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1.
J Chem Inf Model ; 63(8): 2554-2572, 2023 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-36972178

RESUMO

We investigated the capability of internal normal modes to reproduce RNA flexibility and predict observed RNA conformational changes and, notably, those induced by the formation of RNA-protein and RNA-ligand complexes. Here, we extended our iNMA approach developed for proteins to study RNA molecules using a simplified representation of the RNA structure and its potential energy. Three data sets were also created to investigate different aspects. Despite all the approximations, our study shows that iNMA is a suitable method to take into account RNA flexibility and describe its conformational changes opening the route to its applicability in any integrative approach where these properties are crucial.


Assuntos
Proteínas , RNA , Ligantes , Modelos Moleculares , Conformação Proteica , Proteínas/química , Conformação de Ácido Nucleico
2.
Proteins ; 89(5): 531-543, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33349977

RESUMO

Normal mode analysis (NMA) is a fast and inexpensive approach that is largely used to gain insight into functional protein motions, and more recently to create conformations for further computational studies. However, when the protein structure is unknown, the use of computational models is necessary. Here, we analyze the capacity of NMA in internal coordinate space to predict protein motion, its intrinsic flexibility, and atomic displacements, using protein models instead of native structures, and the possibility to use it for model refinement. Our results show that NMA is quite insensitive to modeling errors, but that calculations are strictly reliable only for very accurate models. Our study also suggests that internal NMA is a more suitable tool for the improvement of structural models, and for integrating them with experimental data or in other computational techniques, such as protein docking or more refined molecular dynamics simulations.


Assuntos
Algoritmos , Proteínas/química , Ligantes , Simulação de Dinâmica Molecular , Movimento (Física) , Conformação Proteica , Proteínas/ultraestrutura
3.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33916983

RESUMO

Transcription factors regulate gene activity by binding specific regions of genomic DNA thanks to a subtle interplay of specific and nonspecific interactions that is challenging to quantify. Here, we exploit Reflective Phantom Interface (RPI), a label-free biosensor based on optical reflectivity, to investigate the binding of the N-terminal domain of Gal4, a well-known gene regulator, to double-stranded DNA fragments containing or not its consensus sequence. The analysis of RPI-binding curves provides interaction strength and kinetics and their dependence on temperature and ionic strength. We found that the binding of Gal4 to its cognate site is stronger, as expected, but also markedly slower. We performed a combined analysis of specific and nonspecific binding-equilibrium and kinetics-by means of a simple model based on nested potential wells and found that the free energy gap between specific and nonspecific binding is of the order of one kcal/mol only. We investigated the origin of such a small value by performing all-atom molecular dynamics simulations of Gal4-DNA interactions. We found a strong enthalpy-entropy compensation, by which the binding of Gal4 to its cognate sequence entails a DNA bending and a striking conformational freezing, which could be instrumental in the biological function of Gal4.


Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Algoritmos , Sequência de Bases , Sítios de Ligação , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Cinética , Modelos Moleculares , Modelos Teóricos , Conformação Molecular , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo
4.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445542

RESUMO

Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/- mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/- TNFα and +/- LIM kinase inhibitors (LIMKi). In silico modeling of TNFα-Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin-tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p < 0.05) and an increase in pseudo-tube width (41 ± 4.5%; p < 0.001) compared to control. Upon TNFα stimulation, ECFC Eng-siRNA displayed a significant higher permeability compared to ctr-siRNA (p < 0.01), which is associated to a higher Ca2+ mobilization (p < 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/-, and MLEC+/- compared to controls (p < 0.001, p < 0.01, and p < 0.01, respectively) as well as actin/tubulin distribution (p < 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p < 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Permeabilidade da Membrana Celular , Endoglina/metabolismo , Células Endoteliais/patologia , Inflamação/patologia , Quinases Lim/metabolismo , Neovascularização Patológica/patologia , Fatores de Despolimerização de Actina/genética , Animais , Endoglina/genética , Células Endoteliais/metabolismo , Inflamação/genética , Inflamação/metabolismo , Quinases Lim/genética , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo
5.
Methods ; 162-163: 108-127, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31145972

