References of "Galleni, Moreno"
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See detailExploring the suitability of RanBP2-type Zinc Fingers for RNA-binding protein design
De Franco, Simona ULiege; Vandenameele, Julie ULiege; Brans, Alain ULiege et al

in Scientific Reports (2019), 9(1), 2484

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See detailHomology modeling and in vivo functional characterization of the zinc permeation pathway in a heavy metal P-type ATPase
Lekeux, Gilles ULiege; Crowet, Jean-Marc; Nouet, Cécile ULiege et al

in Journal of Experimental Botany (2019), 70

The P1B ATPase Heavy Metal ATPase 4 (HMA4) is responsible for zinc and cadmium translocation from roots to shoots in the plant Arabidopsis thaliana. It couples ATP hydrolysis to cytosolic domain movements ... [more ▼]

The P1B ATPase Heavy Metal ATPase 4 (HMA4) is responsible for zinc and cadmium translocation from roots to shoots in the plant Arabidopsis thaliana. It couples ATP hydrolysis to cytosolic domain movements enabling metal transport across the membrane. Thanks to high conservation level within the P-type ATPase family, the role of the HMA4 cytoplasmic catalytic domains can be inferred from well characterized pumps. In contrast, the function of its terminal cytosolic extensions as well as the metal permeation mechanism through the membrane remains elusive. Here, homology modeling of the HMA4 transmembrane region was conducted based on the crystal structure of a ZntA bacterial homolog. The analysis highlighted amino acids forming a metal permeation pathway, whose importance was subsequently investigated functionally through mutagenesis and complementation experiments in plants. Although the zinc pathway displayed overall conservation among the two proteins, significant differences were observed, especially in the entrance area with altered electronegativity and the presence of a salt bridge/H-bond network. The analysis also newly identified amino acids whose mutation results in total or partial loss of the protein function. In addition, comparison of zinc and cadmium accumulation in shoots of A. thaliana complemented lines revealed a number of HMA4 mutants exhibiting different abilities in zinc and cadmium translocation. These observations could be instrumental to design low cadmium accumulating crops, hence decreasing human cadmium exposure . [less ▲]

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See detailUnderstanding the significance and implications of antibody numbering and antigen-binding surface/residue definition
Dondelinger, Mathieu ULiege; Filée, Patrice; Sauvage, Eric et al

in Frontiers in Immunology (2018), 9(OCT),

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See detailCombinatorial Design of a Nanobody that Specifically Targets Structured RNAs
Cawez, Frédéric ULiege; Duray, Elodie ULiege; Hu, Y et al

in Journal of Molecular Biology (2018), 430(11), 1652-1670

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See detailThe use of a β-lactamase – based conductimetric biosensor assay to detect biomolecular interactions
Vandevenne, Marylène ULiege; Dondelinger, Mathieu; Yunus, Sami et al

in Journal of Visualized Experiments (2018), (132),

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See detaildi-Cysteine motifs in the C-terminus of plant HMA4 proteins confer nanomolar affinity for zinc and are essential for HMA4 function in vivo.
Lekeux, Gilles ULiege; Laurent, Clémentine ULiege; Joris, Marine ULiege et al

in Journal of Experimental Botany (2018), 69(22), 5547-5560

The PIB ATPase heavy metal ATPase 4 (HMA4) has a central role in the zinc homeostasis network of Arabidopsis thaliana. This membrane protein loads metal from the pericycle cells into the xylem in roots ... [more ▼]

