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Binding and Elution Properties of Mixed-Mode Chromatography and Its Applications for Purification
Current protein & peptide science, 2019, 20, 3-3
2 […]
SANTARELLI, Xavier; CABANNE, Charlotte;
Mixed Mode Chromatography: A Novel Way Toward New Selectivity
Current protein & peptide science, 2019, 20, 14-21
Mixed mode chromatography offers a diversity of ligands, each providing a new selectivity. This allows the design of novel purification processes with reduced column steps. Structure of ligands is based on both hydrophobic and ionic groups. Thanks to its salt tolerance, crude extracts or post-IEX samples can be loaded directly without conditioning. The selectivity could be enhanced by modulating elution parameters or by using additives. More importantly, mixed mode chromatography could be as effective as affinity chromatography for mAb purification processes. Mixed mode chromatography opens the way to short and economical processes.
3 […]
CABANNE, Charlotte; SANTARELLI, Xavier;
Mixed Mode Chromatography, Complex Development for Large Opportunities
Current protein & peptide science, 2019, 20, 22-27
Mixed mode chromatography resins with salt tolerance, large design space and orthogonal selectivity requires a slightly more complex development than traditional resins. It is important to screen several ligands and several binding and elution conditions. This allows taking full advantage of these resins. High-Throughput Screening (HTS) for Process Development should be done with the help of Design of Experiment (DoE). It could be performed in filter plates or Robocolumns, and assisted by liquid handling automated workstation. Modeling of the results allows the choice of optimal parameters that can then be validated and scaled up. All this leads to a better knowledge and robustness of the purification step.
4 […]
OUK, Tan Sothea;
Synthesis, characterization, and antibacterial activities of a new lignocellulosic material carrying aryl triazole moiety
Polymers for Advanced Technologies, 2019, 30, 344-350
Contamination of surfaces by bacteria and the emergence of antimicrobial resistant strains are very worrying issues. One of ways to limit the bacterial proliferation is to develop antimicrobial materials. We reported herein the development of new antimicrobial support by grafting different aryl azide onto propargylated Kraft Pulp, using the copper (I)-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction, forming 1,2,3-triazole aryl. These novel materials have been investigated for their antibacterial properties against Escherichia coli and Staphylococcus aureus. The developed materials acquire its antimicrobial potential only after grafting via triazole link. Depending on the substituent of the grafted aryl, the elaborated materials showed a bacteriostatic or even bactericidal activity.
5 […]
Identification and role of Opuntia ficus indica constituents in the flocculation mechanism of colloidal solutions
Separation and Purification Technology, 2019, 209, 892-899
Opuntia ficus indica has been identified for its bioflocculant properties in water treatment; however, its underlying mechanism and active compounds have not been clearly identified. Flocculent molecules of cactus solid material (CSM) under alkaline conditions were extracted at pH 10 and then precipitated under neutral conditions (pH 7). The precipitate was fractionated by ultrafiltration systems and analyzed using inverted phase chromatography and enzymatic treatments. This approach revealed that quercetin and starch constitute the active agents found in the fractionated parts at <= 3,000 and >= 10,000 Da, respectively. The use of quercetin or (potato) starch alone at 18 mg/L yielded 72% +/- 2% and 54% +/- 3% of turbidity removal, respectively. With a combination of both these components, a higher flocculation activity (84% +/- 2%) could be obtained. From these experimental results, a flocculation model based on identified active constituents is being proposed in order to improve process knowledge.
6 […]
SIMON, Anne; FAURE, Chrystel;
Cu2+-loaded cellulose micro-beads applied to the direct patterning of metallic surfaces using a fast and convenient process
Carbohydrate Polymers, 2019, 207, 492-501
In this paper, we propose both a new application for cellulose micro-beads and a new concept in colloidal lithography to directly deposit and template a metal from ions transported by the organized colloidal particles, using the colloidal particles themselves. To do so, 5 mu m-sized cellulose micro-beads (C mu Bs) were first surface-functionalized by trimellitic anhydride to introduce carboxylate ligands before decorating them with Cu2+ ions by complexation of the carboxylate groups with a CuCl2 solution. The Cu2+-loaded C mu Bs, dispersed in an aqueous phase, were organized in compact monolayer at the vicinity of a planar electrode. The release of cupric ions and subsequent copper deposition were triggered by an electric field delivered by a tension generator. 2D non-close-packing arrays of copper dots assemblies displaying hexagonal symmetry were generated below or around the micro-beads - depending on the ions concentration in the aqueous phase - leading respectively to copper dots deposited circularly or concentrated in rings. The Cu2+-loaded cellulose beads allowed the covering of 2 cm(2)-surfaces by copper patterns in less than 45 min, using an easy and cheap process.