RESUMO

Determination of the tridimensional structure of ribonucleic acid molecules is fundamental for understanding their function in the cell. A common method to investigate RNA structures of large molecules is the use of chemical probes such as SHAPE (2'-hydroxyl acylation analyzed by primer extension) reagents, DMS (dimethyl sulfate) and CMCT (1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfate), the reaction of which is dependent on the local structural properties of each nucleotide. In order to understand the interplay between local flexibility, sugar pucker, canonical pairing and chemical reactivity of the probes, we performed all-atom molecular dynamics simulations on a set of RNA molecules for which both tridimensional structure and chemical probing data are available and we analyzed the correlations between geometrical parameters and the chemical reactivity. Our study confirms that SHAPE reactivity is guided by the local flexibility of the different chemical moieties but suggests that a combination of multiple parameters is needed to better understand the implications of the reactivity at the molecular level. This is also the case for DMS and CMCT for which the reactivity appears to be more complex than commonly accepted.


Assuntos
Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Nucleotídeos/química , RNA/química , Acilação , CME-Carbodi-Imida/análogos & derivados , CME-Carbodi-Imida/química , Ligação de Hidrogênio , Radical Hidroxila/química , Indicadores e Reagentes/química , RNA/genética , RNA/metabolismo , Ésteres do Ácido Sulfúrico/química
6.
Soft Matter ; 12(23): 5188-98, 2016 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-27198729

RESUMO

Recent experiments have evidenced some unconventional features in the elasticity of nematics, which cannot be explained by standard microscopic theories. Here, in the framework of a second-virial density functional theory, we have developed a general approach, relaxing the usual assumption that the angular distribution of particles with respect to their local director is unaffected by the deformation. We show that, for particles with polar symmetry, a new contribution to the splay and bend deformation free energy arises, associated with the onset of polar order. Calculations for conical and bent-shaped particles reveal dramatic softening of the splay and the bend mode, respectively, which eventually may lead to spontaneous deformation.

7.
Org Biomol Chem ; 14(40): 9568-9577, 2016 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-27722414

RESUMO

The solvent-promoted aggregation of porphyrins covalently linked to medium length peptides occurs with the formation of chiral supramolecular structures if the peptide chain can adopt an α-helical secondary structure. The circular dichroism spectra of different porphyrin-peptide conjugates show that the chiral arrangement of the porphyrins in the aggregates does not depend on the screw-sense of the peptide helix. Experimental evidence and molecular dynamic simulations suggest that the linker between the porphyrin and the peptide helix is responsible for the overall chirality of supramolecular structures. In particular when the linker is a chiral α-amino acid it is possible to tune the morphology of the chiral aggregates by inverting the configuration of the chiral center.


Assuntos
Peptídeos/química , Porfirinas/química , Solventes/química , Sequência de Aminoácidos , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice
8.
Chemphyschem ; 15(7): 1336-44, 2014 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-24470302

RESUMO

We present a computational investigation of the nematic phase of the bent-core liquid crystal A131. We use an integrated approach that bridges density functional theory calculations of molecular geometry and torsional potentials to elastic properties through the molecular conformational and orientational distribution function. This unique capability to simultaneously access different length scales enables us to consistently describe molecular and material properties. We can reassign (13)C NMR chemical shifts and analyze the dependence of phase properties on molecular shape. Focusing on the elastic constants we can draw some general conclusions on the unconventional behavior of bent-core nematics and highlight the crucial role of a properly-bent shape.

9.
Soft Matter ; 10(41): 8171-87, 2014 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-25164281

RESUMO

Hard helices can be regarded as a paradigmatic elementary model for a number of natural and synthetic soft matter systems, all featuring the helix as their basic structural unit, from natural polynucleotides and polypeptides to synthetic helical polymers, and from bacterial flagella to colloidal helices. Here we present an extensive investigation of the phase diagram of hard helices using a variety of methods. Isobaric Monte Carlo numerical simulations are used to trace the phase diagram; on going from the low-density isotropic to the high-density compact phases a rich polymorphism is observed, exhibiting a special chiral screw-like nematic phase and a number of chiral and/or polar smectic phases. We present full characterization of the latter, showing that they have unconventional features, ascribable to the helical shape of the constituent particles. Equal area construction is used to locate the isotropic-to-nematic phase transition, and the results are compared with those stemming from an Onsager-like theory. Density functional theory is also used to study the nematic-to-screw-nematic phase transition; within the simplifying assumption of perfectly parallel helices, we compare different levels of approximation, that is second- and third-virial expansions and a Parsons-Lee correction.

