Publications

2021

• 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
• Rapid Evaluation of Novel Therapeutic Strategies Using a 3D Collagen-Based Tissue-Like Model
  
  • Maury Pauline
  • Porcel Erika
  • Mau Adrien
  • Lux François
  • Tillement Olivier
  • Mahou Pierre
  • Schanne-Klein Marie-Claire
  • Lacombe Sandrine
  Frontiers in Bioengineering and Biotechnology, Frontiers, 2021. 2D cell cultures are commonly used to rapidly evaluate the therapeutic potential of various treatments on living cells. However, the effects of the extracellular matrix (ECM) including the 3D arrangement of cells and the complex physiology of native environment are missing, which makes these models far from in vivo conditions. 3D cell models have emerged in preclinical studies to simulate the impact of the ECM and partially bridge the gap between monolayer cultures and in vivo tissues. To date, the difficulty to handle the existing 3D models, the cost of their production and their poor reproducibility have hindered their use. Here, we present a reproducible and commercially available “3D cell collagen-based model” (3D-CCM) that allows to study the influence of the matrix on nanoagent uptake and radiation effects. The cell density in these samples is homogeneous. The oxygen concentration in the 3D-CCM is tunable, which opens the opportunity to investigate hypoxic effects. In addition, thanks to the intrinsic properties of the collagen, the second harmonic imaging microscopy may be used to probe the whole volume and visualize living cells in real-time. Thus, the architecture and composition of 3D-CCMs as well as the impact of various therapeutic strategies on cells embedded in the ECM is observed directly. Moreover, the disaggregation of the collagen matrix allows recovering of cells without damaging them. It is a major advantage that makes possible single cell analysis and quantification of treatment effects using clonogenic assay. In this work, 3D-CCMs were used to evaluate the correlative efficacies of nanodrug exposure and medical radiation on cells contained in a tumor like sample. A comparison with monolayer cell cultures was performed showing the advantageous outcome and the higher potential of 3D-CCMs. This cheap and easy to handle approach is more ethical than in vivo experiments, thus, giving a fast evaluation of cellular responses to various treatments. (10.3389/fbioe.2021.574035)
  DOI : 10.3389/fbioe.2021.574035
• Euryarchaeal genomes are folded into SMC-dependent loops and domains, but lack transcription-mediated compartmentalization
  
  • Cockram Charlotte
  • Thierry Agnès
  • Gorlas Aurore
  • Lestini Roxane
  • Koszul Romain
  Molecular Cell, Cell Press, 2021, 81 (3), pp.459-472.e10. Hi-C has become a routine method for probing the 3D organization of genomes. However, when applied to prokaryotes and archaea, the current protocols are expensive and limited in their resolution. We develop a cost-effective Hi-C protocol to explore chromosome conformations of these two kingdoms at the gene or operon level. We first validate it on E. coli and V. cholera, generating sub-kilobase-resolution contact maps, and then apply it to the euryarchaeota H. volcanii, Hbt. salinarum, and T. kodakaraensis. With a resolution of up to 1 kb, we explore the diversity of chromosome folding in this phylum. In contrast to crenarchaeota, these euryarchaeota lack (active/inactive) compartment-like structures. Instead, their genomes are composed of self-interacting domains and chromatin loops. In H. volcanii, these structures are regulated by transcription and the archaeal structural maintenance of chromosomes (SMC) protein, further supporting the ubiquitous role of these processes in shaping the higher-order organization of genomes. (10.1016/j.molcel.2020.12.013)
  DOI : 10.1016/j.molcel.2020.12.013
• Insights into G-Quadruplex–Hemin Dynamics Using Atomistic Simulations: Implications for Reactivity and Folding
  