7 […]
JOSEPH, Cécile; SAVOIRE, Raphaelle; MONTEIL, Julien; LEAL-CALDERON, Fernando; FAURE, Chrystel;
O/W Pickering emulsions stabilized by cocoa powder: Role of the emulsification process and of composition parameters
Food research international (Ottawa, Ont.), 2019, 116, 755-766
We fabricated oil-in-water emulsions stabilized by delipidated commercial cocoa powder. The emulsions were characterized in terms of droplets and particles size distribution and interfacial coverage by cocoa powder by developing methods to separate droplets from adsorbed and unadsorbed cocoa particles. Three different processes were compared for their ability to produce fine and stable emulsions: rotor/stator turbulent mixing, sonication and microfluidization. Among those techniques, microfluidization was the most performing one. In this case, micron-sized emulsions with narrow size distributions could be obtained with >90 wt% of the powder insoluble material anchored to the interfaces, and they were still stable after 90 days. It was demonstrated that the mixing process did not generate finer cocoa particles but provoked disentanglement of the large primary particles, providing them an open, expanded structure that facilitated emulsification. It was also shown that the finer insoluble fraction of the powder and the soluble fraction had no significant impact on emulsification and on kinetic stability. In the poor particles regime, the oil-water interfacial area varied linearly with the amount of adsorbed powder, suggesting that the final droplet size was controlled by the so-called limited coalescence process, as already observed in conventional Pickering emulsions stabilized by spherical solid particles.
8 […]
HARTE, Etienne; ALVES, Isabel; ELEZGARAY, Juan;
Thickness determination in anisotropic media with plasmon waveguide resonance imaging
Opt Express, 2019, 27, 3264-3275
This paper describes a simple procedure to determine the local thickness of a thin anisotropic layer. It also discriminates between isotropic and anisotropic regions, provided a smoothness hypothesis on the refractive index distribution is satisfied. The procedure is based on the analysis of surface plasmon resonance (SPR) data acquired in an imaging mode. The general arrangement of the setup is the Kretschmann configuration. We show, on an azobenzene modified polymer layer, good agreement between atomic force microscopy and optical measurements of thickness variation.
9 […]
SALVADOR, Dimitri; GLAVIER, Marie; TAVEAU, Jean-Christophe; LECOMTE, Sophie; LAMBERT, Olivier;
Minimal nanodisc without exogenous lipids for stabilizing membrane proteins in detergent-free buffer
Biochimica et biophysica acta. Biomembranes, 2019, 1861, 852-860
Membrane protein stabilization after detergent solubilization presents drawbacks for structural and biophysical studies, in particular that of a reduced stability in detergent micelles. Therefore, alternative methods are required for efficient stabilization. Lipid nanodisc made with the membrane scaffold protein MSP is a valuable system but requires a fine optimization of the lipid to protein ratio. We present here the use of the scaffold protein MSP without added lipids as a minimal system to stabilize membrane proteins. We show that this method is applicable to alpha-helical and beta-strands transmembrane proteins. This method allowed cryo-electron microscopy structural study of the bacterial transporter MexB. A protein quantification indicates that MexB is stabilized by two MSP proteins. This simplified and efficient method proposes a new advance in harnessing the MSP potential to stabilize membrane proteins.
10 […]
Metabolomics and proteomics identify the toxic form and the associated cellular binding targets of the anti-proliferative drug AICAR
The Journal of biological chemistry, 2019, 294, 805-815
5-Aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR, or acadesine) is a precursor of the monophosphate derivative 5-amino-4-imidazole carboxamide ribonucleoside 5'-phosphate (ZMP), an intermediate in de novo purine biosynthesis. AICAR proved to have promising anti-proliferative properties, although the molecular basis of its toxicity is poorly understood. To exert cytotoxicity, AICAR needs to be metabolized, but the AICAR-derived toxic metabolite was not identified. Here, we show that ZMP is the major toxic derivative of AICAR in yeast and establish that its metabolization to succinyl-ZMP, ZDP, or ZTP (di- and triphosphate derivatives of AICAR) strongly reduced its toxicity. Affinity chromatography identified 74 ZMP-binding proteins, including 41 that were found neither as AMP nor as AICAR or succinyl-ZMP binders. Overexpression of karyopherin-beta Kap123, one of the ZMP-specific binders, partially rescued AICAR toxicity. Quantitative proteomic analyses revealed 57 proteins significantly less abundant on nuclei-enriched fractions from AICAR-fed cells, this effect being compensated by overexpression of KAP123 for 15 of them. These results reveal nuclear protein trafficking as a function affected by AICAR.
