Articles

Probing living cells permeabilization dynamics by terahertz attenuated total reflection
- Gallot Guilhem
Proceedings of SPIE, the International Society for Optical Engineering, SPIE, The International Society for Optical Engineering , 2021 . Using attenuated total reflection (ATR) in the terahertz domain, we demonstrate non-invasive, non-staining real time measurements of cytoplasm leakage during permeabilization of live epithelial cells by saponin detergent and after electropermeabilization. The origin of the contrast observed between cells and culture medium is addressed by both experimental and theoretical approaches, and gives access to permeabilization dynamics of live cells in real time. We show that terahertz modalities are more sensitive than fluorescence microscopy which is the reference optical technique for electropermeabilization. We propose analytical models for the influx and efflux of non-permeant molecules through permeabilized cell membranes. (10.1117/12.2596166)
DOI : 10.1117/12.2596166
The role of G-Quadruplex DNA in Paraspeckle formation in cancer
- Bhatt Uditi
- Kretzmann Amy
- Guédin Aurore
- Ou Arnold
- Kobelke Simon
- Bond Charles
- Evans Cameron
- Hurley Laurence
- Mergny Jean-Louis
- Iyer K. Swaminathan
- Fox Archa
- Smith Nicole
Biochimie, Elsevier , 2021 . (10.1016/j.biochi.2021.07.008)
DOI : 10.1016/j.biochi.2021.07.008
Noninvasive quantitative assessment of collagen degradation in parchments by polarization-resolved SHG microscopy
- Schmeltz Margaux
- Robinet Laurianne
- Heu-Thao Sylvie
- Sintès Jean-Marc
- Teulon Claire
- Ducourthial Guillaume
- Mahou Pierre
- Schanne-Klein Marie-Claire
- Latour Gaël
Science Advances, American Association for the Advancement of Science (AAAS) , 2021, 7 (29), pp.eabg1090 . Nondestructive and noninvasive investigation techniques are highly sought-after to establish the degradation state of historical parchments, which is up to now assessed by thermal techniques that are invasive and destructive. We show that advanced nonlinear optical (NLO) microscopy enables quantitative in situ mapping of parchment degradation at the micrometer scale. We introduce two parameters that are sensitive to different degradation stages: the ratio of two-photon excited fluorescence to second harmonic generation (SHG) signals probes severe degradation, while the anisotropy parameter extracted from polarization-resolved SHG measurements is sensitive to early degradation. This approach is first validated by comparing NLO quantitative parameters to thermal measurements on artificially altered contemporary parchments. We then analyze invaluable parchments from the Middle Ages and show that we can map their conservation state and assess the impact of a restoration process. NLO quantitative microscopy should therefore help to identify parchments most at risk and optimize restoration methods. (10.1126/sciadv.abg1090)
DOI : 10.1126/sciadv.abg1090
Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula
- Boutillon Arthur
- Escot Sophie
- David Nicolas B.
Journal of visualized experiments : JoVE, JoVE , 2021 (173) . Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly coordinated, and accumulating evidence suggests this is achieved, at least in part, through mechanical interactions. Testing this in the embryo requires direct physical perturbations. Laser ablations are an increasingly used option that allows relieving mechanical constraints or physically isolating two cell populations from each other. However, many ablations are performed with an ultraviolet (UV) laser, which offers limited axial resolution and tissue penetration. A method is described here to ablate deep, significant, and spatially well-defined volumes using a two-photon microscope. Ablations are demonstrated in a transgenic zebrafish line expressing the green fluorescent protein in the axial mesendoderm and used to sever the axial mesendoderm without affecting the overlying ectoderm or the underlying yolk cell. Cell behavior is monitored by live imaging before and after the ablation. The ablation protocol can be used at different developmental stages, on any cell type or tissue, at scales ranging from a few microns to more than a hundred microns. (10.3791/62815)
DOI : 10.3791/62815
Folding Dynamics of DNA G-Quadruplexes Probed by Millisecond Temperature Jump Circular Dichroism
- Laouer K
- Schmid M
- Wien F
- Changenet Pascale
- Hache François
Journal of Physical Chemistry B, American Chemical Society , 2021 . (10.1021/acs.jpcb.1c01993)
