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Articles

  • Photocycle alteration and increased enzymatic activity in genetically modified photoactivable adenylate cyclase OaPAC
    • Raics Katalin
    • Pirisi Katalin
    • Zhuang Bo
    • Fekete Zsuzsanna
    • Kis-Bicskei Nikolett
    • Pecsi Ildiko
    • Ujfalusi Kinga Pozsonyi
    • Telek Elek
    • Li Yin
    • Collado Jinnette Tolentino
    • Tonge Peter
    • Meech Stephen
    • Vos Marten
    • Bodis Emoke
    • Lukacs Andras
    Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology , 2023, 299 (8), pp.105056 . Photoactivated adenylate cyclases (PACs) are light activated enzymes that combine blue light sensing capacity with the ability to convert ATP to cAMP and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a blue light sensing domain using flavin which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated toward the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, PACs are of great interest in optogenetic studies. The optimal optogenetic device must be “silent” in the dark and highly responsive upon light illumination. PAC from Oscillatoria acuminata is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. We studied the effect of replacing D67 to N, in the blue light using flavin domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signaling state, which was formed with a lower quantum yield. Our studies show that the overall effects of the D67N mutation lead to a slightly higher conversion of ATP to cAMP, which points in the direction that by fine tuning the kinetic properties more responsive PACs and optogenetic devices can be generated (10.1016/j.jbc.2023.105056)
    DOI : 10.1016/j.jbc.2023.105056
  • Topology of DNA G-Quadruplexes Can Be Harnessed in Holliday Junction-Based DNA Suprastructures to Control and Optimize Their Biocatalytic Properties
    • Qiu Dehui
    • Cheng Mingpan
    • Stadlbauer Petr
    • Chen Jielin
    • Langer Michal
    • Zhang Xiaobo
    • Gao Qiang
    • Ju Huangxian
    • Šponer Jiří
    • Mergny Jean-Louis
    • Monchaud David
    • Zhou Jun
    ACS Catalysis, American Chemical Society , 2023, 12, pp.10722-10733 . The nature, composition, and topology of the active sites of both natural and artificial enzymes are key determinants of their catalytic performance. While interesting structural insights have been obtained for natural enzymes (e.g., horseradish peroxidase, HRP), the accurate catalytic microenvironment of HRP-mimicking DNA-based catalysts known as G-quadruplex (GQ)/hemin DNAzymes is still unclear. Herein, we report on a strategy allowing for fully controlling the nature of the active site of GQ DNAzyme, precisely manipulating the composition and topology of the hemin (Fe(III)protoporphyrin IX) cofactor binding site. This was achieved by introducing GQ within a Holliday junction (HJ) suprastructure that enables to seize control of both the GQ folding topology (parallel, antiparallel, hybrid) and the GQ strand directionality (clockwise, counterclockwise). By doing so, we demonstrate that the different GQ topologies are equivalent for both hemin binding and activation and that the flanking nucleotides (dA or dTC) modulate the activation of hemin in a GQ topology-dependent manner. Our experimental findings are supported by the most extensive molecular dynamics simulations ever been done on GQ DNAzyme, thus providing unique mechanistic insights into the biocatalytic activity of GQs. (10.1021/acscatal.3c02818)
    DOI : 10.1021/acscatal.3c02818
  • Impact of corpus callosum fiber tract crossing on polarimetric images of human brain histological sections: ex vivo studies in transmission configuration
    • Ivanov Deyan
    • Si Lu
    • Felger Leonard
    • Maragkou Theoni
    • Schucht Philippe
    • Schanne-Klein Marie-Claire
    • Ma Hui
    • Ossikovski Razvigor
    • Novikova Tatiana
    Journal of Biomedical Optics, Society of Photo-optical Instrumentation Engineers , 2023, 28 (10), pp.102908 . Significance: Imaging Mueller polarimetry is capable to trace in-plane orientation of brain fiber tracts by detecting the optical anisotropy of white matter of healthy brain. Brain tumor cells grow chaotically and destroy this anisotropy. Hence, the drop in scalar retardance values and randomization of the azimuth of the optical axis could serve as the optical marker for brain tumor zone delineation. Aim: The presence of underlying crossing fibers can also affect the values of scalar retardance and the azimuth of the optical axis. We studied and analyzed the impact of fiber crossing on the polarimetric images of thin histological sections of brain corpus callosum. Approach: We used the transmission Mueller microscope for imaging of two-layered stacks of thin sections of corpus callosum tissue to mimic the overlapping brain fiber tracts with different fiber orientations. The decomposition of the measured Mueller matrices was performed with differential and Lu–Chipman algorithms and completed by the statistical analysis of the maps of scalar retardance, azimuth of the optical axis, and depolarization. Results: Our results indicate the sensitivity of Mueller polarimetry to different spatial arrangement of brain fiber tracts as seen in the maps of scalar retardance and azimuth of optical axis of two-layered stacks of corpus callosum sections The depolarization varies slightly (<15 % ) with the orientation of the optical axes in both corpus callosum stripes, but its value increases by 2.5 to 3 times with the stack thickness. Conclusions: The crossing brain fiber tracts measured in transmission induce the drop in values of scalar retardance and randomization of the azimuth of the optical axis at optical path length of 15 μm. It suggests that the presence of nerve fibers crossing within the depth of few microns will be also detected in polarimetric maps of brain white matter measured in reflection configuration. (10.1117/1.JBO.28.10.102908)