11 […]
VALLADE, Maëlle; JEWGINSKI, Michal; BURATTO, Jérémie; BATHANY, Katell; SCHMITTER, Jean-Marie; STUPFEL, Marine; GODDE, Frédéric; HUC, Ivan;
Assessing Interactions between Helical Aromatic Oligoamide Foldamers and Protein Surfaces: A Tethering Approach
Bioconjugate Chemistry, 2019, 30, 54-62
Helically folded aromatic foldamers may constitute suitable candidates for the ab initio design of ligands for protein surfaces. As preliminary steps toward the exploration of this hypothesis, a tethering approach was developed to detect interactions between a protein and a foldamer by confining the former at the surface of the latter. Cysteine mutants of two therapeutically relevant enzymes, CypA and IL4, were produced. Two series of ten foldamers were synthesized bearing different proteinogenic side chains and either a long or a short linker functionalized with an activated disulfide. Disulfide exchange between the mutated cysteines and the activated disulfides yielded 20 foldamer-IL4 and 20 foldamer-CypA adducts. Effectiveness of the reaction was demonstrated by LC-MS, by MS analysis after proteolytic digestion, and by 2D NMR. Circular dichroism then revealed diastereoselective interactions between the proteins and the foldamers confined at their surface which resulted in a preferred handedness of the foldamer helix. Helix sense bias occurred sometimes with both the short and the long linkers and sometimes with only one of them. In a few cases, helix handedness preference is found to be close to quantitative. These cases constitute valid candidates for structural elucidation of the interactions involved.
12 […]
HASTOY, Benoit; SCOTTI, Pier; MILOCHAU, Alexandra; BATHANY, Katell; DESBAT, Bernard; CASTANO, Sabine; ODA, Reiko; LANG, Jochen;
The transmembrane domain of the SNARE protein VAMP2 is highly sensitive to its lipid environment
Biochimica et biophysica acta. Biomembranes, 2019, 1861, 670-676
Neurotransmitter and hormone exocytosis depends on SNARE protein transmembrane domains and membrane lipids but their interplay is poorly understood. We investigated the interaction of the structure of VAMP2, a vesicular transmembrane SNARE protein, and membrane lipid composition by infrared spectroscopy using either the wild-type transmembrane domain (TMD), VAMP2TM22, or a peptide mutated at the central residues G100/C103 (VAMP2TM22VV) previously identified by us as being critical for exocytosis. Our data show that the structure of VAMP2TM22, in terms of alpha-helices and beta-sheets is strongly influenced by peptide/lipid ratios, by lipid species including cholesterol and by membrane surface charges. Differences observed in acyl chain alignments further underscore the role of the two central small amino acid residues G100/C103 within the transmembrane domain during lipid rearrangements in membrane fusion. Copyright © 2018 Elsevier B.V. All rights reserved.
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Avidity-driven polarity establishment via multivalent lipid-GTPase module interactions
The EMBO journal, 2019, 38,
While Rho GTPases are indispensible regulators of cellular polarity, the mechanisms underlying their anisotropic activation at membranes have been elusive. Using the budding yeast Cdc42 GTPase module, which includes a guanine nucleotide exchange factor (GEF) Cdc24 and the scaffold Bem1, we find that avidity generated via multivalent anionic lipid interactions is a critical mechanistic constituent of polarity establishment. We identify basic cluster (BC) motifs in Bem1 that drive the interaction of the scaffold-GEF complex with anionic lipids at the cell pole. This interaction appears to influence lipid acyl chain ordering, thus regulating membrane rigidity and feedback between Cdc42 and the membrane environment. Sequential mutation of the Bem1 BC motifs, PX domain, and the PH domain of Cdc24 lead to a progressive loss of cellular polarity stemming from defective Cdc42 nanoclustering on the plasma membrane and perturbed signaling. Our work demonstrates the importance of avidity via multivalent anionic lipid interactions in the spatial control of GTPase activation.