The PIB ATPase heavy metal ATPase 4 (HMA4) has a central role in the zinc homeostasis network of Arabidopsis thaliana. This membrane protein loads metal from the pericycle cells into the xylem in roots, thereby allowing root to shoot metal translocation. Moreover, HMA4 is key for zinc hyperaccumulation as well as zinc and cadmium hypertolerance in the pseudometallophyte Arabidopsis halleri. The plant-specific cytosolic C-terminal extension of HMA4 is rich in putative metal-binding residues and has substantially diverged between A. thaliana and A. halleri. To clarify the function of the domain in both species, protein variants with truncated C-terminal extension, as well as with mutated di-Cys motifs and/or a His-stretch, were functionally characterized. We show that di-Cys motifs, but not the His-stretch, contribute to high affinity zinc binding and function in planta. We suggest that the HMA4 C-terminal extension is at least partly responsible for protein targeting to the plasma membrane. Finally, we reveal that the C-terminal extensions of both A. thaliana and A. halleri HMA4 proteins share similar function, despite marginally different zinc-binding capacity. [less ▲]

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See detailA Kinetic Study of the Replacement by Site Saturation Mutagenesis of Residue 119 in NDM-1 Metallo- beta-Lactamase
Marcoccia, Francesca; Mercuri, Paola ULiege; Galleni, Moreno ULiege et al

in Antimicrobial Agents and Chemotherapy (2018), 62(8),

New Delhi metallo- beta-lactamase 1 (NDM-1) is a subclass B1 metallo-beta - lactamase that exhibits a broad spectrum of activity against beta -lactam antibiotics. Here we report the kinetic study of 6 ... [more ▼]

New Delhi metallo- beta-lactamase 1 (NDM-1) is a subclass B1 metallo-beta - lactamase that exhibits a broad spectrum of activity against beta -lactam antibiotics. Here we report the kinetic study of 6 Q119X variants obtained by site-directed mutagenesis of NDM-1. All Q119X variants were able to hydrolyze carbapenems, penicillins and first-, second-, third-, and fourth-generation cephalosporins very efficiently. In particular, Q119E, Q119Y, Q119V, and Q119K mutants showed improvements in kcat/Km values for penicillins, compared with NDM-1. The catalytic efficiencies of the Q119K variant for benzylpenicillin and carbenicillin were about 65- and 70-fold higher, respectively, than those of NDM-1. The Q119K and Q119Y enzymes had kcat/Km values for ceftazidime about 25- and 89-fold higher, respectively, than that of NDM-1. [less ▲]

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See detailP174E Substitution in GES-1 and GES-5 beta-Lactamases Improves Catalytic Efficiency toward Carbapenems.
Piccirilli, Alessandra; Mercuri, Paola ULiege; Galleni, Moreno ULiege et al

in Antimicrobial Agents and Chemotherapy (2018), 62(5),

GES-type beta-lactamases are a group of enzymes that have evolved their hydrolytic activity against carbapenems. In this study, the role of residue 174 inside the Omega-loop of GES-1 and GES-5 was ... [more ▼]

GES-type beta-lactamases are a group of enzymes that have evolved their hydrolytic activity against carbapenems. In this study, the role of residue 174 inside the Omega-loop of GES-1 and GES-5 was investigated. GES-1(P174E) and GES-5(P174E) mutants, selected by site saturation mutagenesis, were purified and kinetically characterized. In comparison with GES-1 and GES-5 wild-type enzymes, GES-1(P174E) and GES-5(P174E) mutants exhibited lower kcat and kcat/Km values for cephalosporins and penicillins. Concerning carbapenems, GES-1(P174E) shared higher kcat values but lower Km values than those calculated for GES-1. The GES-1(P174E) and GES-5(P174E) mutants showed high hydrolytic efficiency for imipenem, with kcat/Km values 100- and 660-fold higher, respectively, than those of GES-1. Clavulanic acid and tazobactam are good inhibitors for both GES-1(P174E) and GES-5(P174E) Molecular dynamic (MD) simulations carried out for GES-1, GES-5, GES-1(P174E), and GES-5(P174E) complexed with imipenem and meropenem have shown that mutation at position 174 induces a drastic increase of enzyme flexibility, in particular in the Omega-loop. The circular dichroism (CD) spectroscopy spectra of the four enzymes indicate that the P174E substitution in GES-1 and GES-5 does not affect the secondary structural content of the enzymes. [less ▲]

