Publications

2020

nAdder: A scale-space approach for the 3D analysis of neuronal traces
- Phan Minh Son
- Matho Katherine
- Beaurepaire Emmanuel
- Livet Jean
- Chessel Anatole
, 2020. Tridimensional microscopy and algorithms for automated segmentation and tracing are revolutionizing neuroscience through the generation of growing libraries of neuron reconstructions. Innovative computational methods are needed to analyze these neural traces. In particular, means to analyse the geometric properties of traced neurites along their trajectory have been lacking. Here, we propose a local tridimensional (3D) scale metric derived from differential geometry, which is the distance in micrometers along which a curve is fully 3D as opposed to being embedded in a 2D plane or 1D line. We apply this metric to various neuronal traces ranging from single neurons to whole brain data. By providing a local readout of the geometric complexity, it offers a new mean of describing and comparing axonal and dendritic arbors from individual neurons or the behavior of axonal projections in different brain regions. This broadly applicable approach termed nAdder is available through the GeNePy3D open-source Python quantitative geometry library. (10.1101/2020.06.01.127035)
DOI : 10.1101/2020.06.01.127035
Mechanism of Naphthoquinone Selectivity of Thymidylate Synthase ThyX
- Myllykallio Hannu
- Becker Hubert F
- Aleksandrov Alexey
Biophysical Journal, Biophysical Society, 2020, 119 (12), pp.2508-2516. Naphthoquinones (NQs) are natural and synthetic compounds with a wide range of biological activities commonly attributed to their redox activity and/or chemical reactivity. However, genetic and biochemical experiments have recently demonstrated that 2-hydroxy-NQs (2-OH-NQs) act as highly specific non-covalent inhibitors of the essential bacterial thymidylate synthase ThyX in a cellular context. We used biochemical experiments and molecular dynamics simulations to elucidate the selective inhibition mechanism of NQ inhibitors of ThyX from Mycobacterium tuberculosis (Mtb). Free energy simulations rationalized how ThyX recognizes the natural substrate dUMP in the N3 ionized form using an arginine, Arg199 in Mtb. The results further demonstrated that 2-OH-NQ, similarly to dUMP, binds to ThyX in the ionized form and the strong and selective binding of 2-OH-NQ to ThyX is also explained by electrostatic interactions with Arg199. The stronger binding of the close analog 5F-dUMP to ThyX and its inhibitory properties compared to dUMP were explained by the stronger acidity of the uracil N3 atom. Our results, therefore, revealed that the ionization of 2-OH-NQs drives their biological activities by mimicking the interactions with the natural substrate. Our observations encourage the rational design of optimized ThyX inhibitors that ultimately may serve as antibiotics. (10.1016/j.bpj.2020.10.042)
DOI : 10.1016/j.bpj.2020.10.042
Electric field measurements in plasmas: how focusing strongly distorts the E-FISH signal
- Chng Tat Loon
- Starikovskaia Svetlana
- Schanne-Klein Marie-Claire
Plasma Sources Science and Technology, IOP Publishing, 2020, 29 (12), pp.125002. (10.1088/1361-6595/abbf93)
DOI : 10.1088/1361-6595/abbf93
CRISPR-mediated biocontainment
- Castanon Oscar
- Smith Cory J
- Khoshakhlagh Parastoo
- Ferreira Raphael
- Güell Marc
- Said Khaled
- Yildiz Ramazan
- Dysart Matthew
- Wang Stan
- Thompson David
- Myllykallio Hannu
- Church George M
, 2020. We have exploited the repetitive nature of transposable elements of the human genome to generate synthetic circuits. Transposable elements such as LINE-1 and Alu have successfully replicated in mammalian genomes throughout evolution to reach a copy number ranging from thousands to more than a million. Targeting these repetitive elements with programmable DNA nucleases such as CRISPR-Cas9 rapidly induce extremely high levels of cell death. We use this genotoxic feature to build synthetic biocontainment circuits: CRISPR defense system (CRISPR-DS) capable of preventing CRISPR genome editing, and we introduce the proof-of-concept of CRISPR Safety-Switch, an inducible, stringent and non-leaky kill-switch capable of clearing out cell lines resistant to DNA breaks. . (10.1101/2020.02.03.922146)
DOI : 10.1101/2020.02.03.922146
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
Probing living cells by terahertz Attenuated Total Reflection : permeabilization dynamics of the cell membrane
- Zheng Xiujun
