Publications

2022

• Spectroscopie infrarouge pompe-sonde de la picoseconde à la microseconde dans une photoenzyme
  
  • Vos Marten H.
  • Joffre M.
  • Antonucci Laura
  • Sorigué Damien
  • Aleksandrov Alexey
  • Frederic Beisson
  , 2022. Nous avons développé une méthode de spectroscopie pompe sonde permettant la mesure de spectres différentiels sur des échelles de temps allant de la sub-picoseconde à la milliseconde, méthode qui s’implémente facilement sur deux lasers femtosecondes amplifiés pré-existants sans besoin d’asservir les oscillateurs [1-3]. Cette méthode dénommée AD-ASOPS pour Arbitrary Detuning Asynchronous Optical Sampling, a été appliquée à une photoenzyme récemment découverte [4], Fatty Acid Photodecarboxylase (FAP). La mesure des spectres infrarouges pompe-sonde a mis en évidence la dynamique de formation du CO2 au cours d’un processus complexe qui transforme un acide gras en hydrocarbure, et permis d’élucider une partie du mécanisme à l’œuvre dans la biomolécule [5]. [1] L. Antonucci, A. Bonvalet, X. Solinas, L. Daniault, M. Joffre, Opt. Express 23, 27931 (2015), https://doi.org/10.1364/OE.23.027931 [2] X. Solinas, L. Antonucci, A. Bonvalet, M. Joffre, Opt. Express 25, 17811 (2017), https://doi.org/10.1364/OE.25.017811 [3] L. Antonucci, X. Solinas, A. Bonvalet, M. Joffre, Opt. Express 28, 18251 (2020), https://doi.org/10.1364/OE.393887 [4] D. Sorigué et al., Science 357, 903 (2017), https://doi.org/10.1126/science.aan6349 [5] D. Sorigué et al., Science 372, 6538 (2021), https://doi.org/10.1126/science.abd5687
• 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
• Bacterial origin of thymidylate and folate metabolism in Asgard Archaea
  
  • Filée Jonathan
  • Becker Hubert F
  • Mellottee Lucille
  • Li Zhihui
  • Lambry Jean-Christophe
  • Liebl Ursula
  • Myllykallio Hannu
  , 2022. Abstract Little is known about the evolution and biosynthetic function of DNA precursor and the folate metabolism in the Asgard group of archaea. As Asgard occupy a key position in the archaeal and eukaryotic phylogenetic trees, we have exploited very recently emerged genome and metagenome sequence information to investigate these central metabolic pathways. Our genome-wide analyses revealed that the recently cultured Asgard archaeon Candidatus Prometheoarchaeum syntrophicum strain MK-D1 ( Psyn ) contains a complete folate-dependent network for the biosynthesis of DNA/RNA precursors, amino acids and syntrophic amino acid utilization. Altogether our experimental and computational data suggest that phylogenetic incongruences of functional folate-dependent enzymes from Asgard archaea reflect their persistent horizontal transmission from various bacterial groups, which has rewired the key metabolic reactions in an important and recently identified archaeal phylogenetic group. We also experimentally validated the functionality of the lateral gene transfer of Psyn thymidylate synthase ThyX. This enzyme uses bacterial-like folates efficiently and is inhibited by mycobacterial ThyX inhibitors. Our data raise the possibility that the thymidylate metabolism, required for de novo DNA synthesis, originated in bacteria and has been independently transferred to archaea and eukaryotes. In conclusion, our study has revealed that recent prevalent lateral gene transfer has markedly shaped the evolution of Asgard archaea by allowing them to adapt to specific ecological niches. (10.1101/2021.12.15.472764)
  DOI : 10.1101/2021.12.15.472764
• 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
• Non-invasive characterization of varnish on gilt leather by high-resolution Optical Coherence Tomography
  
  • Galante Giulia
  • Vilbert Maëlle
  • Bonnot-Diconne Céline
  • Schanne-Klein Marie-Claire
  • Robinet Laurianne
  • Latour Gaël
  , 2025.
• 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
• Diagnostic optique in vivo de la transparence cornéenne par tomographie par cohérence optique (OCT)
  
