, we show how details of atomic motions and energy redistribution can be unveiled. (10.1021/jacs.9b10560)
DOI : 10.1021/jacs.9b10560
Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy
- Karadi Kristof
- Kapetanaki Sofia
- Raics Katalin
- Pecsi Ildiko
- Kapronczai Robert
- Fekete Zsuzsanna
- Iuliano James
- Collado Jinnette Tolentino
- Gil Agnieszka
- Orban Jozsef
- Greetham Greg
- Vos Marten H.
- Tonge Peter
- Meech Stephen
- Lukacs Andras
Scientific Reports, Nature Publishing Group , 2020, 10, pp.2061 . Blue Light Using flavin (BLUf) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light-and darkadapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule. (10.1038/s41598-020-59073-5)
DOI : 10.1038/s41598-020-59073-5
Differentiation of neural-type cells on multi-scale ordered collagen-silica bionanocomposites
- Debons Nicolas
- Dems Dounia
- Hélary Christophe
- Le Grill Sylvain
- Picaut Lise
- Renaud Flore
- Delsuc Nicolas
- Schanne-Klein Marie-Claire
- Coradin Thibaud
- Aimé Carole
Biomaterials Science, Royal Society of Chemistry (RSC) , 2020 . Cells respond to biophysical and biochemical signals. We developed a composite filament from collagen and silica particles modified to interact with collagen and/or present a laminin epitope (IKVAV) crucial for cell–matrix adhesion and signal transduction. This combines scaffolding and signaling and shows that local tuning of collagen organization enhances cell differentiation. (10.1039/C9BM01029G)
DOI : 10.1039/C9BM01029G