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See detailProtein-polysaccharide complexes, a tool for protein delivery in CaCO3 microparticles
Ramalapa, Bathabile ULiege; Crasson, Oscar ULiege; Vandevenne, Marylène ULiege et al

Poster (2017, September 06)

INTRODUCTION The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated. The success of these carriers has been driven by the ionic interactions ... [more ▼]

INTRODUCTION The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated. The success of these carriers has been driven by the ionic interactions between proteins and particles making the encapsulation of proteins highly dependent on the pH of reaction solutions and the isoelectric point of the protein.1 This poses a great limitation on the successful loading of proteins into microparticles. In this study, we explored the use of polysaccharide-protein interactions to strongly enhance the encapsulation of proteins in CaCO3 microparticles. EXPERIMENTAL METHODS Previously, Vandevenne and colleagues2 inserted a human chitin binding domain (ChBD) that has intrinsic affinity for hyaluronic acid (HA) into β-lactamase (BlaP). This generated chimeric protein, named BlaPChBD, was shown to be fully bifunctional. In this study this hybrid protein (BlapChBD) was associated to HA and successfully loaded into CaCO3 microparticles using super critical CO2 technology aided by the templating effect of HA on CaCO3. Furthermore, thrombin cleavage sites were engineered on both sides of the inserted ChBD in the chimeric BlaP so that release of the protein from the microparticles could be easily achieved by protease cleavage. The microparticles were characterised for size, surface charge, poly-morphism and protein loading and in-vitro release assays were performed. RESULTS AND DISCUSSION The presence of ChBD inserted into the β-lactamase increased the encapsulation of the protein by 6-fold when complexed with HA (Fig. 1). In addition, we also showed that the encapsulated BlaP remains stable during this process using kinetic reaction of β-lactam hydrolysis. Our data showed that vaterite CaCO3 microparticles of sizes ranging between 4 and 7 µm were produced. We were also able to demonstrate that thrombin cleavage increased the release of the protein from the microparticles within 36 hours from <25% to 87% (Fig. 2). In conclusion, the presence of ChBD successfully improved the encapsulation yield of the protein while retaining up to 81% of its activity. CONCLUSION Protein-polysaccharide complexation demonstrates an excellent approach for the delivery of sensitive biomacromolecules which can otherwise be complicated due to electrostatic hindrances. Future prospects include using the methods we have developed for encapsulation of therapeutic proteins and using calcium carbonate as a carrier and scaffold in bone regeneration for example. [less ▲]

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See detailProtein–polysaccharide complexes for enhanced protein delivery in hyaluronic acid templated calcium carbonate microparticles
Ramalapa, Bathabile ULiege; Crasson, Oscar ULiege; Vandevenne, Marylène ULiege et al

in Journal of Materials Chemistry B (2017), 5

The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated. The success of these carriers is driven by ionic interactions between the encapsulated ... [more ▼]

The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated. The success of these carriers is driven by ionic interactions between the encapsulated proteins and the particles. This poses a great limitation on the successful loading of proteins that have no ionic affinity to CaCO3. In this study, we explored the use of polysaccharide–protein interactions to strongly enhance the encapsulation of proteins in CaCO3 microparticles. Previously, Vandevenne and colleagues inserted a human chitin binding domain (ChBD) that has intrinsic affinity for hyaluronic acid (HA) into a β-lactamase (BlaP). This generated chimeric protein, named BlaPChBD, was shown to be fully bifunctional. In this study we showed that this hybrid protein can associate with HA and be successfully loaded into vaterite CaCO3 microparticles using supercritical CO2 (ScCO2) technology aided by the templating effect of HA on CaCO3. The presence of ChBD inserted into BlaP increased the encapsulation of the protein by 6-fold when complexed with HA. Furthermore, thrombin cleavage sites were engineered on both sides of the inserted ChBD in the chimeric BlaP to achieve release of the protein from the microparticles by protease cleavage. Our results showed that thrombin cleavage increased the release of the protein from the microparticles within 36 hours from <20% to 87%. In conclusion, the presence of ChBD successfully improved the encapsulation yield of the protein while retaining up to 82% of its activity and efficient release of the protein from the microparticles was achieved by protease cleavage. [less ▲]