DOI : 10.1021/acs.jpcb.1c01993
Modeling nonlinear microscopy near index-mismatched interfaces
- Morizet Josephine
- Sartorello Giovanni
- Dray Nicolas
- Stringari Chiara
- Beaurepaire Emmanuel
- Olivier Nicolas
Optica, Optical Society of America - OSA Publishing , 2021, 8 (7), pp.944 . Nonlinear microscopy is widely used to characterize thick, optically heterogeneous biological samples. While quantitative image analysis requires accurately describing the contrast mechanisms at play, the majority of established numerical models neglect the influence of field distortion caused by sample heterogeneity near focus. In this work, we show experimentally and numerically that finite-difference time-domain (FDTD) methods are applicable to model focused fields interactions in the presence of heterogeneities, typical of nonlinear microscopy. We analyze the ubiquitous geometry of a vertical interface between index-mismatched media (water, glass, and lipids) and consider the cases of two-photon-excited fluorescence (2PEF), third-harmonic generation (THG) and polarized THG contrasts. We show that FDTD simulations can accurately reproduce experimental images obtained on model samples and in live adult zebrafish, in contrast with previous models neglecting field distortions caused by index mismatch at the micrometer scale. Accounting for these effects appears to be particularly critical when interpreting coherent and polarization-resolved microscopy data. (10.1364/OPTICA.421257)
DOI : 10.1364/OPTICA.421257
Quantitative structural imaging of keratoconic corneas using polarization-resolved SHG microscopy
- Raoux Clothilde
- Schmeltz Margaux
- Bied Marion
- Alnawaiseh Maged
- Hansen Uwe
- Latour Gaël
- Schanne-Klein Marie-Claire
Biomedical optics express, Optical Society of America - OSA Publishing , 2021, 12 . The human cornea is mainly composed of collagen fibrils aligned together within stacked lamellae. This lamellar structure can be affected in pathologies such as keratoconus, which is characterized by progressive corneal thinning and local steepening. In this study, we use polarization-resolved second harmonic generation (P-SHG) microscopy to characterize 8 control and 6 keratoconic human corneas. Automated processing of P-SHG images of transverse sections provides the collagen orientation in every pixel with sub-micrometer resolution. Series of P-SHG images recorded in the most anterior region of the stroma evidence sutural lamellae inclined at 22°± 5°to the corneal surface, but show no significant difference between control and keratoconic corneas. In contrast, series of P-SHG images acquired along the full thickness of the stroma show a loss of order in the lamellar structure of keratoconic corneas, in agreement with their defective mechanical properties. This structural difference is analyzed quantitatively by computing the entropy and the orientation index of the collagen orientation distribution and significant differences are obtained along the full thickness of the stroma. This study shows that P-SHG is an effective tool for automatic quantitative analysis of structural defects of human corneas and should be applied to other collagen-rich tissues. (10.1364/boe.426145)
DOI : 10.1364/boe.426145
Additive CHARMM36 Force Field for Nonstandard Amino Acids
- Croitoru Anastasia
- Park Sang-Jun
- Kumar Anmol
- Lee Jumin
- Im Wonpil
- Mackerell Alexander
- Aleksandrov Alexey
Journal of Chemical Theory and Computation, American Chemical Society , 2021, 17 (6), pp.3554-3570 . (10.1021/acs.jctc.1c00254)
DOI : 10.1021/acs.jctc.1c00254
Exposure to the Methylselenol Precursor Dimethyldiselenide Induces a Reductive Endoplasmic Reticulum Stress in Saccharomyces cerevisiae
- Dauplais Marc
- Mahou Pierre
- Plateau Pierre
- Lazard Myriam
International Journal of Molecular Sciences, MDPI , 2021, 22 (11), pp.5467 . Methylselenol (MeSeH) is a major cytotoxic metabolite of selenium, causing apoptosis in cancer cells through mechanisms that remain to be fully established. Previously, we demonstrated that, in Saccharomyces cerevisiae, MeSeH toxicity was mediated by its metabolization into selenomethionine by O-acetylhomoserine (OAH)-sulfhydrylase, an enzyme that is absent in higher eukaryotes. In this report, we used a mutant met17 yeast strain, devoid of OAH- sulfhydrylase activity, to identify alternative targets of MeSeH. Exposure to dimethyldiselenide (DMDSe), a direct precursor of MeSeH, caused an endoplasmic reticulum (ER) stress, as evidenced by increased expression