10.
Phys Chem Chem Phys ; 16(30): 16225-32, 2014 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-24969095

RESUMO

Using an Onsager-like theory, we have investigated the relationship between the morphology of hard helical particles and the features (pitch and handedness) of the cholesteric phase that they form. We show that right-handed helices can assemble into right- (R) and left-handed (L) cholesterics, depending on their curliness, and that the cholesteric pitch is a non-monotonic function of the intrinsic pitch of particles. The theory leads to the definition of a hierarchy of pseudoscalars, which quantify the difference in the average excluded volume between pair configurations of helices having (R) and (L)-skewed axes. The predictions of the Onsager-like theory are supported by Monte Carlo simulations of the isotropic phase of hard helices, showing how the cholesteric organization, which develops on scales longer than hundreds of molecular sizes, is encoded in the short-range chiral correlations between the helical axes.

11.
J Chem Phys ; 140(8): 081101, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24588140

RESUMO

Evidence of a special chiral nematic phase is provided using numerical simulation and Onsager theory for systems of hard helical particles. This phase appears at the high density end of the nematic phase, when helices are well aligned, and is characterized by the C2 symmetry axes of the helices spiraling around the nematic director with periodicity equal to the particle pitch. This coupling between translational and rotational degrees of freedom allows a more efficient packing and hence an increase of translational entropy. Suitable order parameters and correlation functions are introduced to identify this screw-like phase, whose main features are then studied as a function of radius and pitch of the helical particles. Our study highlights the physical mechanism underlying a similar ordering observed in colloidal helical flagella [E. Barry, Z. Hensel, Z. Dogic, M. Shribak, and R. Oldenbourg, Phys. Rev. Lett. 96, 018305 (2006)] and raises the question of whether it could be observed in other helical particle systems, such as DNA, at sufficiently high densities.


Assuntos
Simulação de Dinâmica Molecular , Coloides/química , DNA/química , Método de Monte Carlo , Polímeros/química
12.
J Phys Chem Lett ; 15(16): 4351-4358, 2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38619551

RESUMO

Water molecules are essential to determine the structure of nucleic acids and mediate their interactions with other biomolecules. Here, we characterize the hydration dynamics of analogous DNA and RNA double helices with unprecedented resolution and elucidate the molecular origin of their differences: first, the localization of the slowest hydration water molecules─in the minor groove in DNA, next to phosphates in RNA─and second, the markedly distinct hydration dynamics of the two phosphate oxygen atoms OR and OS in RNA. Using our Extended Jump Model for water reorientation, we assess the relative importance of previously proposed factors, including the local topography, water bridges, and the presence of ions. We show that the slow hydration dynamics at RNA OR sites is not due to bridging water molecules but is caused by both the larger excluded volume and the stronger initial H-bond next to OR, due to the different phosphate orientations in A-form double helical RNA.


Assuntos
DNA , Ligação de Hidrogênio , Conformação de Ácido Nucleico , RNA , Água , DNA/química , RNA/química , Água/química , Fosfatos/química , Simulação de Dinâmica Molecular
13.
J Phys Chem B ; 128(20): 4865-4886, 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38740056

RESUMO

Facing the current challenges posed by human health diseases requires the understanding of cell machinery at a molecular level. The interplay between proteins and RNA is key for any physiological phenomenon, as well protein-RNA interactions. To understand these interactions, many experimental techniques have been developed, spanning a very wide range of spatial and temporal resolutions. In particular, the knowledge of tridimensional structures of protein-RNA complexes provides structural, mechanical, and dynamical pieces of information essential to understand their functions. To get insights into the dynamics of protein-RNA complexes, we carried out all-atom molecular dynamics simulations in explicit solvent on nine different protein-RNA complexes with different functions and interface size by taking into account the bound and unbound forms. First, we characterized structural changes upon binding and, for the RNA part, the change in the puckering. Second, we extensively analyzed the interfaces, their dynamics and structural properties, and the structural waters involved in the binding, as well as the contacts mediated by them. Based on our analysis, the interfaces rearranged during the simulation time showing alternative and stable residue-residue contacts with respect to the experimental structure.