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See detailFunctional and structural characterisation of the Arabidopsis thaliana HMA4 protein
Lekeux, Gilles ULiege; Laurent, Clémentine; Xiao, Zhiguang et al

Poster (2017, July 24)

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See detailProtein-polysaccharide complexes for improved protein delivery within CaCO3 microparticles
Ramalapa, Bathabile ULiege; Crasson, Oscar ULiege; Vandevenne, Marylène ULiege et al

Conference (2017, July 17)

Introduction: The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated due to accessibility, low cost, safety, pH-sensitive properties, high surface ... [more ▼]

Introduction: The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated due to accessibility, low cost, safety, pH-sensitive properties, high surface area and high porosity. The success of these carriers has been driven by the ionic interactions between proteins and particles making the encapsulation of proteins highly dependent on the pH of reaction solutions and the isoelectric point of the protein. This poses a great limitation on the successful loading of proteins into microparticles. In this study we explored the use of polysaccharide-protein complexes to enhance the encapsulation of otherwise poorly encapsulated proteins in CaCO3 microparticles. Methods: A chitin binding domain (ChBD) was inserted on the protein β-lactamase to form a chimeric protein. A protein-polysaccharide complex was formed between the protein and hyaluronic acid (HA) owing to the intrinsic affinity of the ChBD to HA. The chimeric protein was then loaded into CaCO3 microparticles using super critical CO2 technology aided by the templating effect of HA on CaCO3. The microparticles were characterised for size, surface charge, polymorphism and protein loading. Bioactivity and stability of the encapsulated β-lactamase was characterised by kinetic reaction with nitrocefin. A thrombin cleavage site was inserted onto the gene sequence of the protein to achieve release of the protein from the microparticles by proteases mediation using thrombin. Results: Vaterite CaCO3 microparticles of sizes ranging between 6 and 8 µm were produced. The presence of the ChBD on the β-lactamase increased the encapsulation of the protein by 6 fold when complexed with HA. Thrombin mediated release increased the release of the protein from the microparticles within 36 hours from <25% to 87%. The protein-polysaccharide complex demonstrated success in encapsulation of the protein while retaining up to 81% activity of the protein and allowing controlled release by proteases. Conclusion: Protein-polysaccharide complexation demonstrates an excellent approach for the delivery of sensitive biomacromolecules which can otherwise be complicated due to electrostatic hindrances. Future prospects include using the methods we have developed for encapsulation of therapeutic proteins and using calcium carbonate as a carrier and scaffold for example in bone regeneration. [less ▲]

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See detailCrystal Structure and Kinetic Analysis of the Class B3 Di-Zinc Metallo-β-Lactamase LRA-12 from an Alaskan Soil Metagenome
Rodríguez, María Margarita; Herman, Raphaël ULiege; Ghiglione, Barbara et al

in PLoS ONE (2017), 12(7), 0182043

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See detailIncreased antimicrobial resistance in a novel CMY-54 AmpC-type enzyme with a GluLeu217-218 insertion in the Ω-loop
Pérez-Llarena, Francisco José; Vázquez-Ucha, Juan Carlos; Kerff, Frédéric ULiege et al

in Microbial Drug Resistance: Mechanism, Epidemiology, and Disease (2017)

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See detailStudy of the outer membrane permeability of Pseudomonas aeruginosa to ß-lactam antibiotics
Amisano, Francesco ULiege; Silvestri, Mauro ULiege; Mercuri, Paola ULiege et al

Poster (2017, June 17)

Background The resistance of Gram negative bacteria toward β-lactam antibiotics is caused by the interplay between four independent factors: i) the alteration of the sensitivity of the target enzymes, the ... [more ▼]