14 […]
Molecular mechanisms for the destabilization of model membranes by islet amyloid polypeptide
Biophysical chemistry, 2019, 245, 34-40
Misfolding of human islet amyloid polypeptide (hIAPP) into insoluble aggregates is associated with Type 2 diabetes. It has been suggested that hIAPP toxicity may be due to its accumulation in pancreatic islets, causing membrane disruption and cell permeabilization, however the molecular basis underlying its lipid association are still unclear. Here, we combine solid-state NMR, fluorescence and bright field microscopy to investigate hIAPP - lipid membrane interactions. Real-time microscopy highlights a time-dependent penetration of hIAPP oligomers toward the most buried layers of the lipid vesicles until the membrane disrupts. Deuterium NMR was conducted on liposomes at different hIAPP concentration to probe lipid internal order and thermotropism. The gel-to-fluid phase transition of the lipids is decreased by the presence of hIAPP, and site-specific analysis of the order parameter showed a significant increase of lipid order for the first eight positions of the acyl chain, suggesting a partial insertion of the peptide inside the bilayer. These results offer experimental insight into the membrane destabilization of hIAPP on model membrane vesicles.
15 […]
GRELARD, Axelle; DUFOURC, Erick;
Heat-Triggered Crystallization of Liquid Crystalline Macrocycles Allowing for Conductance Switching through Hysteretic Thermal Phase Transitions
Chemistry-an Asian Journal, 2019, 14, 141-148
A polymesomorphic thermal phase-transition of a macrocyclic amphiphile consisting of aromatic groups and oligoethylene glycol (OEG) chains is reported. The macrocyclic amphiphile exists in a highly-ordered liquid crystal (LC) phase at room temperature. Upon heating, this macrocycle shows phase-transition from columnar-lamellar to nematic LC phases followed by crystallization before melting. Spectroscopic studies suggest that the thermally induced crystallization is triggered by a conformational change at the OEG chains. Interestingly, while the macrocycle returns to the columnar-lamellar phase after cooling from the isotropic liquid, it retains the crystallinity after cooling from the thermally-induced crystal. Thanks to this bistability, conductance switching was successfully demonstrated. A different macrocyclic amphiphile also shows an analogous phase-transition behavior, suggesting that this molecular design is universal for developing switchable and memorizable materials, by means of hysteretic phase-transition processes.
16 […]
MOLINARI, Michael;
Real Time and Quantitative Imaging of Lignocellulosic Films Hydrolysis by Atomic Force Microscopy Reveals Lignin Recalcitrance at Nanoscale
Biomacromolecules, 2019, 20, 515-527
Lignocellulosic biomass is considered as a sustainable source of energy and chemicals, but its recalcitrance to bioconversion still limits its use. In this paper, a strategy based on two aspects was developed to improve our knowledge on the lignin recalcitrance to enzymatic hydrolysis. First, lignocellulosic films of cellulose nanofibrils (CNFs) with increasing content of lignin (up to 40%) were prepared. Thanks to in situ real time Atomic Force Microscopy (AFM) measurements during the hydrolysis and by comparison with biochemical assays, the use of such films allows to fully assess the importance of the lignin content and of the arrangement between CNFs and lignin on the hydrolysis efficiency. In a second time, contrary to other studies by AFM which only followed a specific structure during enzymatic processes mostly on simple systems (CNFs or cellulose nanocrystals), a quantitative analysis of in-situ time-lapse measurements was developed. It enables to accurately address lignocellulosic biomass recalcitrance mechanisms mediated by lignin at nanoscale. Such analysis could pave the way for the use of a quantitative criteria to visualize in situ deconstruction of complex lignocellulosic substrates. Coupling the use of lignocellulosic films and dynamical AFM quantitative analysis to follow the evolution of the structure at nanoscale might lead to an effective targeting of new promising bioconversion strategies.
17 […]
URDACI, Maria;
(EN) Probiotic Strains for Treating and/or Preventing Diarrhea
, 2019, 0,
The present invention relates to a method of selecting or identifying probiotic strains capable of acting on the absorption of water in the colon, and use thereof as medicinal products in the treatment and/or prevention of diarrhea. The invention relates in particular to the strain of Bacillus subtilis CU1 for use in the treatment and/or prevention of diarrhea.