    DOI : 10.1117/1.JBO.28.10.102908
  • Individual heme a and heme a3 contributions to the Soret absorption spectrum of the reduced bovine cytochrome c oxidase
    • Diuba Artem
    • Vygodina Tatiana
    • Azarkina Natalia
    • Arutyunyan Alexander
    • Soulimane Tewfik
    • Vos Marten
    • Konstantinov Alexander
    Biochimica biophysica acta (BBA) - Bioenergetics, Elsevier , 2023, 1864, pp.148937 . Bovine cytochrome c oxidase (CcO) contains two hemes, a and a3, chemically identical but differing in coordination and spin state. The Soret absorption band of reduced aa3-type cytochrome c oxidase consists of overlapping bands of the hemes a2+ and a32+. It shows a peak at ∼444 nm and a distinct shoulder at ∼425 nm. However, attribution of individual spectral lineshapes to hemes a2+ and a32+ in the Soret is controversial. In the present work, we characterized spectral contributions of hemes a2+ and a32+ using two approaches. First, we reconstructed bovine CcO heme a2+ spectrum using a selective Ca2+-induced spectral shift of the heme a2+. Second, we investigated photobleaching of the reduced Thermus thermophilus ba3- and bovine aa3-oxidases in the Soret induced by femtosecond laser pulses in the Q-band. The resolved spectra show splitting of the electronic B0x-, B0y-transitions of both reduced hemes. The heme a2+ spectrum is shifted to the red relative to heme a32+ spectrum. The ∼425 nm shoulder is mostly attributed to heme a32+. (10.1016/j.bbabio.2022.148937)
    DOI : 10.1016/j.bbabio.2022.148937
  • Artifact-free balanced detection for the measurement of circular dichroism with a sub-picosecond time resolution
    • Changenet Pascale
    • Hache François
    Optics Express, Optical Society of America - OSA Publishing , 2023, 31 (13), pp.21296 . Here we present the development of a subpicosecond spectropolarimeter enabling high sensitivity balanced detection of time-resolved circular dichroism (TRCD) signals from chiral sample in solution. The signals are measured with a conventional femtosecond pump-probe set-up using the combination of a quarter-waveplate and a Wollaston prism. This simple and robust method allows access to TRCD signals with improved signal-to-noise ratio and very short acquisition times. We provide a theoretical analysis of the artifacts of such detection geometry and the strategy to eliminate them. We illustrate the potential of this new detection with the study of the [Ru(phen) 3 ]·2PF 6 complexes in acetonitrile. (10.1364/OE.489468)
    DOI : 10.1364/OE.489468
  • Excited-State Properties of Fully Reduced Flavins in Ferredoxin–NADP + Oxidoreductase
    • Zhuang Bo
    • Aleksandrov Alexey
    • Seo Daisuke
    • Vos Marten
    Journal of Physical Chemistry Letters, American Chemical Society , 2023, 14 (4), pp.1096-1102 . The fully reduced flavin cofactor (FADred) in ferredoxin–NADP+ oxidoreductase (FNR) is a functional intermediate that displays different catalytic and steady-state spectral properties for enzymes from Bacillus subtilis (BsFNR), Chlorobaculum tepidum (CtFNR), and Rhodopseudomonas palustris (RpFNR). Using ultrafast spectroscopy, we reveal that at physiological pH, photoexcited FADred in BsFNR and RpFNR exhibits unprecedentedly fast decays (dominantly in 6 and 8 ps, respectively), whereas in CtFNR the decay is much slower (∼400 ps), as in other flavoproteins. Correlating these observations with the protonation states of FADred and the dynamic properties of the protein environment, we conclude that the excited state of neutral FADred can be intrinsically short-lived even in proteins, contrasting with the well-documented behavior of the anionic form that systematically displays markedly increased excited-state lifetime upon binding to proteins. This work provides new insight into the photochemistry of fully reduced flavins, which are emerging as functional initial states in bioengineered photocatalysts (10.1021/acs.jpclett.2c03741)
    DOI : 10.1021/acs.jpclett.2c03741
  • Accurate calibration of optical tweezers close to a glass surface using interference rings in backscattered light
    • Gillant Flavie
    • Moreau Julien
    • Richly Maximilian U
    • Alexandrou Antigoni
    • Perronet Karen
    • Westbrook Nathalie