Background The resistance of Gram negative bacteria toward β-lactam antibiotics is caused by the interplay between four independent factors: i) the alteration of the sensitivity of the target enzymes, the penicillin-binding proteins, ii) the properties and concentration of the periplasmic β-lactamases, iii) the permeability of the outer membrane, iv) the efficiency of the active efflux system. On this basis, Zimmermann and Rosselet [1] proposed a model yelding a quantitative prediction of the MICs for gram-negative bacteria which was successfully applied to Escherichia coli and Enterobacter cloacae. This model seems to be less suitable in Pseudomonas aeruginosa due to its low outer membrane permeability which is mostly influenced by both a remarkable reduction of functional porins expression and an over-expression of efflux systems [2]. This decreased permeability causes difficulties in obtaining permeability coefficient direct measures. Moreover, the few published coefficients for P. aeruginosa are highly variable. For this purpose, BlaR-CTD, the C-terminal domain of a highly sensitive penicillin binding protein from Bacillus licheniformis, expressed in the periplasmic space, has been used in order to directly determinate of the concentrations of different β-lactams in this cell compartment and, consequently to obtain reliable measures of the permeability coefficient [3]. Methods P. aeruginosa PAO1 cells were incubated with different β-lactams, whose penetration into the periplasm is rapidly followed by the formation of a stable complex with BlaR-CTD. This latter was quantified in cells lysate by densitometric analysis, countermarking the free BlaR-CTD with a fluorescent β-lactam. The excess of the antibiotics will be hydrolysed by the addition of a class B β-lactamase. We used the same protocol for P. aeruginosa TNP004 [4], a PAO1 strain with a selective deletion of OprD porin, in order to study the influence of this single mutation for the antibiotic permeability. Results By the approach described above we determined the permeability coefficients of the external membrane of P. aeruginosa for different antibiotics belonging to the penicillin, cephalosporin and carbapenem sub-families. The comparison with the porin mutant strain showed similar coefficients for all the antibiotic tested except, as expected, for Imipenem Conclusion This work allowed a preliminary characterization of antibiotic permeability in P. aeruginosa which was poorly studied until now. Furthermore, we could apply this method to correlate the permeability with the role of porin deletions and/or efflux pumps overexpression in antibiotic resistant strains of clinical relevance. References 1 Zimmermann, W. and A. Rosselet. 1977. Function of the outer membrane of Escherichia coli as a permeability barrier to beta-lactam antibiotics. Antimicrob. Agents Chemother. 12:368–372. 2 Livermore D. M., and K. W. M. Davy. 1991. Invalidity for Pseudomonas aeruginosa of an accepted model of bacterial permeability to β-lactam antibiotics. Antimicrob. Agents Chemother. 35:916-921. 3 Lakaye B., Dubus A., Joris B., and J.M. Frère. 2002. Method for estimation of low outer membrane permeability to β-lactam antibiotics. Antimicrob. Agents Chemother. 46:2901-2907. 4 Yoneyama H., Yamano Y and T. Nakae. 1995 Role of porins in the antibiotic susceptibility of Pseudomonas aeruginosa: construction of mutants with deletions in the multiple porin genes. Biochem Biophys Res Commun. 213:88-95. [less ▲]

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See detailCharacterisation of the sub-class B2 metallo-β-lactamase of Yersinia mollaretii Wauters
Mercuri, Paola ULiege; Blétard, Sylvie; Kerff, Frédéric ULiege et al

Poster (2017, June)

Background. The sub class B2 metallo-β- lactamases are Zn2+-dependent enzymes that efficiently hydrolyzes only carbapenem antibiotics. We study the metallo-β-lactamase (MBL) produced by a soil bacteria ... [more ▼]