18 […]
, 2019, 0,
(EN) The invention concerns a method for simultaneously extracting at least one lipophilic molecule and at least one polyphenolic compound from Pseudotsuga menziesii, comprising at least one step of extraction by percolation diffusion of a pressurised solvent in a fixed bed reactor carried out from a raw material derived from Pseudotsuga menziesii. The invention also concerns the concentrate obtained during the implementation of the method, and the extracts obtained and the uses of same. (FR) L'objet de l'invention est un procédé d'extraction simultanée d'au moins une molécule lipophile et d'au moins un composé polyphénolique de Pseudotsuga menziesii, comprenant au moins une étape d'extraction par percolation diffusion d'un solvant sous pression en réacteur à lit fixe réalisée à partir d'une matière première dérivée de Pseudotsuga menziesii. L'invention vise aussi le concentrât obtenu pendant la mise en œuvre du procédé, ainsi que les extraits obtenus et leurs utilisations.
19 […]
FAURE, Chrystel; JOSEPH, Cécile; LEAL-CALDERON, Fernando; CANSELL, Maud;
, 2019, 0,
(EN) The present invention relates to a method for producing a Pickering emulsion, comprising the steps of: a) producing a plant powder of at least one oleaginous species; b) adding said plant powder, either to an aqueous phase to obtain a suspension (S) or to an oil phase to obtain a suspension (S'), followed by incorporating either oil into suspension (S) to obtain an oil-in-water type emulsion, or water into suspension (S') to obtain a water-in-oil type emulsion; and c) stirring the emulsion that was obtained at the end of the previous step. (FR) La présente invention concerne un procédé de préparation d'une émulsion de Pickering comprenant les étapes suivantes : a) la préparation d'une poudre végétale d'au moins une espèce oléagineuse; b) l'addition de ladite poudre végétale - soit dans une phase aqueuse pour obtenir une suspension (S), - soit dans une phase huileuse pour obtenir une suspension (S'), suivie de l'incorporation - soit d'huile dans la suspension (S) pour obtenir une émulsion de type huile- dans-eau, - soit d'eau dans la suspension (S') pour obtenir une émulsion de type eau- dans-huile; et c) l'agitation de l'émulsion obtenue à l'issue de l'étape précédente.
20 […]
FAURE, Chrystel; JOSEPH, Cécile; LEAL-CALDERON, Fernando; CANSELL, Maud;
, 2019, 0,
(EN) The present invention relates to a method of producing a dry emulsion, involving a step of drying an oil-in-water Pickering emulsion that contains particles of plant powder. The invention also relates to the dry emulsions which can be obtained by following said method. (FR) La présente invention concerne un procédé de préparation d'une émulsion sèche, comprenant une étape de séchage d'une émulsion de Pickering huile-dans- eau contenant des particules de poudre végétale. Elle concerne également les émulsions sèches susceptibles d'être obtenues selon ledit procédé.
21 […]
GUICHARD, Gilles; FREMAUX, Juliette; VENIN, Claire;
, 2019, 0,
The present disclosure relates to a pro-drug peptide, or a salt thereof, having improvement for at least one biological property relative to a parent peptide or peptidomimetic, wherein the biological property is selected from the group consisting of therapeutic index, stability, solubility, toxicity, adsorption, and pre-systemic metabolism. The pro-drug peptide comprising the following structure: Z-pep, wherein: pep is the parent peptide or peptidomimetic; Z is a sequence of n amino acids, Z is cleaved in vivo releasing pep; n≥2 amino acids. The present disclosure also relates to methods of making and using the pro-drug peptide of the present disclosure. For example, the present disclosure describes a pro-drug peptide that may be used to prevent, treat, or ameliorate at least one symptom of hypoglycemia or a hypoglycemia-related disease or disorder.
22 […]
PEDROSA, Catarina; KHAN, Irfan; DURRIEU, Marie-Christine;
Controlled Nanoscale Topographies for Osteogenic Differentiation of Mesenchymal Stem Cells
Acs Applied Materials & Interfaces, 2019, 11, 8858-8866
Nanotopography with length scales of the order of extracellular matrix elements offers the possibility of regulating cell behavior. Investigation of the impact of nanotopography on cell response has been limited by the inability to precisely control geometries, especially at high spatial resolutions and across practically large areas. In this paper, we demonstrate well-controlled and periodic nanopillar arrays of silicon and investigate their impact on osteogenic differentiation of human mesenchymal stem cells (hMSCs). Silicon nanopillar arrays with critical dimensions in the range of 40-200 nm, exhibiting standard deviations below 15% across full wafers, were realized using the self-assembly of block copolymer colloids. Immunofluorescence and quantitative polymerase chain reaction measurements reveal clear dependence of osteogenic differentiation of hMSCs on the diameter and periodicity of the arrays. Further, the differentiation of hMSCs was found to be dependent on the age of the donor. While osteoblastic differentiation was found to be promoted by the pillars with larger diameters and heights independent of donor age, they were found to be different for different spacings. Pillar arrays with smaller pitch promoted differentiation from a young donor, while a larger spacing promoted those of an old donor. These findings can contribute for the development of personalized treatments of bone diseases, namely, novel implant nanostructuring depending on patient age.