of the ER chaperone Kar2p. Mutant strains (∆ire1 and ∆hac1) unable to activate the unfolded protein response were hypersensitive to MeSeH precursors but not to selenomethionine. In contrast, deletion of YAP1 or SKN7, required to activate the oxidative stress response, did not affect cell growth in the presence of DMDSe. ER maturation of newly synthesized carboxypeptidase Y was impaired, indicating that MeSeH/DMDSe caused protein misfolding in the ER. Exposure to DMDSe resulted in induction of the expression of the ER oxidoreductase Ero1p with concomitant reduction of its regulatory disulfide bonds. These results suggest that MeSeH disturbs protein folding in the ER by generating a reductive stress in this compartment. (10.3390/ijms22115467)
DOI : 10.3390/ijms22115467
GeNePy3D: a quantitative geometry python toolbox for bioimaging
- Phan Minh-Son
- Chessel Anatole
F1000Research, Faculty of 1000 , 2021 . The advent of large-scale fluorescence and electronic microscopy techniques along with maturing image analysis is giving life sciences a deluge of geometrical objects in 2D/3D(+t) to deal with. These objects take the form of large scale, localised, precise, single cell, quantitative data such as cells’ positions, shapes, trajectories or lineages, axon traces in whole brains atlases or varied intracellular protein localisations, often in multiple experimental conditions. The data mining of those geometrical objects requires a variety of mathematical and computational tools of diverse accessibility and complexity. Here we present a new Python library for quantitative 3D geometry called GeNePy3D which helps handle and mine information and knowledge from geometric data, providing a unified application programming interface (API) to methods from several domains including computational geometry, scale space methods or spatial statistics. By framing this library as generically as possible, and by linking it to as many state-of-the-art reference algorithms and projects as needed, we help render those often specialist methods accessible to a larger community. We exemplify the usefulness of the GeNePy3D toolbox by re-analysing a recently published whole-brain zebrafish neuronal atlas, with other applications and examples available online. Along with an open source, documented and exemplified code, we release reusable containers to allow for convenient and wide usability and increased reproducibility. (10.12688/f1000research.27395.2)
DOI : 10.12688/f1000research.27395.2
Drivers of i-DNA Formation in a Variety of Environments Revealed by Four-Dimensional UV Melting and Annealing
- Cheng Mingpan
- Chen Jielin
- Ju Huangxian
- Zhou Jun
- Mergny Jean-Louis
Journal of the American Chemical Society, American Chemical Society , 2021, 143, pp.7792-7807 . i-DNA is a four-stranded, pH-sensitive structure formed by cytosine-rich DNA sequences. Previous reports have addressed the conditions for formation of this motif in DNA in vitro and validated its existence in human cells. Unfortunately, these in vitro studies have often been performed under different experimental conditions, making comparisons difficult. To overcome this, we developed a four-dimensional UV melting and annealing (4DUVMA) approach to analyze i-DNA formation under a variety of conditions (e.g., pH, temperature, salt, crowding). Analysis of 25 sequences provided a global understanding of i-DNA formation under disparate conditions, which should ultimately allow the design of accurate prediction tools. For example, we found reliable linear correlations between the mid-point of pH transition and temperature (-0.04 ± 0.003 pH unit per 1.0 °C temperature increment) and between the melting temperature and pH (-23.8 ± 1.1 °C per pH unit increment). In addition, by analyzing the hysteresis between denaturing and renaturing profiles in both pH and thermal transitions, we found that loop length, nature of the C-tracts, pH, temperature, and crowding agents all play roles in i-DNA folding kinetics. Interestingly, our data indicate which conformer is more favorable for the sequences with an odd number of cytosine base pairs. Then the h m l pH l f " "-DNAs from human promoter genes were measured under near physiological conditions (pH 7.0, 37 °C). The 4DUVMA method can become a universal resource to analysis the properties of any i-DNA-prone sequence. (10.1021/jacs.1c02209)
DOI : 10.1021/jacs.1c02209
Fluorescent iron‑sulfur centers: Photochemistry of the PetA Rieske protein from Aquifex aeolicus
- Vos Marten H.