Background. The sub class B2 metallo-β- lactamases are Zn2+-dependent enzymes that efficiently hydrolyzes only carbapenem antibiotics. We study the metallo-β-lactamase (MBL) produced by a soil bacteria named Yersinia mollaretii Wauters DMS 18520 (1). The enzyme exhibits two potential zinc-binding sites, the conserved residues Zn1 site (N116-H118-H196), and Zn2 site (D120-C221-H263), tipycals of the sub-class B2 metallo-β- lactamases. Compared to CphA, we found the similar α3 helix located near the active-site and three prolines of the "rich prolines loop" were conserved (2). Materials&methods. The gene coding for the metallo-β-lactamase was isolated by PCR from the genomic DNA as template. The MBL was produced in E. coli Rosetta (DE3) pLysS with the help of overexpression vector as pET26b. The enzyme was produced in TB medium at 18°C in presence of 100 µM IPTG. The metallo-β- lactamases was purified by three steps, an ion exchange chromatography (Sepharose SP-HP column), a Pentadentate Chelator (PDC) Zn2+ column, followed by a molecular sieve column (Superdex 75). pH dependence of activity was study. The enzymatic profile was studied on a representative numbers of carbapenems and measured in the presence of increasing concentrations of zinc. The influence of chelating agents on the β-lactamase activity was also assessed. Results. The MBL from Yersinia mollaretii was overproduced in E. coli Rosetta (DE3) pLysS and purified in three chromatography steps as described in Materials & Methods. The active fractions were pooled and concentrated. The enzyme showed the best activity in MES buffer at pH 6.0. The steady state kinetic parameters were determined for a representative set of carbapenems antibiotics. We showed that Imipenem was the best substrate among the tested substrates. Conclusion. Compared to CphA the new sub class B2 MBL shows a narrow antibiotics profile, a reduced susceptibility toward high zinc concentration and to zinc chelators. [less ▲]

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See detailNDM-1 could still evolve: analysis of a random mutagenesis in position 123
Marcoccia, Francesca; Melchiorre, C; Robert, Charly ULiege et al

Poster (2017, June)

Detailed reference viewed: 30 (10 ULiège)
See detailImproved encapsulation of proteins within calcium carbonate microparticles by means of protein-polysaccharide complexes
Ramalapa, Bathabile ULiege; Crasson, Oscar ULiege; Vandevenne, Marylène ULiege et al

Poster (2017, May 30)

The controlled delivery of proteins within calcium carbonate particles is currently widely investigated. The success of these carriers has been driven by the ionic interactions between proteins and ... [more ▼]

The controlled delivery of proteins within calcium carbonate particles is currently widely investigated. The success of these carriers has been driven by the ionic interactions between proteins and particles making the encapsulation of proteins highly dependent on the pH of reaction solutions and the isoelectric point of the protein. This poses a great limitation on the successful loading of proteins into microparticles. In this study we explored the use of polysaccharide-protein complexes to enhance the encapsulation of otherwise poorly encapsulated proteins in CaCO3 microparticles. A chitin binding domain (ChBD) was inserted on the protein β-lactamase to form a chimeric protein. A protein-polysaccharide complex was formed between the protein and hyaluronic acid (HA) owing to the intrinsic affinity of the ChBD to HA. The chimeric protein was then loaded into calcium carbonate (CaCO3) microparticles using super critical CO2 technology aided by the templating effect of HA on CaCO3. The microparticles were characterised for size, surface charge, poly-morphism and protein loading. Biochemical stability of the encapsulated β-lactamase was characterized by kinetic reaction with nitrocefin. The presence of the ChBD on the β-lactamase increased the encapsulation of the protein by 6 fold when complexed with HA. Thrombin mediated release increased the release of the protein from the microparticles within 36 hours from <25% to 87%. The protein-polysaccharide complex proved successful in enhancing the encapsulation of protein while retaining up to 81% activity and allowing controlled release of the protein by proteases. [less ▲]

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See detailHuman chitotriosidase: Catalytic domain or carbohydrate binding module, who's leading HCHT's biological function
Crasson, Oscar; Courtade, Gaston; Léonard, Raphaël ULiege et al

in Scientific Reports (2017), 7(1),

Detailed reference viewed: 36 (8 ULiège)