  • Vilbert Maëlle
  , 2022. Le handicap visuel par perte de la transparence cornéenne affecte plus de 10 millions de personnes dans le monde, dans un contexte de pénurie globale de greffons cornéens. La détection précoce des pathologies et un suivi quantitatif permettraient une meilleure prise en charge des patient·es. Or, les outils cliniques actuels pour le diagnostic de la transparence cornéenne sont limités à des évaluations subjectives ou non standardisées.L’objectif de cette thèse est le transfert à la pratique clinique d’une méthode de quantification de la transparence cornéenne par l’analyse d’images de tomographie par cohérence optique (OCT) acquises via des dispositifs ophtalmiques standards. Nous avons implémenté un algorithme convivial à destination des praticien·nes pour standardiser les images cornéennes, identifier et corriger les artefacts récurrents puis extraire le signal d’intérêt analysé en termes de paramètres physiques et statistiques.L’application de la méthode à un groupe de 83 cornées normales nous a permis de délimiter le périmètre d’utilisation des images cliniques à des fins d’analyse quantitative.La robustesse du prétraitement et l’excellente répétabilité des mesures nous ont permis d’établir une base de données normative des paramètres de transparence pour les cornées normales. La comparaison avec un groupe de cornées atteintes de dystrophie de Fuchs montre une séparation significative entre les cornées saines et les cornées pathologiques. L’évolution de nos paramètres après greffe endothéliale est cohérente avec celle des variables cliniques.Nous avons entraîné un modèle de classification automatique des images OCT cornéennes à partir de quantificateurs morphologiques d’une fibrose symptomatique de la dystrophie de Fuchs. Appliqué au diagnostic du haze cornéen post-chirurgie réfractive, le modèle est précis à 97%.Enfin, nous avons exploré le potentiel d’un dispositif OCT portable à bas coût pour l’imagerie in situ de greffons cornéens à travers leur flacon de conservation scellé, pour le suivi de l’œdème cornéen lors d’expérimentations sur cornées fraîches et pour la cartographie d’épaisseur non invasive d’objets patrimoniaux vernis.
• Coordinated metabolic transitions and gene expression by NAD+ during adipogenesis
  
  • Sánchez-Ramírez Edgar
  • Ung Thi Phuong Lien
  • Alarcón del Carmen Alejandro
  • del Toro-Ríos Ximena
  • Fajardo-Orduña Guadalupe R
  • Noriega Lilia G
  • Cortés-Morales Victor A
  • Tovar Armando R
  • Montesinos Juan José
  • Orozco-Solís Ricardo
  • Stringari Chiara
  • Aguilar-Arnal Lorena
  Journal of Cell Biology, Rockefeller University Press, 2022, 221 (12). Adipocytes are the main cell type in adipose tissue, which is a critical regulator of metabolism, highly specialized in storing energy as fat. Adipocytes differentiate from multipotent mesenchymal stromal cells (hMSCs) through adipogenesis, a tightly controlled differentiation process involving close interplay between metabolic transitions and sequential programs of gene expression. However, the specific gears driving this interplay remain largely obscure. Additionally, the metabolite nicotinamide adenine dinucleotide (NAD +) is becoming increasingly recognized as a regulator of lipid metabolism, and a promising therapeutic target for dyslipidemia and obesity. Here, we explored how NAD + bioavailability controls adipogenic differentiation from hMSC. We found a previously unappreciated repressive role for NAD + on adipocyte commitment, while a functional NAD +-dependent deacetylase SIRT1 appeared crucial for terminal differentiation of pre-adipocytes. Repressing NAD + biosynthesis during adipogenesis promoted the adipogenic transcriptional program, while two-photon microscopy and extracellular flux analyses suggest that SIRT1 activity mostly relies on the metabolic switch. Interestingly, SIRT1 controls subcellular compartmentalization of redox metabolism during adipogenesis. (10.1083/jcb.202111137)
  DOI : 10.1083/jcb.202111137
• Synthesis and structure–activity relationship studies of pyrido [1,2-e]purine-2,4(1H,3H)-dione derivatives targeting Flavin-Dependent $Thymidylate\ Synthase$ in $Mycobacterium\ tuberculosis$
  