- Salman Mayla
- Ramodiharilafy Rivo
- Liebl Ursula
Biochimica biophysica acta (BBA) - Bioenergetics, Elsevier , 2021, 1862 (5), pp.148385 . Cytochrome bc1 complexes are energy-transducing enzymes and key components of respiratory electron chains. They contain Rieske 2Fe-2S proteins that absorb very weakly in the visible absorption region compared to the heme cofactors of the cytochromes, but are known to yield photoproducts. Here, the photoreactions of isolated Rieske proteins from the hyperthermophilic bacterium Aquifex aeolicus are studied in two redox states using ultrafast transient fluorescence and absorption spectroscopy. We provide evidence, for the first time in iron-sulfur proteins, of very weak fluorescence of the excited state, in the oxidized as well as the reduced state. The excited states of the oxidized and reduced forms decay in 1.5 ps and 30 picoseconds, respectively. In both cases they give rise to product states with lifetimes beyond 1 nanosecond, reflecting photo-reduction of oxidized centers as well as photo-oxidation of reduced centers. Potential reaction partners are discussed and studied using site-directed mutagenesis. For the reduced state, a nearby disulfide bridge is suggested as an electron acceptor. The resulting photoproducts in either state may play a role in photoactivation processes. (10.1016/j.bbabio.2021.148385)
DOI : 10.1016/j.bbabio.2021.148385
Mechanism and dynamics of fatty acid photodecarboxylase
- Sorigué Damien
- Hadjidemetriou Kyprianos
- Blangy S.
- Gotthard G.
- Bonvalet A.
- Coquelle Nicolas
- Samire P.
- Aleksandrov Alexey
- Antonucci L.
- Benachir A.
- Boutet S.
- Byrdin Martin
- Cammarata Marco
- Carbajo S.
- Cuine Stephan
- Doak R.
- Foucar L.
- Gorel A.
- Grünbein M.
- Hartmann E.
- Hienerwadel Rainer
- Hilpert M.
- Kloos M.
- Lane T.
- Légeret B.
- Legrand P.
- Li-Beisson Y.
- Moulin S.
- Nurizzo D.
- Peltier G.
- Schirò Giorgio
- Shoeman R.
- Sliwa M.
- Solinas X.
- Zhuang B.
- Barends T.
- Colletier Jacques-Philippe
- Joffre M.
- Royant Antoine
- Berthomieu C.
- Weik Martin
- Domratcheva T.
- Brettel K.
- Vos Marten H.
- Schlichting I.
- Arnoux Pascal
- Müller P.
- Beisson F.
Science, American Association for the Advancement of Science (AAAS) , 2021, 372 (6538), pp.eabd5687 . Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer. (10.1126/science.abd5687)
DOI : 10.1126/science.abd5687
Dynamic spatiotemporal coordination of neural stem cell fate decisions occurs through local feedback in the adult vertebrate brain
- Dray Nicolas
- Mancini Laure
- Binshtok Udi
- Cheysson Felix
- Supatto Willy
- Mahou Pierre
- Bedu Sébastien
- Ortica Sara
- Than-Trong Emmanuel
- Krecsmarik Monika
- Herbert Sébastien
- Masson Jean-Baptiste
- Tinevez Jean-Yves
- Lang Gabriel
- Beaurepaire Emmanuel
- Sprinzak David
- Bally-Cuif Laure
Cell Stem Cell, Cambridge, MA : Cell Press , 2021, 28 (8), pp.1-16 . Neural stem cell (NSC) populations persist in the adult vertebrate brain over a lifetime, and their homeostasis is controlled at the population level through unknown mechanisms. Here, we combine dynamic imaging of entire NSC populations in their in vivo niche over several weeks with pharmacological manipulations, mathematical modeling, and spatial statistics and demonstrate that NSCs use spatiotemporally resolved local feedback signals to coordinate their decision to divide in adult zebrafish brains. These involve Notch-mediated short-range inhibition from transient neural progenitors and a dispersion effect from the dividing NSCs themselves exerted with a delay of 9–12 days. Simulations from a stochastic NSC lattice model capturing these interactions demonstrate that these signals 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 and coordinate specific spatiotemporal correlations. (10.1016/j.stem.2021.03.014)
DOI : 10.1016/j.stem.2021.03.014
Analyses of viral genomes for G-quadruplex forming sequences reveal their correlation with the type of infection
- Bohálová Natália
- Cantara Alessio
- Bartas Martin
- Kaura Patrik
- Šťastný Jiří
- Pečinka Petr
- Fojta Miroslav
- Mergny Jean‐louis
- Brázda Václav
Biochimie, Elsevier , 2021, 186, pp.13-27 . (10.1016/j.biochi.2021.03.017)
DOI : 10.1016/j.biochi.2021.03.017
Exploration of head-to-tail and head-to-head isomers of a guanine quadruplex platinum-based binder
- Carson Jacob Joel Kirsh
- Miron Caitlin Elizabeth
- Luo Jingwei
- Mergny Jean‐louis
- van Staalduinen Laura
- Jia Zongchao
- Petitjean Anne
Inorganica Chimica Acta Reviews, Elsevier , 2021, 518, pp.120236 . (10.1016/j.ica.2020.120236)