  • Stadlbauer Petr
  • Islam Barira
  • Otyepka Michal
  • Chen Jielin
  • Monchaud David
  • Zhou Jun
  • Mergny Jean-Louis
  • Šponer Jiří
  Journal of Chemical Theory and Computation, American Chemical Society, 2021, 17 (3), pp.1883 - 1899. Guanine quadruplex nucleic acids (G4s) are involved in key biological processes such as replication or transcription. Beyond their biological relevance, G4s find applications as biotechnological tools since they readily bind hemin and enhance its peroxidase activity, creating a G4-DNAzyme. The biocatalytic properties of G4-DNAzymes have been thoroughly studied and used for biosensing purposes. Despite hundreds of applications and massive experimental efforts, the atomistic details of the reaction mechanism remain unclear. To help select between the different hypotheses currently under investigation, we use extended explicit-solvent molecular dynamics (MD) simulations to scrutinize the G4/hemin interaction. We find that besides the dominant conformation in which hemin is stacked atop the external G-quartets, hemin can also transiently bind to the loops and be brought to the external G-quartets through diverse delivery mechanisms. The simulations do not support the catalytic mechanism relying on a wobbling guanine. Similarly, the catalytic role of the iron-bound water molecule is not in line with our results; however, given the simulation limitations, this observation should be considered with some caution. The simulations rather suggest tentative mechanisms in which the external G-quartet itself could be responsible for the unique H 2 O 2-promoted biocatalytic properties of the G4/hemin complexes. Once stacked atop a terminal G-quartet, hemin rotates about its vertical axis while readily sampling shifted geometries where the iron transiently contacts oxygen atoms of the adjacent G-quartet. This dynamics is not apparent from the ensemble-averaged structure. We also visualize transient interactions between the stacked hemin and the G4 loops. Finally, we investigated interactions between hemin and on-pathway folding intermediates of the parallel-stranded G4 fold. The simulations suggest that hemin drives the folding of parallel-stranded G4s from slip-stranded intermediates, acting as a G4 chaperone. Limitations of the MD technique are briefly discussed. (10.1021/acs.jctc.0c01176)
  DOI : 10.1021/acs.jctc.0c01176
• Human Papillomavirus G-Rich Regions as Potential Antiviral Drug Targets
  
  • Carvalho Josué
  • Lopes-Nunes Jéssica
  • Campello Maria Paula Cabral
  • Paulo António
  • Milici Janice
  • Meyers Craig
  • Mergny Jean-Louis
  • Salgado Gilmar
  • Queiroz João
  • Cruz Carla
  Nucleic Acid Therapeutics, Mary Ann Liebert, Inc. publishers, 2021, 31 (1), pp.68-81. Abstract G‐quadruplexes (G4) play crucial roles in biology, analytical chemistry and nanotechnology. The stability of G4 structures is impacted by the number of G‐quartets, the length and positions of loops, flanking motifs, as well as additional structural elements such as bulges, capping base pairs, or triads. Algorithms such as G4Hunter or Quadparser may predict if a given sequence is G4‐prone by calculating a quadruplex propensity score; however, experimental validation is still required. We previously demonstrated that this validation is not always straightforward, and that a combination of techniques is often required to unambiguously establish whether a sequence forms a G‐quadruplex or not. In this article, we adapted the well‐known FRET‐melting assay to characterize G4 in batch, where the sequence to be tested is added, as an unlabeled competitor, to a system composed of a dual‐labeled probe (F21T) and a specific quadruplex ligand. PhenDC3 was preferred over TMPyP4 because of its better selectivity for G‐quadruplexes. In this so‐called FRET‐MC (melting competition) assay, G4‐forming competitors lead to a marked decrease of the ligand‐induced stabilization effect (∆ T m ), while non‐specific competitors (e.g., single‐ or double‐stranded sequences) have little effect. Sixty‐five known sequences with different typical secondary structures were used to validate the assay, which was subsequently employed to assess eight novel sequences that were not previously characterized. (10.1089/nat.2020.0869)
  DOI : 10.1089/nat.2020.0869
• Targeting nucleolin by RNA G-quadruplex-forming motif
  