23 […]
GERBOD-GIANNONE, Marie-Christine; DALLET, Laurence; NAUDIN, Grégoire; SAHIN, Annelise; POUSSARD, Sylvie; LAMBERT, Olivier;
Involvement of caveolin-1 and CD36 in native LDL endocytosis by endothelial cells
Biochimica et biophysica acta. General subjects, 2019, 1863, 830-838
Atherosclerosis is a lipid disease characterized by accumulation of low density lipoprotein (LDL) in the artery wall. The transport of LDL across the endothelium of coronary artery is an initiating event of atherosclerosis, whose mechanism remains poorly understood. In the last decade, it has been shown that in caveolin-1 (Cav-1) deficient mice, LDL infiltration in aorta wall is decreased and CD36 expression in aortas is down-regulated, leading to regression of atherosclerotic lesions. In the present study, we show that native LDL endocytosis is decreased in endothelial cells deficient in Cav-1 or CD36. We demonstrate that Cav-1 and CD36 interact in caveolae-rich domains by different biochemical approaches. In addition, confocal microscopy reveals some colocalization of Cav-1 with CD36. These findings indicate that caveolae and CD36 are involved in native LDL endocytosis and suggest that CD36 might be a good candidate for the transport of native LDL across the endothelium, an early event in atherosclerosis.
24 […]
WANG, Xiang; GAN, Quan; WICHER, Barbara; FERRAND, Yann; HUC, Ivan;
Directional Threading and Sliding of a Dissymmetrical Foldamer Helix on Dissymmetrical Axles
Angewandte Chemie (International ed. in English), 2019, 58, 4205-4209
We have investigated the self-assembly of a dissymmetrical aromatic oligoamide helix on linear amido-carbamate rods. A dissymmetric sequence bearing two differentiated ends is able to wrap around dissymmetric dumbbell guest molecules. Structural and thermodynamic investigations allowed us to decipher the mode of binding of the helix that can bind specifically to the amide and carbamate groups of the rod. In parallel kinetic studies of threading and sliding of the helix along linear axles were also monitored by 1 H NMR. Results show that threading of a dissymmetrical host can be kinetically biased by the nature of the guest terminus allowing a preferential sense of sliding of the helix. The study presented below further demonstrates the valuable potential of foldaxanes to combine designed molecular recognition patterns with fine control of self-assembly kinetics to conceive complex supramolecular events. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
25 […]
LOMBARDO, Caterina Maria; DOUAT, Céline; GUICHARD, Gilles;
Design and Structure Determination of a Composite Zinc Finger Containing a Nonpeptide Foldamer Helical Domain
Journal of the American Chemical Society, 2019, 141, 2516-2525
A number of foldamer backbones have been described as useful mimics of protein secondary structure elements, enabling for example the design of synthetic oligomers with the ability to engage specific protein surfaces. Synthetic folded backbones can also be used to create artificial proteins in which a folded peptide segment (e.g., an alpha-helix, a loop) is replaced by its unnatural counterpart, with the expectation that the resulting molecule would maintain its ability to fold while manifesting new exploitable features. The similarities in screw sense, pitch, and polarity between peptide alpha-helices and oligourea 2.5-helices suggest that a tertiary structure could be retained when swapping the two backbones in a protein sequence. In the present work, we move a step toward the creation of such composite proteins by replacing the 10-residue long original alpha-helical segment in the Cys2His2 zinc finger 3 of transcription factor Egrl (also known as Zif268) by an oligourea sequence bearing two appropriately spaced imidazole side chains for zinc coordination. We show by spectroscopic techniques and mass spectrometry analysis under native conditions that the ability of the peptide/oligourea hybrid to coordinate the zinc ion is not affected by the foldamer replacement. Moreover, detailed NMR analysis provides evidence that the engineered zinc finger motif adopts a folded structure in which the native beta-sheet arrangement of the peptide region and global arrangement of DNA-binding side chains are preserved. Titration in the presence of the Egrl target DNA sequence supports binding to GC bases as reported for the wild type zinc finger.