DOI : 10.1016/j.ica.2020.120236
Mueller polarimetric imaging for fast macroscopic mapping of microscopic collagen matrix remodeling by smooth muscle cells
- Chashchina Olga
- Mezouar Hachem
- Vizet Jérémy
- Raoux Clothilde
- Park Junha
- Ramón-Lozano Clara
- Schanne-Klein Marie-Claire
- Barakat Abdul I
- Pierangelo Angelo
Scientific Reports, Nature Publishing Group , 2021, 11 . Smooth muscle cells (SMCs) are critical players in cardiovascular disease development and undergo complex phenotype switching during disease progression. However, SMC phenotype is difficult to assess and track in co-culture studies. To determine the contractility of SMCs embedded within collagen hydrogels, we performed polarized light imaging and subsequent analysis based on Mueller matrices. Measurements were made both in the absence and presence of endothelial cells (ECs) in order to establish the impact of EC-SMC communication on SMC contractility. The results demonstrated that Mueller polarimetric imaging is indeed an appropriate tool for assessing SMC activity which significantly modifies the hydrogel retardance in the presence of ECs. These findings are consistent with the idea that EC-SMC communication promotes a more contractile SMC phenotype. More broadly, our findings suggest that Mueller polarimetry can be a useful tool for studies of spatial heterogeneities in hydrogel remodeling by SMCs. (10.1038/s41598-021-85164-y)
DOI : 10.1038/s41598-021-85164-y
Ultrafast dynamics of heme distortion in the O2-sensor of a thermophilic anaerobe bacterium
- Petrova Olga N
- Yoo Byung-Kuk
- Lamarre Isabelle
- Selles Julien
- Nioche Pierre
- Negrerie Michel
Communications Chemistry, Nature Research , 2021, 4 (1) . Heme-Nitric oxide and Oxygen binding protein domains (H-NOX) are found in signaling pathways of both prokaryotes and eukaryotes and share sequence homology with soluble guanylate cyclase, the mammalian NO receptor. In bacteria, H-NOX is associated with kinase or methyl accepting chemotaxis domains. In the O2-sensor of the strict anaerobe Caldanaerobacter tengcongensis (Ct H-NOX) the heme appears highly distorted after O2 binding, but the role of heme distortion in allosteric transitions was not yet evidenced. Here, we measure the dynamics of the heme distortion triggered by the dissociation of diatomics from Ct H-NOX using transient electronic absorption spectroscopy in the picosecond to millisecond time range. We obtained a spectroscopic signature of the heme flattening upon O2 dissociation. The heme distortion is immediately (<1 ps) released after O2 dissociation to produce a relaxed state. This heme conformational change occurs with different proportions depending on diatomics as follows: CO < NO < O2. Our time-resolved data demonstrate that the primary structural event of allostery is the heme distortion in the Ct H-NOX sensor, contrastingly with hemoglobin and the human NO receptor, in which the primary structural events are respectively the motion of the proximal histidine and the rupture of the iron-histidine bond. (10.1038/s42004-021-00471-9)
DOI : 10.1038/s42004-021-00471-9
Ligand Binding to Dynamically Populated G‐Quadruplex DNA
- Aznauryan Mikayel
- Noer Sofie Louise
- Pedersen Camilla
- Mergny Jean‐louis
- Teulade-Fichou Marie‐paule
- Birkedal Victoria
ChemBioChem, Wiley-VCH Verlag , 2021, 22 (10), pp.1811-1817 . Several small‐molecule ligands specifically bind and stabilize G‐quadruplex (G4) nucleic acid structures, which are considered to be promising therapeutic targets. G4s are polymorphic structures of varying stability, and their formation is dynamic. Here, we investigate the mechanisms of ligand binding to dynamically populated human telomere G4 DNA by using the bisquinolinium based ligand Phen‐DC3 and a combination of single‐molecule FRET microscopy, ensemble FRET and CD spectroscopies. Different cations are used to tune G4 polymorphism and folding dynamics. We find that ligand binding occurs to pre‐folded G4 structures and that Phen‐DC3 also induces G4 formation in unfolded single strands. Following ligand binding to dynamically populated G4s, the DNA undergoes pronounced conformational redistributions that do not involve direct ligand‐induced G4 conformational interconversion. On the contrary, the redistribution is driven by ligand‐induced G4 folding and trapping of dynamically populated short‐lived conformation states. Thus, ligand‐induced stabilization does not necessarily require the initial presence of stably folded G4s. (10.1002/cbic.202000792)