  • Biteau Nicolas G.
  • Roy Vincent
  • Nicolas Cyril
  • Becker Hubert F
  • Lambry Jean-Christophe
  • Myllykallio Hannu
  • Agrofoglio Luigi A.
  Molecules, MDPI, 2022, 27, pp.6216. In 2002, a new class of thymidylate synthase (TS) involved in the de novo synthesis of dTMP named Flavin-Dependent Thymidylate Synthase (FDTS) encoded by the thyX gene was discovered; FDTS is present only in 30% of prokaryote pathogens and not in human pathogens, which makes it an attractive target for the development of new antibacterial agents, especially against multi-resistant pathogens. We report herein the synthesis and structure-activity relationship of a novel series of hitherto unknown pyrido[1,2-E]purine-2,4(1H,3H)-dione analogues. Several synthetics efforts were done to optimize regioselective N1-alkylation through organopalladium cross-coupling. Modelling of potential hits were performed to generate a model of interaction into the active pocket of FDTS to understand and guide further synthetic modification. All those compounds were evaluated on an in-house in vitro NADPH oxidase assays screening as well as against Mycobacterium tuberculosis ThyX. The highest inhibition was obtained for compound 23a with 84.3% at 200 μM without significant cytotoxicity (CC50 > 100 micromolar) on PBM cells. (10.3390/molecules27196216)
  DOI : 10.3390/molecules27196216
• BrdU Incorporation and Labeling of Nascent DNA to Investigate Archaeal Replication Using Super-Resolution Imaging
  
  • Lestini Roxane
  • Collien Yoann
  • Olivier Debora
  • Olivier Nicolas
  • Myllykallio Hannu
  , 2022, 2522, pp.419-434. The labeling and specific detection of nascent DNA by the incorporation of thymidine analogs provide crucial information about DNA replication dynamics without requiring the intracellular expression of fluorescent proteins. After cell fixation and permeabilization, specific detection of thymidine analogs by antibodies can be performed using super-resolution imaging techniques. Here we describe a protocol to label nascent DNA using 5'-bromo-2'-deoxyuridine (BrdU) in Haloferax volcanii cells and generate super-resolved images of neo-synthesized DNA foci either by 3D Structured illumination microscopy (3D-SIM) or Stochastic Optical Reconstruction Microscopy (STORM). (10.1007/978-1-0716-2445-6_29)
  DOI : 10.1007/978-1-0716-2445-6_29
• Parametrization of Force Field Bonded Terms under Structural Inconsistency
  
  • Croitoru Anastasia
  • Aleksandrov Alexey
  Journal of Chemical Information and Modeling, American Chemical Society, 2022, pp.4471-4482. (10.1021/acs.jcim.2c00950)
  DOI : 10.1021/acs.jcim.2c00950
• The syndecan-1 binding domain released by cleavage of laminin-332 impacts dermal repair during wound healing
  
  • Castillo Alejandro
  • Vincent Silène
  • Gross Viktoriia
  • Chessel Anatole
  • Montmasson Marine
  • Ragaru Bernard
  • Herbage Benjamin
  • Pin Didier
  • Schanne-Klein Marie-Claire
  • Rousselle Patricia
  , 2022, 30 (5). (10.1111/wrr.13041)
  DOI : 10.1111/wrr.13041
• Development of force field methods
  