  • Figueiredo Joana
  • Miranda André
  • Lopes-Nunes Jéssica
  • Carvalho Josué
  • Alexandre Daniela
  • Valente Salete
  • Mergny Jean‐louis
  • Cruz Carla
  Biochemical Pharmacology, Elsevier, 2021, 189, pp.114418. (10.1016/j.bcp.2021.114418)
  DOI : 10.1016/j.bcp.2021.114418
• Cyclodipeptide synthases of the NYH subfamily recognize tRNA using an α-helix enriched with positive residues
  
  • Croitoru Anastasia
  • Babin Morgan
  • Myllykallio Hannu
  • Gondry Muriel
  • Aleksandrov Alexey
  Biochemistry, American Chemical Society, 2021, 60 (1), pp.64-76. Cyclodipeptide synthases (CDPSs) perform nonribosomal protein synthesis using two aminoacyl-tRNA substrates to produce cyclodipeptides. There is no available structural detail on the CDPS:tRNA interaction to date. Using AlbC, a CDPS that produces cyclo(L-Phe-L-Phe), the interaction between AlbC with its Phe-tRNA substrate was investigated. Simulations of models of the AlbC:tRNA complex, proposed by rigid body docking or homology modeling, demonstrated that interactions with residues of an AlbC alpha helix, α4, significantly contribute to the binding free energy of AlbC to tRNA. Individual residue contributions to the tRNA binding free energy of the discovered binding mode explain well available biochemical data, and the results of in vivo assay experiments performed in this work and guided by simulations. In molecular dynamics simulations the phenylalanyl group predominantly occupied the two positions observed in the experimental structure of AlbC in the dipeptide intermediate state, suggesting that tRNAs of the first and second substrates interact with AlbC in a similar manner. Overall, given the high sequence and structural similarity among the members of the CDPS NYH protein subfamily, the mechanism of the protein:tRNA interaction is expected to be pertinent to a wide range of tRNA interacting proteins. (10.1021/acs.biochem.0c00761)
  DOI : 10.1021/acs.biochem.0c00761
• Gene level resolution of bacteria and archaea genome folding
  
  • Cockram Charlotte
  • Thierry Agnès
  • Lestini Roxane
  • Koszul Romain
  , 2021.
• Core–Shell Pure Collagen Threads Extruded from Highly Concentrated Solutions Promote Colonization and Differentiation of C3H10T1/2 Cells
  
  • Picaut Lise
  • Trichet Lea
  • Hélary Christophe
  • Ducourthial Guillaume
  • Bonnin Marie-Ange
  • Haye Bernard
  • Ronsin Olivier
  • Schanne-Klein Marie-Claire
  • Duprez Delphine
  • Baumberger Tristan
  • Mosser Gervaise
  ACS Biomaterials Science and Engineering, ACS, 2021. The elaboration of scaffolds able to efficiently promote cell differentiation toward a given cell type remains challenging. Here, we engineered dense type I collagen threads with the aim of providing scaffolds with specific morphological and mechanical properties for C3H10T1/2 mesenchymal stem cells. Extrusion of pure collagen solutions at different concentrations (15, 30, and 60 mg/mL) in a PBS 5× buffer generated dense fibrillated collagen threads. For the two highest concentrations, threads displayed a core-shell structure with a marked fibril orientation of the outer layer along the longitudinal axis of the threads. Young's modulus and ultimate tensile stress as high as 1 and 0.3 MPa, respectively, were obtained for the most concentrated collagen threads without addition of any cross-linkers. C3H10T1/2 cells oriented themselves with a mean angle of 15-24° with respect to the longitudinal axis of the threads. Cells penetrated the 30 mg/mL scaffolds but remained on the surface of the 60 mg/mL ones. After three weeks of culture, cells displayed strong expression of the tendon differentiation marker Tnmd, especially for the 30 mg/mL threads. These results suggest that both the morphological and mechanical characteristics of collagen threads are key factors in promoting C3H10T1/2 differentiation into tenocytes, offering promising levers to optimize tissue engineering scaffolds for tendon regeneration. (10.1021/acsbiomaterials.0c01273)
  DOI : 10.1021/acsbiomaterials.0c01273