DOI : 10.1002/cbic.202000792
Influence of core extension and side chain nature in targeting G-quadruplex structures with perylene monoimide derivatives
- Busto Natalia
- García-Calvo José
- Cuevas José Vicente
- Herrera Antonio
- Mergny Jean-Louis
- Pons Sebastian
- Torroba Tomás
- García Begoña
Bioorganic Chemistry, Elsevier , 2021, 108 (18), pp.104660 . Abstract Recent studies indicate that i‐DNA, a four‐stranded cytosine‐rich DNA also known as the i‐motif, is actually formed in vivo; however, a systematic study on sequence effects on stability has been missing. Herein, an unprecedented number of different sequences (271) bearing four runs of 3–6 cytosines with different spacer lengths has been tested. While i‐DNA stability is nearly independent on total spacer length, the central spacer plays a special role on stability. Stability also depends on the length of the C‐tracts at both acidic and neutral pHs. This study provides a global picture on i‐DNA stability thanks to the large size of the introduced data set; it reveals unexpected features and allows to conclude that determinants of i‐DNA stability do not mirror those of G‐quadruplexes. Our results illustrate the structural roles of loops and C‐tracts on i‐DNA stability, confirm its formation in cells, and allow establishing rules to predict its stability. (10.1016/j.bioorg.2021.104660)
DOI : 10.1016/j.bioorg.2021.104660
Characterization of Light-Induced, Short-Lived Interacting Radicals in the Active Site of Flavoprotein Ferredoxin-NADP + Oxidoreductase
- Zhuang Bo
- Seo Daisuke
- Aleksandrov Alexey
- Vos Marten H.
Journal of the American Chemical Society, American Chemical Society , 2021, 143 (7), pp.2757-2768 . Radicals of flavin adenine dinucleotide (FAD), as well as tyrosine and tryptophan, are widely involved as key reactive intermediates during electron transfer (ET) reactions in flavoproteins. Due to the high reactivity of these species, and their corresponding short lifetime, characterization of these intermediates in functional processes of flavoproteins is usually challenging, but can be achieved by ultrafast spectroscopic studies of light-activatable flavoproteins. In ferredoxin-NADP + oxidoreductase from Bacillus subtilis (BsFNR), fluorescence of the FAD cofactor that very closely interacts with a neighboring tyrosine residue (Tyr50), is strongly quenched. Here we study short-lived photoproducts of this enzyme and its variants with Tyr50 replaced by tryptophan or glycine. Using time-resolved fluorescence and absorption spectroscopies, we show that upon the excitation of WT BsFNR, ultrafast ET from Tyr50 to the excited FAD cofactor occurs in ~260 fs, an order of magnitude faster than the decay by charge recombination, facilitating the characterization of the reaction intermediates in the charge-separated state with respect to other recently studied systems. These studies are corroborated by experiments on the Y50W mutant protein, which yield photoproducts qualitatively similar to those observed in other tryptophan bearing flavoproteins. By combining the experimental results with molecular dynamics simulations and quantum mechanics calculations, we investigate in detail the effect of protein environment and relaxations on the spectral properties of those radical intermediates, and demonstrate that the spectral features of radical anionic FAD are highly sensitive to its environment, and in particular to the dynamics and nature of the counter-ions formed in the photoproducts. Altogether, comprehensive characterizations are provided for important radical intermediates that are generally involved in functional processes of flavoproteins. (10.1021/jacs.0c09627)
DOI : 10.1021/jacs.0c09627
Effects of sequence and base composition on the CD and TDS profiles of i-DNA
- Iaccarino Nunzia
- Cheng Mingpan
- Qiu Dehui
- Pagano Bruno
- Amato Jussara
- Porzio Anna Di
- Zhou Jun
- Randazzo Antonio
- Mergny Jean‐louis
Angewandte Chemie International Edition, Wiley-VCH Verlag , 2021, 60, pp.10295-10303 . (10.1002/anie.202016822)
DOI : 10.1002/anie.202016822
Identification of the vibrational marker of tyrosine cation radical using ultrafast transient infrared spectroscopy of flavoprotein systems
- Pirisi Katalin
- Nag Lipsa
- Fekete Zsuzsanna
- Iuliano James N
- Tolentino Collado Jinnette
- Clark Ian P
- Pécsi Ildikó
- Sournia Pierre
- Liebl Ursula
- Greetham Gregory M
- Tonge Peter J
- Meech Stephen R
- Vos Marten H.