Assuntos
Simulação de Dinâmica Molecular , RNA , RNA/química , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas/química , Conformação de Ácido Nucleico
14.
J Chem Phys ; 138(16): 164906, 2013 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-23635170

RESUMO

We investigate the isotropic-to-nematic phase transition in systems of hard helical particles, using Onsager theory and Monte Carlo computer simulations. Motivation of this work resides in the ubiquity of the helical shape motif in many natural and synthetic polymers, as well as in the well known importance that the details of size and shape have in determining the phase behaviour and properties of (soft) condensed matter systems. We discuss the differences with the corresponding spherocylinder phase diagram and find that the helix parameters affect the phase behaviour and the existence of the nematic phase. We find that for high helicity Onsager theory significantly departs from numerical simulations even when a modified form of the Parsons-Lee rescaling is included to account for the non-convexity of particles.


Assuntos
Simulação de Dinâmica Molecular , Polímeros/química , Teoria Quântica , Método de Monte Carlo , Transição de Fase , Polímeros/síntese química
15.
Beilstein J Org Chem ; 8: 155-63, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22423282

RESUMO

The chirality transfer in liquid crystals induced by two helical perylenequinones (namely, the natural compounds cercosporin and phleichrome) was investigated by integrating measurements of helical twisting power with a conformational analysis by DFT calculations and with the prediction of their twisting ability by the surface-chirality method. The two quasi-enantiomeric derivatives induce oppositely handed cholesteric phases when introduced as dopants in nematic solvents. We evaluated the role of the different conformations of the chiral hydroxyalkyl side chains in determining the helical twisting power: They were found to affect the strength of the chirality transfer, although the handedness of the induced cholesteric phase is essentially determined by the axial chirality (helicity) of the core of the perylenequinones.

16.
Front Mol Biosci ; 9: 970109, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36275619

RESUMO

Protein-protein interactions are at the basis of many protein functions, and the knowledge of 3D structures of protein-protein complexes provides structural, mechanical and dynamical pieces of information essential to understand these functions. Protein-protein interfaces can be seen as stable, organized regions where residues from different partners form non-covalent interactions that are responsible for interaction specificity and strength. They are commonly described as a peripheral region, whose role is to protect the core region that concentrates the most contributing interactions, from the solvent. To get insights into the dynamics of protein-protein complexes, we carried out all-atom molecular dynamics simulations in explicit solvent on eight different protein-protein complexes of different functional class and interface size by taking into account the bound and unbound forms. On the one hand, we characterized structural changes upon binding of the proteins, and on the other hand we extensively analyzed the interfaces and the structural waters involved in the binding. Based on our analysis, in 6 cases out of 8, the interfaces rearranged during the simulation time, in stable and long-lived substates with alternative residue-residue contacts. These rearrangements are not restricted to side-chain fluctuations in the periphery but also affect the core interface. Finally, the analysis of the waters at the interface and involved in the binding pointed out the importance to take into account their role in the estimation of the interaction strength.

17.
J Chem Theory Comput ; 17(10): 6509-6521, 2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34506136

RESUMO

RNA molecules can easily adopt alternative structures in response to different environmental conditions. As a result, a molecule's energy landscape is rough and can exhibit a multitude of deep basins. In the absence of a high-resolution structure, small-angle X-ray scattering data (SAXS) can narrow down the conformational space available to the molecule and be used in conjunction with physical modeling to obtain high-resolution putative structures to be further tested by experiments. Because of the low resolution of these data, it is natural to implement the integration of SAXS data into simulations using a coarse-grained representation of the molecule, allowing for much wider searches and faster evaluation of SAXS theoretical intensity curves than with atomistic models. We present here the theoretical framework and the implementation of a simulation approach based on our coarse-grained model HiRE-RNA combined with SAXS evaluations "on-the-fly" leading the simulation toward conformations agreeing with the scattering data, starting from partially folded structures as the ones that can easily be obtained from secondary structure prediction-based tools. We show on three benchmark systems how our approach can successfully achieve high-resolution structures with remarkable similarity with the native structure recovering not only the overall shape, as imposed by SAXS data, but also the details of initially missing base pairs.