26 […]
FREMAUX, Juliette; VENIN, Claire; MAURAN, Laura; GUICHARD, Gilles;
Peptide-oligourea hybrids analogue of GLP-1 with improved action in vivo
Nature Communications, 2019, 10,
Peptides have gained so much attention in the last decade that they are now part of the main strategies, with small molecules and biologics, for developing new medicines. Despite substantial progress, the successful development of peptides as drugs still requires a number of limitations to be addressed, including short in vivo half-lives and poor membrane permeability. Here, we describe the use of oligourea foldamers as tool to improve the pharmaceutical properties of GLP-1, a 31 amino acid peptide hormone involved in metabolism and glycemic control. Our strategy consists in replacing four consecutive amino acids of GLP-1 by three consecutive ureido residues by capitalizing on the structural resemblance of oligourea and alpha-peptide helices. The efficacy of the approach is demonstrated with three GLP-1-oligourea hybrids showing prolonged activity in vivo. Our findings should enable the use of oligoureas in other peptides to improve their pharmaceutical properties and may provide new therapeutic applications.
27 […]
JOBIN, Marie-Lise; ALVES, Isabel;
The Contribution of Differential Scanning Calorimetry for the Study of Peptide/Lipid Interactions
Methods Mol Biol, 2019, 1964, 3-15
Membrane-active peptides include a variety of molecules such as antimicrobial (AMP), cell-penetrating (CPP), viral, and amyloid peptides that are implicated in several pathologies. They constitute important targets because they are either at the basis of novel therapies (drug delivery for CPPs or antimicrobial activity for AMPs) or they are the agents causing these pathologies (viral and amyloid peptides). They all share the common property of interacting with the cellular lipid membrane in their mode of action. Therefore, a better understanding of the peptide/lipid (P/L) interaction is essential to help decipher their mechanism of action. Among the different biophysical methods that can be used to fully characterize P/L interactions, differential scanning calorimetry (DSC) allows determining the peptide effect on the lipid phase transitions, a property that reflects the P/L interaction mode. A general protocol for classical DSC experiments for P/L studies will be provided.
28 […]
POUGET, Emilie; SCALABRE, Antoine;
Adsorption of Proteins on Dual Loaded Silica Nanocapsules
Journal of Physical Chemistry B, 2019, 123, 1708-1717
The design of nanocarriers containing hydrophobic and hydrophilic compounds represents a powerful tool for cocktail delivery. Water-in-oil-in-water emulsions constitute an attractive approach, as they offer dual encapsulation and provide a template for the constitution of a capsule. A limitation in the preparation of nano double emulsions is their instability resulting from high curvature radii. In this work, silica nanocapsules (NCs) stable over several months were synthesized. This was achieved by exploiting a double emulsion in which the oil phase is constituted by a combination of oils presenting several volatilities. The decrease of oil droplet size by evaporation favored the deposition of a silica layer at the nanoscale interface. The release of the payload obtained by drying the capsules was investigated by fluorescence spectroscopy. Understanding the interactions between proteins and nanocapsules is a fundamental point for many biological applications. Nanocapsules were exposed to two model proteins, which were bovine serum albumin (BSA) and lysozyme (Ly). These proteins, presenting differences in charges and size, showed distinctive arrangements onto the nanocapsules. Moreover, we have studied changes in alpha-helix and beta-sheet content, which divulged the interactions between the proteins and the nanocapsules.