    Journal of the European Optical Society : Rapid publications, European Optical Society , 2023, 19 . Mechanical forces play an important role in the behaviour of cells, from differentiation to migration and the development of diseases. Optical tweezers provide a quantitative tool to study these forces and must be combined with other tools, such as phase contrast and fluorescence microscopy. Detecting the retro-reflected trap beam is a convenient way to monitor the force applied by optical tweezers, while freeing top access to the sample. Accurate in situ calibration is required especially for single cells close to a surface where viscosity varies rapidly with height. Here, we take advantage of the well contrasted interference rings in the back focal plane of the objective to find the height of a trapped bead above a cover slip. We thus map the viscous drag dependence close to the surface and find agreement between four different measurement techniques for the trap stiffness down to 2 lm above the surface. Combining this detection scheme with phase contrast microscopy, we show that the phase ring in the back focal plane of the objective must be deported in a conjugate plane on the imaging path. This simplifies implementation of optical tweezers in combination with other techniques for biomechanical studies. (10.1051/jeos/2023026)
    DOI : 10.1051/jeos/2023026
  • FLUTE: a Python GUI for interactive phasor analysis of FLIM data
    • Gottlieb Dale
    • Asadipour Bahar
    • Kostina Polina
    • Ung Thi Phuong Lien
    • Stringari Chiara
    Biological Imaging, Cambridge University Press , 2023, 3, pp.e21 . Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique used to probe the local environment of fluorophores. The fit-free phasor approach to FLIM data is increasingly being used due to its ease of interpretation. To date, no open-source graphical user interface (GUI) for phasor analysis of FLIM data is available in Python, thus limiting the widespread use of phasor analysis in biomedical research. Here, we present Fluorescence Lifetime Ultimate Explorer (FLUTE), a Python GUI that is designed to fill this gap. FLUTE simplifies and automates many aspects of the analysis of FLIM data acquired in the time domain, such as calibrating the FLIM data, performing interactive exploration of the phasor plot, displaying phasor plots and FLIM images with different lifetime contrasts simultaneously, and calculating the distance from known molecular species. After applying desired filters and thresholds, the final edited datasets can be exported for further user-specific analysis. FLUTE has been tested using several FLIM datasets including autofluorescence of zebrafish embryos and in vitro cells. In summary, our user-friendly GUI extends the advantages of phasor plotting by making the data visualization and analysis easy and interactive, allows for analysis of large FLIM datasets, and accelerates FLIM analysis for non-specialized labs. (10.1017/s2633903x23000211)
    DOI : 10.1017/s2633903x23000211
  • Second harmonic generation in the presence of walk-off and group velocity mismatch
    • Hanna Marc
    • Natile Michele
    • Zaouter Yoann
    • Joffre Manuel
    • Georges Patrick
    Journal of the Optical Society of America B, Optical Society of America , 2023, 40 (5), pp.930-938 . We study a second harmonic generation interaction geometry in the case where both group velocity mismatch and walk-off have significant impacts. This results in a frequency-converted beam exhibiting a pulse front tilt. Using the global response function of the crystal, we provide an analytical model that allows to predict the spatiotemporal structure of the second harmonic wave packet and verify its validity using numerical simulations and a simple experiment. Distinctive features of this geometry are the suppression of back-conversion and the ability to conserve the fundamental bandwidth in space and time domains. Subsequent compensation of the pulse front tilt should allow efficient generation of ultrashort pulses in the deep ultraviolet. (10.1364/josab.485597)
    DOI : 10.1364/josab.485597
  • The paradoxes of Mycobacterium tuberculosis molecular evolution and consequences for the inference of tuberculosis emergence date
    • Zein Eddine Rima
    • Hak F.
    • Le Meur A.
    • Genestet C.
    • Dumitrescu O.
    • Guyeux C.
    • Senelle G.
    • Sola C.
    • Refrégier G.
    Tuberculosis, Elsevier , 2023, 143, pp.102378 . The date of Mycobacterium tuberculosis complex emergence has been the subject of long debates. New studies joining archaeological efforts with sequencing methods raise high hopes for solving whether this emergence is closer to 70,000 or to 6000 years before present. Inferring the date of emergence of this pathogen based on sequence data requires a molecular clock. Several clocks inferred from different types of loci and/or different samples, using both sound reasoning and reliable data, are actually very different, which we refer to as the paradoxes of M. tuberculosis molecular evolution. After having presented these paradoxes, we will remind the limits of the molecular clocks used in the different studies such as the assumption of homogeneous substitution rate. We will then review recent results that shed new light on the characteristics of M. tuberculosis molecular evolution: traces of diverse selection pressures, the impact of host dynamics, etc. We provide some ideas on what to do next to get nearer to a reliable dating of Mycobacterium tuberculosis complex emergence. Among them, the collection of additional remains from ancient tuberculosis seems still essential. (10.1016/j.tube.2023.102378)
    DOI : 10.1016/j.tube.2023.102378
  • Quadruplexes and aging: G4-binding proteins regulate the presence of miRNA in small extracellular vesicles (sEVs)
    • Brázda Václav
    • Mergny Jean-Louis