Assuntos
Dobramento de RNA , RNA , Espalhamento a Baixo Ângulo , Raios X
18.
Noncoding RNA ; 7(4)2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34842779

RESUMO

As more sequencing data accumulate and novel puzzling genetic regulations are discovered, the need for accurate automated modeling of RNA structure increases. RNA structure modeling from chemical probing experiments has made tremendous progress, however accurately predicting large RNA structures is still challenging for several reasons: RNA are inherently flexible and often adopt many energetically similar structures, which are not reliably distinguished by the available, incomplete thermodynamic model. Moreover, computationally, the problem is aggravated by the relevance of pseudoknots and non-canonical base pairs, which are hardly predicted efficiently. To identify nucleotides involved in pseudoknots and non-canonical interactions, we scrutinized the SHAPE reactivity of each nucleotide of the 188 nt long lariat-capping ribozyme under multiple conditions. Reactivities analyzed in the light of the X-ray structure were shown to report accurately the nucleotide status. Those that seemed paradoxical were rationalized by the nucleotide behavior along molecular dynamic simulations. We show that valuable information on intricate interactions can be deduced from probing with different reagents, and in the presence or absence of Mg2+. Furthermore, probing at increasing temperature was remarkably efficient at pointing to non-canonical interactions and pseudoknot pairings. The possibilities of following such strategies to inform structure modeling software are discussed.

19.
Chem Sci ; 12(40): 13492-13505, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34777769

RESUMO

The RNA helicase (non-structural protein 13, NSP13) of SARS-CoV-2 is essential for viral replication, and it is highly conserved among the coronaviridae family, thus a prominent drug target to treat COVID-19. We present here structural models and dynamics of the helicase in complex with its native substrates based on thorough analysis of homologous sequences and existing experimental structures. We performed and analysed microseconds of molecular dynamics (MD) simulations, and our model provides valuable insights to the binding of the ATP and ssRNA at the atomic level. We identify the principal motions characterising the enzyme and highlight the effect of the natural substrates on this dynamics. Furthermore, allosteric binding sites are suggested by our pocket analysis. Our obtained structural and dynamical insights are important for subsequent studies of the catalytic function and for the development of specific inhibitors at our characterised binding pockets for this promising COVID-19 drug target.

20.
Int J Biol Macromol ; 184: 209-217, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34126147

RESUMO

Alpha2-macroglobulin (α2M) is a physiological macromolecule that facilitates the clearance of many proteinases, cytokines and growth factors in human. Here, we explored the effect of induced forms of α2M on anticoagulant drugs. Gla-domainless factor Xa (GDFXa) and methylamine (MA)-induced α2M were prepared and characterized by electrophoresis, immunonephelometry, chromogenic, clot waveform and rotational thromboelastometry assays. Samples from healthy volunteers and anticoagulated patients were included. In vivo neutralization of anticoagulants was evaluated in C57Bl/6JRj mouse bleeding-model. Anticoagulant binding sites on induced α2M were depicted by computer-aided energy minimization modeling. GDFXa-induced α2M neutralized dabigatran and heparins in plasma and whole blood. In mice, a single IV dose of GDFXa-induced α2M following anticoagulant administration significantly reduced blood loss and bleeding time. Being far easier to prepare, we investigated the efficacy of MA-induced α2M. It neutralized rivaroxaban, apixaban, dabigatran and heparins in spiked samples in a concentration-dependent manner and in samples from treated patients. Molecular docking analysis evidenced the ability of MA-induced α2M to bind non-covalently these compounds via some deeply buried binding sites. Induced forms of α2M have the potential to neutralize direct oral anticoagulants and heparins, and might be developed as a universal antidote in case of major bleeding or urgent surgery.


Assuntos
Inibidores do Fator Xa/efeitos adversos , Fator Xa/química , Hemorragia/tratamento farmacológico , Heparina/efeitos adversos , alfa 2-Macroglobulinas Associadas à Gravidez/administração & dosagem , Administração Oral , Animais , Modelos Animais de Doenças , Feminino , Voluntários Saudáveis , Hemorragia/induzido quimicamente , Humanos , Metilaminas/farmacologia , Camundongos , Simulação de Acoplamento Molecular , Gravidez , alfa 2-Macroglobulinas Associadas à Gravidez/química , alfa 2-Macroglobulinas Associadas à Gravidez/farmacologia , Domínios Proteicos
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