  • Croitoru Anastasia
  , 2022. Drug discovery and development are very time and resources consuming processes, which can be significantly facilitated by computer-aided drug design methods. Among such methods force field-based are arguably the most used. The goal of this PhD was to develop the force field (FF) model for a large number of biologically important molecules. In the first part, I focused on extending the CHARMM force field to a large set of 333 nonstandard amino acids. These nonstandard amino acids are frequent in the protein structures available in the protein data bank. These are biologically important molecules produced as a result of post-translational modifications (PTMs) in the cell, but also can be synthetized and incorporated in labs. For parametrization, amino acids with nonstandard sidechains as well as amino acids with modified backbone groups were considered. Amino acids were parametrized for the most important protonation states at physiological pH and, for some more common residues, in both D- and L-stereoisomers. Both inter- and intramolecular terms were parametrized targeting quantum mechanics (QM) data. Validation was performed by molecular dynamics simulations of 20 protein systems.To improve the force field development method, I developed and tested a new method for bond and valence angle terms parametrization to improve transferability and robustness of developed parameters. The novelty of the method is that it allows explicitly structural deviations between QM and CHARMM structures during optimization. The results demonstrate that without any need for additional restraints the new method produces robust and transferable force field parameters. The new method also improves the agreement for the QM normal modes for all molecules in the set. Thus, the new method will allow parametrization of molecules under the structural deviation, common for force fields for small molecules, producing robust and transferable parameters.In the final part of the project, I am performing a large-scale parametrization of drug-like molecules. Around 300,000 ligands were selected from the ZINC20 database to cover a broad region of the chemical space. The selection criteria accounted for the drug-likeness and chemical diversity of molecules, which are not parametrized in the current CHARMM force field. Based on the sorting method to find molecules containing common atom groups, 7000 molecules were subjected to the force field development. A special attention was given to the optimization of non-planar rings, which can exist in different puckering states.Overall, the current PhD project represents a significant step forward in extending the CHARMM FF to a wide variety of chemical entities. The FF model for both, nonstandard amino acids and the ligand library, apart from structure/function studies can be used for virtual screening studies. To make available for the scientific community, the FFs developed in this work are included into the standard CHARMM package of FF.
• Iso-FRET: an isothermal competition assay to analyze quadruplex formation in vitro
  
  • Luo Yu
  • Verga Daniela
  • Mergny Jean-Louis
  Nucleic Acids Research, Oxford University Press, 2022, 50 (16), pp.e93-e93. Algorithms have been widely used to predict G-quadruplexes (G4s)-prone sequences. However, an experimental validation of these predictions is generally required. We previously reported a high-throughput technique to evidence G4 formation in vitro called FRET-MC. This method, while convenient and reproducible, has one known weakness: its inability to pin point G4 motifs of low thermal stability. As such quadruplexes may still be biologically relevant if formed at physiological temperature, we wanted to develop an independent assay to overcome this limitation. To this aim, we introduced an isothermal version of the competition assay, called iso-FRET, based on a duplex-quadruplex competition and a well-characterized bis-quinolinium G4 ligand, PhenDC3. G4-forming competitors act as decoys for PhenDC3, lowering its ability to stabilize the G4-forming motif reporter oligonucleotide conjugated to a fluorescence quencher (37Q). The decrease in available G4 ligand concentration restores the ability of 37Q to hybridize to its FAM-labeled short complementary C-rich strand (F22), leading to a decrease in fluorescence signal. In contrast, when no G4-forming competitor is present, PhenDC3 remains available to stabilize the 37Q quadruplex, preventing the formation of the F22 + 37Q complex. Iso-FRET was first applied to a reference panel of 70 sequences, and then used to investigate 23 different viral sequences. (10.1093/nar/gkac465)
  DOI : 10.1093/nar/gkac465
• Inline Amplification of Mid-Infrared Intrapulse Difference Frequency Generation
  
  • Bournet Quentin
  • Guichard François
  • Natile Michele
  • Zaouter Yoann
  • Zheng A.
  • Joffre M.
  • Bonvalet A.
  • Jonusas M.
  • Druon Frédéric
  • Hanna Marc
  • Georges Patrick
  , 2022.
• Sol-gel transition induced by alumina nanoparticles in a model pulmonary surfactant
  