    Biochimie, Elsevier , 2023, pp.S0300-9084(23)00014-7 . The interaction between proteins and nucleic acids is a core element of life. Many proteins bind nucleic acids via a sequence-specific manner, but there are also many types of proteins that recognize various structural motifs. Researchers have recently found that proteins that can recognize DNA and RNA G- quadruplexes (G4s) are very important for basic cellular processes, particularly in eukaryotes. Some of these proteins are located outside the nucleus and interact with RNA, potentially affecting miRNA functions in intercellular communication, which is facilitated by small extracellular vesicles (sEVs). Im- balances in the production of sEVs are associated with various pathologies and senescence in humans. The distribution of miRNA into sEVs is regulated by two RNA-binding proteins, Alyref and FUS. Both proteins possess G-rich recognition motifs that are compatible with the formation of RNA parallel G4 structures. This lends credence to the new hypothesis that G4-formation in RNAs and their interaction with G4-binding proteins can affect the fate of miRNAs and control their distribution in sEVs that are associated with senescence and aging. (10.1016/j.biochi.2023.01.014)
    DOI : 10.1016/j.biochi.2023.01.014
  • Terahertz Spectroscopy Sheds Light on Real‐Time Exchange Kinetics Occurring through Plasma Membrane during Photodynamic Therapy Treatment
    • Zheng Xiujun
    • Lordon Blandine
    • Mingotaud Anne‐françoise
    • Vicendo Patricia
    • Brival Rachel
    • Fourquaux Isabelle
    • Gibot Laure
    • Gallot Guilhem
    Advanced Science, Wiley Open Access , 2023, 10 (18), pp.e2300589 . Methods to follow in real time complex processes occurring along living cell membranes such as cell permeabilization are rare. Here, the terahertz spectroscopy reveals early events in plasma membrane alteration generated during photodynamic therapy (PDT) protocol, events which are not observable in any other conventional biological techniques performed in parallel as comparison. Photodynamic process is examined in Madin-Darby canine kidney cells using Pheophorbide (Pheo) photosensitizer alone or alternatively encapsulated in poly(ethylene oxide)-block-poly(ε-caprolactone) micelles for drug delivery purpose. Terahertz spectroscopy (THz) reveals that plasma membrane permeabilization starts simultaneously with illumination and is stronger when photosensitizer is encapsulated. In parallel, the exchange of biological species is assessed. Over several hours, this conventional approach demonstrates significant differences between free and encapsulated Pheo, the latter leading to high penetration of propidium iodide, Na+ and Ca2+ ions, and a high level of leakage of K+, ATP, and lactate dehydrogenase. THz spectroscopy provides, in a single measurement, the relative number of defects per membrane surface created after PDT, which is not achieved by any other method, providing early, sensitive real-time information. THz spectroscopy is therefore a promising technique and can be applied to any biological topic requiring the examination of short-term plasma membrane permeabilization. (10.1002/advs.202300589)
    DOI : 10.1002/advs.202300589
  • A sodium / potassium switch for G4-prone G / C-rich sequences
    • Luo Yu
    • Živković Martina Lenarčič
    • Wang Jiawei
    • Ryneš Jan
    • Foldynová-Trantírková Silvie
    • Trantírek Lukáš
    • Verga Daniela
    • Mergny Jean-Louis
    Nucleic Acids Research, Oxford University Press , 2023, 52 (1), pp.448-461 . Metal ions are essential components for the survival of living organisms. For most species, intracellular and extracellular ionic conditions differ significantly. As G-quadruple x es (G4s) are ion-dependent structures, changes in the [Na+]/[K+] ratio may affect the folding of genomic G4s. More than 11000 putative G4 sequences in the human genome (hg19) contain at least two runs of three continuous cytosines, and these mixed G/C-rich sequences may form a quadruplex or a competing hairpin structure based on G-C base pairing. In this study, we examine how the [Na+]/[K+] ratio influences the structures of G/C-rich sequences. The natural G4 structure with a 9-nt long central loop, CEBwt, was chosen as a model sequence, and the loop bases were gradually replaced by cytosines. The series of CEB mutations revealed that the presence of cytosines in G4 loops does not prevent G4 f olding or decrease G4 stability but increases the probability of forming a competing structure, either a hairpin or an intermolecular duplex. Slow conversion to the quadruplex in vitro (in a potassium-rich buffer) and cells was demonstrated by NMR. 'Shape-shifting' sequences may respond to [Na+]/[K+] changes with delayed kinetics. (10.1093/nar/gkad1073)
    DOI : 10.1093/nar/gkad1073
  • Label-free single-cell live imaging reveals fast metabolic switch in T lymphocytes
    • Paillon Noémie
    • Ung Thi Phuong Lien
    • Dogniaux Stéphanie
    • Stringari Chiara
    • Hivroz Claire