- Lukacs Andras
Photochemical & Photobiological Sciences, Springer , 2021 . Tryptophan and tyrosine radical intermediates play crucial roles in many biological charge transfer processes. Particularly in flavoprotein photochemistry, short-lived reaction intermediates can be studied by the complementary techniques of ultrafast visible and infrared spectroscopy. The spectral properties of tryptophan radical are well established, and the formation of neutral tyrosine radicals has been observed in many biological processes. However, only recently, the formation of a cation tyrosine radical was observed by transient visible spectroscopy in a few systems. Here, we assigned the infrared vibrational markers of the cationic and neutral tyrosine radical at 1483 and 1502 cm −1 (in deuterated buffer), respectively, in a variant of the bacterial methyl transferase TrmFO, and in the native glucose oxidase. In addition, we studied a mutant of AppABLUF blue-light sensor domain from Rhodobacter sphaeroides in which only a direct formation of the neutral radical was observed. Our studies highlight the exquisite sensitivity of transient infrared spectroscopy to low concentrations of specific radicals. (10.1007/s43630-021-00024-y)
DOI : 10.1007/s43630-021-00024-y
The lncRNA 44s2 Study Applicability to the Design of 45-55 Exon Skipping Therapeutic Strategy for DMD
- Gargaun Elena
- Falcone Sestina
- Sole Guilhem
- Durigneux Julien
- Urtizberea Andoni
- Cuisset Jean Marie
- Benkhelifa-Ziyyat Sofia
- Julien Laura
- Boland Anne
- Sandron Florian
- Meyer Vincent
- Deleuze Jean François
- Salgado David
- Desvignes Jean-Pierre
- Béroud Christophe
- Chessel Anatole
- Blesius Alexia
- Krahn Martin
- Levy Nicolas
- Leturcq France
- Pietri-Rouxel France
Biomedicines, MDPI , 2021, 9 (2), pp.219 . In skeletal muscle, long noncoding RNAs (lncRNAs) are involved in dystrophin protein stabilization but also in the regulation of myocytes proliferation and differentiation. Hence, they could represent promising therapeutic targets and/or biomarkers for Duchenne and Becker muscular dystrophy (DMD/BMD). DMD and BMD are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy. Two-thirds of DMD gene mutations are represented by deletions, and 63% of patients carrying DMD deletions are eligible for 45 to 55 multi-exons skipping (MES), becoming BMD patients (BMDΔ45-55). We analyzed the genomic lncRNA presence in 38 BMDΔ45-55 patients and characterized the lncRNA localized in introns 44 and 55 of the DMD gene. We highlighted that all four lncRNA are differentially expressed during myogenesis in immortalized and primary human myoblasts. In addition, the lncRNA44s2 was pointed out as a possible accelerator of differentiation. Interestingly, lncRNA44s expression was associated with a favorable clinical phenotype. These findings suggest that lncRNA44s2 could be involved in muscle differentiation process and become a potential disease progression biomarker. Based on these results, we support MES45-55 therapy and propose that the design of the CRISPR/Cas9 MES45-55 assay consider the lncRNA sequences bordering the exonic 45 to 55 deletion. (10.3390/biomedicines9020219)
DOI : 10.3390/biomedicines9020219
Thermal and pH stabilities of i-DNA: confronting in vitro experiments with models and in-cell NMR data
- Cheng Mingpan
- Qiu Dehui
- Tamon Liezel
- Ištvánková Eva
- Víšková Pavlína
- Amrane Samir
- Guédin Aurore
- Chen Jielin
- Lacroix Laurent
- Ju Huangxian
- Trantírek Lukáš
- Sahakyan Aleksandr B
- Zhou Jun
- Mergny Jean‐louis
Angewandte Chemie International Edition, Wiley-VCH Verlag , 2021, 60, pp.10286-10294 . (10.1002/anie.202016801)
DOI : 10.1002/anie.202016801