, 2020. Terahertz radiation is located in the electromagnetic range between far infrared and microwaves, corresponding to frequencies between 0.1 and 10 THz. This spectral range is currently largely under-exploited, but its application to the study of biological objects has already shown a strong potential, in the detection of skin cancer, ion flow monitoring or biosensors. In the field of biology, which is of particular interest to us here, the terahertz range makes it possible to quantify and discriminate solutes of biological interest thanks to the interaction with low-frequency modes of liquid water, and thus to study biomolecules, microorganisms and cells in their physiological environment. The first part of this thesis work consisted in studying the dynamics of membrane permeabilization of living cells by attenuated total reflection (ATR) with our device based on a femtosecond laser and the generation of ultrashort terahertz pulses. Monolayers of MDCK epithelial cells were exposed to varying concentrations of saponin, a detergent that digs holes in the cell membrane. The dynamics obtained were then compared to a theoretical model describing the physical behavior of the cell layer, taking into account the diffusion of detergent molecules and the physical characteristics of the membrane. The good agreement between experiment and theory indicates that membrane permeabilization is limited mainly by the diffusion of detergent molecules and their binding to the membrane.In a second part, we developed a completely new system based on a continuous terahertz QCL source at 2.5 THz. This new instrument is based on a very simplified design, with a single ATR prism and a single detector, and on the dual modulation of the terahertz beam using a mechanical chopper. This chopper synchronizes the dual modulation and defines the measurement and reference zones. The long-term stability of this device has been greatly enhanced by the precise control of temperature and humidity inside the device. Performance is excellent in both the short and long term. A signal-to-noise ratio of 30 dB is achieved over 300ms, and remains above 30 dB for several hours. In addition, a theoretical and experimental study has been carried out to calibrate the instrument. Thus, the ATR reflection coefficients of several solutions of biological interest (ions, sugars and proteins) were obtained over a wide range of concentrations. A sensitivity at least 20 times higher than that of the existing literature was thus obtained. Thanks to this new high-performance system, we studied the dynamics of membrane permeabilization following the action of photodynamic therapy (PDT). The first results showed that the encapsulation of photosensitizers by micellar vectors significantly improves the efficiency of PDT.
Balancing signal and photoperturbation in multiphoton light-sheet microscopy by optimizing laser pulse frequency
- Maioli Vincent
- Boniface Antoine
- Mahou Pierre
- Julia Ferrer Ortas
- Abdeladim Lamiae
- Beaurepaire Emmanuel
- Supatto Willy
, 2020. 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 balance 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. (10.1101/2020.06.02.130377)
DOI : 10.1101/2020.06.02.130377
Dynamic spatiotemporal coordination of neural stem cell fate decisions through local feedback in the adult vertebrate brain
- Dray Nicolas
- Mancini Laure
- Binshtok Udi
- Cheysson Felix
- Supatto Willy
- Mahou Pierre
- Bedu Sébastien
- Ortica Sara
- Krecsmarik Monika
- Herbert Sébastien
- Masson Jean-Baptiste
- Tinevez Jean-Yves
- Lang Gabriel
- Beaurepaire Emmanuel
- Sprinzak David
- Bally-Cuif Laure
, 2020. Neural stem cell (NSC) populations persist in the adult vertebrate brain over a life time, and their homeostasis is controlled at the population level. The nature and properties of these coordination mechanisms remain unknown. Here we combine dynamic imaging of entire NSC populations in their in vivo niche over weeks, pharmacological manipulations, mathematical modeling and spatial statistics, and demonstrate that NSCs use spatiotemporally resolved local feedbacks to coordinate their decision to divide. These involve a Notch-mediated inhibition from transient neural progenitors, and a dispersion effect from dividing NSCs themselves, exerted with a delay of 9-12 days. Simulations from a stochastic NSC lattice model capturing these interactions demonstrate that they are linked by lineage progression and control the spatiotemporal distribution of output neurons. These results highlight how local and temporally delayed interactions occurring between brain germinal cells generate self-propagating dynamics that maintain NSC population homeostasis with specific spatiotemporal correlations. (10.1101/2020.07.15.205021)
DOI : 10.1101/2020.07.15.205021
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
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
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. 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
Relating Nanostructural Properties of the Cornea, its Macroscopic Transparency and Compressibility
- Plamann Karsten
- Bocheux Romain
- Tricoli Ugo
- Peyrot Donald A.
- Savoldelli Michèle
- Irsch Kristina
- Carminati Rémi
, 2020.

Retour aux années