    Molecular Biology of the Cell, American Society for Cell Biology , 2023 . T cell activation induces a metabolic switch generating energy for proliferation, survival, and functions. We used non-invasive label-free two-photon fluorescence lifetime microscopy (2P-FLIM) to map the spatial and temporal dynamics of the metabolic NAD(P)H co-enzyme during T lymphocyte activation. This provides a readout of the OXPHOS and glycolysis rates at a single cell level. Analyzes were performed in the CD4+ leukemic T cell line Jurkat, and in human CD4+ primary T cells. Cells were activated on glass surfaces coated with activating antibodies mimicking immune synapse formation. Comparing the fraction of bound NAD(P)H between resting and activated T cells, we show that T cell activation induces a rapid switch toward glycolysis. This occurs after 10 minutes and remains stable for one hour. Three-dimensional analyzes revealed that the intracellular distribution of fraction of bound NAD(P)H increases at the immune synapse in activated cells. Finally, we show that fraction of bound NAD(P)H tends to negatively correlate with spreading of activated T cells, suggesting a link between actin remodeling and metabolic changes. This study highlights that 2P-FLIM measurement of fraction of bound NAD(P)H is well suited to follow a fast metabolic switch in 3D, in single T lymphocytes with subcellular resolution. (10.1091/mbc.E23-01-0009)
    DOI : 10.1091/mbc.E23-01-0009
  • Simple Postsynthesis Thermal Treatment toward High Luminescence Performance of Rare Earth Vanadate Nanoparticles
    • Perrella Rafael Vieira
    • Mohammedi Rabei
    • Kuhner Robin
    • Cardone Christophe
    • Larquet Eric
    • Alexandrou Antigoni
    • de Sousa Filho Paulo Cesar
    • Gacoin Thierry
    Crystal Growth & Design, American Chemical Society , 2023, 23 (8), pp.5389-5396 . Optical applications of colloidal oxide nanoparticles are often limited by low luminescence efficiencies caused by poor crystallinity and surface quenching. Bulk oxides prepared via conventional high-temperature annealing offer intense luminescence but commonly fail to yield stable colloidal dispersions. Coupling the best of these two situations to afford highly crystalline, dispersible nanoparticles with luminescence performance exceeding bulk solids is still challenging, thus requiring new safe, scalable, and reproducible methodologies. Herein we report a silicate-coating strategy followed by aggregate elimination to recover stable colloids of 40-150 nm single crystalline rare earth vanadates after unprotected annealing (800-1000 °C). Eu3+-doped nanoparticles showed enhanced photostability and ~50% emission quantum yields in water (λexc=280 nm), while Dy3+-, Tm3+-, and Yb3+/Er3+-doped vanadates provided remarkably intense multicolour emissions via downshift or upconversion luminescence. We correlated spectroscopic properties of pristine and annealed solids to microstructural characteristics to explain the superior emission features, opening new perspectives for sensing applications. (10.1021/acs.cgd.3c00308)
    DOI : 10.1021/acs.cgd.3c00308
  • Three-Dimensional Collagen Topology Shapes Cell Morphology, beyond Stiffness
    • Chen Changchong
    • Ibrahim Zeinab
    • Marchand Marion
    • Piolot Tristan
    • Kamboj Sahil
    • Carreiras Franck
    • Yamada Ayako
    • Schanne-Klein Marie-Claire
    • Chen Yong
    • Lambert Ambroise
    • Aimé Carole
    ACS Biomaterials Science and Engineering, ACS , 2022, 8 (12), pp.5284-5294 . Cellular heterogeneity is associated with many physiological processes, including pathological ones, such as morphogenesis and tumorigenesis. The extracellular matrix (ECM) is a key player in the generation of cellular heterogeneity. Advances in our understanding rely on our ability to provide relevant in vitro models. This requires obtainment of the characteristics of the tissues that are essential for controlling cell fate. To do this, we must consider the diversity of tissues, the diversity of physiological contexts, and the constant remodeling of the ECM along these processes. To this aim, we have fabricated a library of ECM models for reproducing the scaffold of connective tissues and the basement membrane by using different biofabrication routes based on the electrospinning and drop casting of biopolymers from the ECM. Using a combination of electron microscopy, multiphoton imaging, and AFM nanoindentation, we show that we can vary independently protein composition, topology, and stiffness of ECM models. This in turns allows one to generate the in vivo complexity of the phenotypic landscape of ovarian cancer cells. We show that, while this phenotypic shift cannot be directly correlated with a unique ECM feature, the three-dimensional collagen fibril topology patterns cell shape, beyond protein composition and stiffness of the ECM. On this line, this work is a further step toward the development of ECM models recapitulating the constantly remodeled environment that cells face and thus provides new insights for cancer model engineering and drug testing. (10.1021/acsbiomaterials.2c00879)
    DOI : 10.1021/acsbiomaterials.2c00879
  • A Double Hemin Bonded G-Quadruplex Embedded in Metal–Organic Frameworks for Biomimetic Cascade Reaction
    • Mao Xuanxiang
    • Qiu Dehui
    • Wei Shijiong
    • Zhang Xiaobo
    • Lei Jianping
    • Mergny Jean-Louis
    • Ju Huangxian
    • Zhou Jun