  • Berret Jean-François
  • Mousseau Fanny
  • Le Borgne Rémi
  • Oikonomou Evdokia K
  Colloids and Surfaces A: Physicochemical and Engineering Aspects, Elsevier, 2022, 646, pp.128974. Inhaled airborne particles smaller than 100 nm entering the airways have been shown to deposit in significant amount in the alveolar region of the lungs. The interior of the alveoli is covered with a ~ 1 µm thick lining fluid, called pulmonary surfactant. Inhaled nanoparticles are susceptible to interact with the lung fluid and modify pulmonary functions. Here we evaluate the structural and rheological properties of the pulmonary surfactant substitute Curosurf® which is administered to premature babies for the treatment of respiratory distress syndrome. Curosurf® is considered a reliable model of endogenous pulmonary surfactant in terms of composition, structure and function. Using active microrheology based on magnetically actuated wires, we find that Curosurf® dispersions exhibit a Newtonian behavior at lipid concentration from 0 to 80 g L-1 , and that the viscosity follows the Krieger-Dougherty law observed for a wide variety of colloids. Upon addition of 40 nm alumina nanoplatelets, a significant change of the Curosurf® rheology is noticed. The dispersions then enter a soft solid phase characterized by an infinite viscosity and a non-zero equilibrium elastic modulus. The sol-gel transition induced by the nanoparticles is interpreted as the result of the alumina/vesicle interaction, which are illustrated by transmission electron microscopy. It also suggests a potential toxicity associated with the modification of the lung fluid structural and dynamical properties. (10.1016/j.colsurfa.2022.128974)
  DOI : 10.1016/j.colsurfa.2022.128974
• The Newly Sequenced Genome of Pisum sativum Is Replete with Potential G-Quadruplex-Forming Sequences—Implications for Evolution and Biological Regulation
  
  • Dobrovolná Michaela
  • Bohálová Natália
  • Peška Vratislav
  • Wang Jiawei
  • Luo Yu
  • Bartas Martin
  • Volná Adriana
  • Mergny Jean-Louis
  • Brázda Václav
  International Journal of Molecular Sciences, MDPI, 2022, 23 (15), pp.8482. G-quadruplexes (G4s) have been long considered rare and physiologically unimportant in vitro curiosities, but recent methodological advances have proved their presence and functions in vivo. Moreover, in addition to their functional relevance in bacteria and animals, including humans, their importance has been recently demonstrated in evolutionarily distinct plant species. In this study, we analyzed the genome of Pisum sativum (garden pea, or the so-called green pea), a unique member of the Fabaceae family. Our results showed that this genome contained putative G4 sequences (PQSs). Interestingly, these PQSs were located nonrandomly in the nuclear genome. We also found PQSs in mitochondrial (mt) and chloroplast (cp) DNA, and we experimentally confirmed G4 formation for sequences found in these two organelles. The frequency of PQSs for nuclear DNA was 0.42 PQSs per thousand base pairs (kbp), in the same range as for cpDNA (0.53/kbp), but significantly lower than what was found for mitochondrial DNA (1.58/kbp). In the nuclear genome, PQSs were mainly associated with regulatory regions, including 5 UTRs, and upstream of the rRNA region. In contrast to genomic DNA, PQSs were located around RNA genes in cpDNA and mtDNA. Interestingly, PQSs were also associated with specific transposable elements such as TIR and LTR and around them, pointing to their role in their spreading in nuclear DNA. The nonrandom localization of PQSs uncovered their evolutionary and functional significance in the Pisum sativum genome. (10.3390/ijms23158482)
  DOI : 10.3390/ijms23158482
• The Newly Sequenced Genome of Pisum sativum Is Replete with Potential G-Quadruplex-Forming Sequences—Implications for Evolution and Biological Regulation
  
  • Dobrovolná Michaela
  • Bohálová Natália
  • Peška Vratislav
  • Wang Jiawei
  • Luo Yu
  • Bartas Martin
  • Volná Adriana
  • Mergny Jean-Louis
  • Brázda Václav
  International Journal of Molecular Sciences, MDPI, 2022, 23 (15), pp.8482.