    ACS Applied Materials & Interfaces, Washington, D.C. : American Chemical Society , 2022, 14, pp.54598−54606 . Biocatalytic transformations in living cells, such as enzymatic cascades, function effectively in spatially confined microenvironments. However, mimicking enzyme catalytic cascade processes is challenging. Herein, we report a new dual-Hemin-G-quadruplex (dHemin-G4) DNAzyme with high catalytic activity over noncovalent G4/Hemin and monocovalent counterparts (G4-Hemin and Hemin-G4) by covalently linking hemin to both ends of an intramolecular G4. We use MAF-7, a hydrophilic metal-organic framework (MOF), as the protecting scaffold to integrate a biocatalytic cascade consisting of dHemin-G4 DNAzyme and glucose oxidase (GOx), by a simple and mild method with a single-step encapsulation of both enzymes. Such a MAF-7-confined cascade system shows superior activity over not only traditional G4/Hemin but also other MOFs (ZIF-8 and ZIF-90), which was mainly attributed to high-payload enzyme packaging. Notably, the introduction of hydrophilic G4 allows to avoid the accumulation of hydrophobic hemin on the surface of MAF-7, which decreases cascade biocatalytic activity. Furthermore, MAF-7 as protective coatings endowed the enzyme with excellent recyclability and good operational stability in harsh environments, including elevated temperature, urea, protease, and organic solvents, extending its practical application in biocatalysis. In addition, the incorporated enzymes can be replaced on demand to broaden the scope of catalytic substrates. Taking advantages of these features, the feasibility of dHemin-G4/GOx@MAF-7 systems for biosensing was demonstrated. This study is conducive to devise efficient and stable enzyme catalytic cascades to facilitate applications in biosensing and industrial processes. (10.1021/acsami.2c18473)
    DOI : 10.1021/acsami.2c18473
  • Guidelines for G-quadruplexes: I. In vitro characterization
    • Luo Yu
    • Granzhan Anton
    • Marquevielle Julien
    • Cucchiarini Anne
    • Lacroix Laurent
    • Amrane Samir
    • Verga Daniela
    • Mergny Jean-Louis
    Biochimie, Elsevier , 2022, 214, pp.5-23 . Besides the well-known DNA double-helix, non-canonical nucleic acid structures regulate crucial bio- logical activities. Among these oddities, guanine-rich DNA sequences can form unusual four-stranded secondary structures called G-quadruplexes (G4s). G4-prone sequences have been found in the ge- nomes of most species, and G4s play important roles in essential processes such as transcription, replication, genome integrity and epigenetic regulation. Here, we present a short overview of G-quad- ruplexes followed by a detailed description of the biophysical and biochemical methods used to char- acterize G4s in vitro. The principles, experimental details and possible shortcomings of each method are discussed to provide a comprehensive view of the techniques used to study these structures. We aim to provide a set of guidelines for standardizing research on G-quadruplexes; these guidelines are not meant to be a dogmatic set of rules, but should rather provide useful information on the methods currently used to study these fascinating motifs. (10.1016/j.biochi.2022.12.019)
    DOI : 10.1016/j.biochi.2022.12.019
  • Deciphering RNA G-quadruplex function during the early steps of HIV-1 infection
    • Amrane Samir
    • Jaubert Chloé
    • Bedrat Amina
    • Rundstadler Tiffany
    • Recordon-Pinson Patricia
    • Aknin Cindy
    • Guédin Aurore
    • de Rache Aurore
    • Bartolucci Laura
    • Diene Ibra
    • Éric Lemoine Fréd
    • Gascuel Olivier
    • Pratviel Geneviève
    • Mergny Jean-Louis
    • Andreola Marie-Line
    Nucleic Acids Research, Oxford University Press , 2022, 50 (21), pp.12328-12343 . G-quadruplexes (G4s) are four-stranded nucleic acid structures formed by the stacking of G-tetrads. Here we investigated their formation and function during HIV-1 infection. Using bioinformatics and biophysics analyses we first searched for evolutionary conserved G4-forming sequences in HIV-1 genome. We identified 10 G4s with conservation rates higher than those of HIV-1 regulatory sequences such as RRE and TAR. We then used porphyrin-based G4binders to probe the formation of the G4s during infection of human cells by native HIV-1. The G4binders efficiently inhibited HIV-1 infectivity, which is attributed to the formation of G4 structures during HIV-1 replication. Using a qRT-PCR approach, we showed that the formation of viral G4s occurs during the first 2 h post-infection and their stabilization by the G4-binders prevents initiation of reverse transcription. We also used a G4-RNA pull-down approach, based on a G4-specific biotinylated probe, to allow the direct detection and identification of viral G4-RNA in infected cells. Most of the detected G4-RNAs contain crucial regulatory elements such as the PPT and cPPT sequences as well as the U3 region. Hence, these G4s would function in the early stages of infection when the viral RNA genome is being processed for the reverse transcription step. (10.1093/nar/gkac1030)
    DOI : 10.1093/nar/gkac1030
  • Telomeres expand sphere of influence: emerging molecular impact of telomeres in non-telomeric functions
    • Vinayagamurthy Soujanya
    • Bagri Sulochana
    • Mergny Jean-Louis
    • Chowdhury Shantanu
    Trends in Genetics, Elsevier , 2022, 39 (1), pp.59-73 . Although the impact of telomeres on physiology stands well established, a question remains: how do telomeres impact cellular functions at a molecular level? This is because current understanding limits the influence of telomeres to adjacent subtelomeric regions despite the wide-ranging impact of telomeres. Emerging work in two distinct aspects offers opportunities to bridge this gap. First, telomere-binding factors were found with non-telomeric functions. Second, locally induced DNA secondary structures called G-quadruplexes are notably abundant in telomeres, and gene regulatory regions genome wide. Many telomeric factors bind to G-quadruplexes for non-telomeric functions. Here we discuss a more general model of how telomeres impact the non-telomeric genome – through factors that associate at telomeres and genome wide – and influence cell-intrinsic functions, particularly aging, cancer, and pluripotency. (10.1016/j.tig.2022.10.002)
    DOI : 10.1016/j.tig.2022.10.002
  • Gallic Acid-Triethylene Glycol Aptadendrimers Synthesis, Biophysical Characterization and Cellular Evaluation
    • Miranda André
    • Lopez-Blanco Roi
    • Lope-Nunes Jéssica
    • Mel Ana M
    • Cabral Campello Maria Pau
    • Paulo António
    • Oliveira Maria Cristina
    • Mergny Je-Louis
    • Oliveira Paula A
    • Fernandez-Megia Eduardo
    • Cruz Carla
    Pharmaceutics, MDPI , 2022, 14 (11), pp.2456 . Herein, we describe the synthesis of an aptadendrimer by covalent bioconjugation of a gallic acid–triethylene glycol (GATG) dendrimer with the G-quadruplex (G4) AT11 aptamer (a modified version of AS1411) at the surface. We evaluated the loading and interaction of an acridine orange ligand, termed C8, that acts as an anticancer drug and binder/stabilizer of the G4 structure of AT11. Dynamic light scattering experiments demonstrated that the aptadendrimer was approximately 3.1 nm in diameter. Both steady-state and time-resolved fluorescence anisotropy evidenced the interaction between the aptadendrimer and C8. Additionally, we demonstrated that the iodine atom of the C8 ligand acts as an effective intramolecular quencher in solution, while upon complexation with the aptadendrimer, it adopts a more extended conformation. Docking studies support this conclusion. Release experiments show a delivery of C8 after 4 h. The aptadendrimers tend to localize in the cytoplasm of various cell lines studied as demonstrated by confocal microscopy. The internalization of the aptadendrimers is not nucleolin-mediated or by passive diffusion, but via endocytosis. MTT studies with prostate cancer cells and non-malignant cells evidenced high cytotoxicity mainly due to the C8 ligand. The rapid internalization of the aptadendrimers and the fluorescence properties make them attractive for the development of potential nanocarriers. (10.3390/pharmaceutics14112456)
    DOI : 10.3390/pharmaceutics14112456
  • Design, Synthesis, and Antiprotozoal Evaluation of New Promising 2,9-Bis[(substituted-aminomethyl)]-4,7-phenyl-1,10phenanthroline Derivatives, a Potential Alternative Scaffold to Drug Efflux
    • Guillon Jean
    • Cohen Anita
    • Boudot Clotilde
    • Monic Sarah
    • Savrimoutou Solène
    • Moreau Stéphane
    • Albenque-Rubio Sandra
    • Lafon-Schmaltz Camille
    • Dassonville-Klimpt Alexandra
    • Mergny Jean-Louis
    • Ronga Luisa
    • Bernabeu de Maria Mikel
    • Lamarche Jérémy
    • Dal Lago Cristina
    • Largy Eric
    • Gabelica Valérie
    • Moukha Serge
    • Dozolme Pascale
    • Agnamey Patrice
    • Azas Nadine
    • Mullié Catherine
    • Courtioux Bertrand
    • Sonnet Pascal
    Pathogens, MDPI , 2022, 11 (11), pp.1339 . A series of novel 2,9-bis[(substituted-aminomethyl)]-4,7-phenyl-1,10-phenanthroline derivatives was designed, synthesized, and evaluated in vitro against three protozoan parasites (Plasmodium falciparum, Leishmania donovani and Trypanosoma brucei brucei). Pharmacological results showed antiprotozoal activity with IC50 values in the sub and μM range. In addition, the in vitro cytotoxicity of these original molecules was assessed with human HepG2 cells. The substituted diphenylphenanthroline 1l was identified as the most potent antimalarial derivative with a ratio of cytotoxic to antiparasitic activities of 505.7 against the P. falciparum CQ-resistant strain W2. Against the promastigote forms of L. donovani, the phenanthrolines 1h, 1j, 1n and 1o were the most active with IC50 from 2.52 to 4.50 μM. The phenanthroline derivative 1o was also identified as the most potent trypanosomal candidate with a selectivity index (SI) of 91 on T. brucei brucei strain. FRET melting and native mass spectrometry experiments evidenced that the nitrogen heterocyclic derivatives bind the telomeric G-quadruplexes of P. falciparum and Trypanosoma. Moreover, as the telomeres of the parasites P. falciparum and Trypanosoma could be considered to be possible targets of this kind of nitrogen heterocyclic derivatives, their potential ability to stabilize the parasitic telomeric G-quadruplexes have been determined through the FRET melting assay and by native mass spectrometry. (10.3390/pathogens11111339)
    DOI : 10.3390/pathogens11111339
  • Synthesis and evaluation of 2,9-disubstituted-1,10-phenanthroline derivatives as G-quadruplex binders
    • Figueiredo Joana
    • Carreira-Barral Israel
    • Quesada Roberto
    • Mergny Jean-Louis
    • Cruz Carla
    Bioorganic and Medicinal Chemistry Letters, Elsevier , 2022, 73, pp.116971 . G-quadruplex (G4) structures are non-canonical DNA/RNA secondary structures able to form within guanine rich nucleic acids sequences. They are present in several regions of the human genome including gene promoters, untranslated sequences, and telomeres. Due to their biological relevance G4 structures are considered important drug targets, in particular for anticancer therapies, leading to the development of G4 stabilizing small molecules. Telomeric regions have received special attention in this field since they can fold into several distinct intramolecular G-quadruplexes topologies. Herein, we report the synthesis of 2,9-disubstituted-1,10-phenanthroline derivatives and their ability to stabilize different intramolecular telomeric G4 sequences. We evaluated ligand-induced stabilization, selectivity and specificity of ligands using Förster Resonance Energy Transfer (FRET) melting experiments and circular dichroism (CD). In addition, we assessed the cytotoxicity of ligands against two cancer cell lines (A549 and H1299) and one healthy cell line (NHDF). (10.1016/j.bmc.2022.116971)
    DOI : 10.1016/j.bmc.2022.116971
  • Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts
    • Irsch Kristina
    • Grieve Kate
    • Borderie Marie
    • Vilbert Maëlle
    • Plamann Karsten
    • Ghoubay Djida
    • Georgeon Cristina
    • Borderie Vincent
    Journal of visualized experiments : JoVE, JoVE , 2022, 18 (188), pp.e0291613 . Corneal transparency is essential to provide a clear view into and out of the eye, yet clinical means to assess such transparency are extremely limited and usually involve a subjective grading of visible opacities by means of slit-lamp biomicroscopy. Here, we describe an automated algorithm allowing extraction of quantitative corneal transparency parameters with standard clinical spectral-domain optical coherence tomography (SD-OCT). Our algorithm employs a novel pre-processing procedure to standardize SD-OCT image analysis and to numerically correct common instrumental artifacts before extracting mean intensity stromal-depth ( z ) profiles over a 6-mm-wide corneal area. The z -profiles are analyzed using our previously developed objective method that derives quantitative transparency parameters directly related to the physics of light propagation in tissues. Tissular heterogeneity is quantified by the Birge ratio B r and the photon mean-free path ( l s ) is determined for homogeneous tissues (i.e., B r ~1 ). SD-OCT images of 83 normal corneas (ages 22–50 years) from a standard SD-OCT device (RTVue-XR Avanti, Optovue Inc.) were processed to establish a normative dataset of transparency values. After confirming stromal homogeneity ( B r <10), we measured a median l s of 570 μm (interdecile range: 270–2400 μm). By also considering corneal thicknesses, this may be translated into a median fraction of transmitted (coherent) light T coh(stroma) of 51% (interdecile range: 22–83%). Excluding images with central saturation artifact raised our median T coh(stroma) to 73% (interdecile range: 34–84%). These transparency values are slightly lower than those previously reported, which we attribute to the detection configuration of SD-OCT with a relatively small and selective acceptance angle. No statistically significant correlation between transparency and age or thickness was found. In conclusion, our algorithm provides robust and quantitative measurements of corneal transparency from standard SD-OCT images with sufficient quality (such as ‘Line’ and ‘CrossLine’ B-scan modes without central saturation artifact) and addresses the demand for such an objective means in the clinical setting. (10.3791/57104)
    DOI : 10.3791/57104
  • G-Quadruplex Aptamer-Ligand Characterization
    • Moreira David
    • Leitão Daniela
    • Lopes-Nunes Jéssica
    • Santos Tiago
    • Figueiredo Joana
    • Miranda André
    • Alexandre Daniela
    • Tomaz Cândida
    • Mergny Jean-Louis
    • Cruz Carla
    Molecules, MDPI , 2022, 27 (20), pp.6781 . In this work we explore the structure of a G-rich DNA aptamer termed AT11-L2 (TGGTGGTGGTTGTTGTTGGTGGTGGTGGT; derivative of AT11) by evaluating the formation and stability of G-quadruplex (G4) conformation under different experimental conditions such as KCl concentration, temperature, and upon binding with a variety of G4 ligands (360A, BRACO-19, PDS, PhenDC3, TMPyP4). We also determined whether nucleolin (NCL) can be a target of AT11-L2 G4. Firstly, we assessed by circular dichroism, UV and NMR spectroscopies the formation of G4 by AT11-L2. We observed that, for KCl concentrations of 65 mM or less, AT11-L2 adopts hybrid or multiple topologies. In contrast, a parallel topology predominates for buffer containing 100 mM of KCl. The Tm of AT11-L2 in 100 mM of KCl is 38.9 °C, proving the weak stability of this sequence. We also found that upon titration with two molar equivalents of 360A, BRACO-19 and PhenDC3, the G4 is strongly stabilized and its topology is maintained, while the addition of 3.5 molar equivalents of TMPyP4 promotes the disruption of G4. The KD values between AT11-L2 G4, ligands and NCL were obtained by fluorescence titrations and are in the range of µM for ligand complexes and nM when adding NCL. In silico studies suggest that four ligands bind to the AT11-L2 G4 structure by stacking interactions, while the RBD1,2 domains of NCL interact preferentially with the thymines of AT11-L2 G4. Finally, AT11-L2 G4 co-localized with NCL in NCL-positive tongue squamous cell carcinoma cell line. (10.3390/molecules27206781)
    DOI : 10.3390/molecules27206781