29 […]
HARTE, Etienne; ALVES, Isabel; ABDULHALIM, Ibrahim;
Improved Detection of Plasmon Waveguide Resonance Using Diverging Beam, Liquid Crystal Retarder, and Application to Lipid Orientation Determination
Sensors (Basel, Switzerland), 2019, 19,
Plasmon waveguide resonance (PWR) sensors exhibit narrow resonances at the two orthogonal polarizations, transverse electric (TE) and transverse magnetic (TM), which are narrower by almost an order of a magnitude than the standard surface plasmon resonance (SPR), and thus the figure of merit is enhanced. This fact is useful for measuring optical anisotropy of materials on the surface and determining the orientation of molecules with high resolution. Using the diverging beam approach and a liquid crystal retarder, we present experimental results by simultaneous detection of TE and TM polarized resonances as well as using fast higher contrast serial detection with a variable liquid crystal retarder. While simultaneous detection makes the system simpler, a serial one has the advantage of obtaining a larger contrast of the resonances and thus an improved signal-to-noise ratio. Although the sensitivity of the PWR resonances is smaller than the standard SPR, the angular width is much smaller, and thus the figure of merit is improved. When the measurement methodology has a high enough angular resolution, as is the one presented here, the PWR becomes advantageous over other SPR modes. The possibility of carrying out exact numerical simulations for anisotropic molecules using the 4 * 4 matrix approach brings another advantage of the PWR over SPR on the possibility of extracting the orientation of molecules adsorbed to the surface. High sensitivity of the TE and TM signals to the anisotropic molecules orientation is found here, and comparison to the experimental data allowed detection of the orientation of lipids on the sensor surface. The molecular orientations cannot be fully determined from the TM polarization alone as in standard SPR, which underlines the additional advantage of the PWR technique.
30 […]
PLAWINSKI, Laurent; DURRIEU, Marie-Christine;
Remote imaging of single cell 3D morphology with ultrafast coherent phonons and their resonance harmonics
Scientific Reports, 2019, 9,
Cell morphological analysis has long been used in cell biology and physiology for abnormality identification, early cancer detection, and dynamic change analysis under specific environmental stresses. This work reports on the remote mapping of cell 3D morphology with an in-plane resolution limited by optics and an out-of-plane accuracy down to a tenth of the optical wavelength. For this, GHz coherent acoustic phonons and their resonance harmonics were tracked by means of an ultrafast opto-acoustic technique. After illustrating the measurement accuracy with cell-mimetic polymer films we map the 3D morphology of an entire osteosarcoma cell. The resulting image complies with the image obtained by standard atomic force microscopy, and both reveal very close roughness mean values. In addition, while scanning macrophages and monocytes, we demonstrate an enhanced contrast of thickness mapping by taking advantage of the detection of high-frequency resonance harmonics. Illustrations are given with the remote quantitative imaging of the nucleus thickness gradient of migrating monocyte cells.
31 […]
URDACI, Maria;
In vivo screening of multiple bacterial strains identifies Lactobacillus rhamnosus Lb102 and Bifidobacterium animalis ssp. lactis Bf141 as probiotics that improve metabolic disorders in a mouse model of obesity
Faseb Journal, 2019, 33, 4921-4935
Given the growing evidence that gut dysfunction, including changes in gut microbiota composition, plays a critical role in the development of inflammation and metabolic diseases, the identification of novel probiotic bacteria with immunometabolic properties has recently attracted more attention. Herein, bacterial strains were first isolated from dairy products and human feces and then screened in vitro for their immunomodulatory activity. Five selected strains were further analyzed in vivo, using a mouse model of diet-induced obesity. C57BL/6 mice were fed a high-fat high-sucrose diet, in combination with 1 of 3 Lactobacillus strains (Lb38, L. plantarum; L79, L. paracasei/casei; Lb102, L. rhamnosus) or Bifidobacterium strains (Bf26, Bf141, 2 different strains of B. animalis ssp. lactis species) administered for 8 wk at 10(9) colony-forming units/d. Whereas 3 strains showed only modest (Lb38, Bf26) or no (L79) effects, Lb102 and Bf141 reduced diet-induced obesity, visceral fat accretion, and inflammation, concomitant with improvement of glucose tolerance and insulin sensitivity. Further analysis revealed that Lb102 and Bf141 enhanced intestinal integrity markers in association with selective changes in gut microbiota composition. We have thus identified 2 new potential probiotic bacterial strains with immunometabolic properties to alleviate obesity development and associated metabolic disturbances.
32 […]
RENOUARD, Sullivan;
Green Ultrasound Assisted Extraction of trans Rosmarinic Acid from Plectranthus scutellarioides (L.) R.Br. Leaves
Plants-Basel, 2019, 8,
Painted nettle (Plectranthus scutellarioides (L.) R.Br.) is an ornamental plant belonging to Lamiaceae family, native of Asia. Its leaves constitute one of the richest sources of trans-rosmarinic acid, a well-known antioxidant and antimicrobial phenolic compound. These biological activities attract interest from the cosmetic industry and the demand for the development of green sustainable extraction processes. Here, we report on the optimization and validation of an ultrasound-assisted extraction (USAE) method using ethanol as solvent. Following preliminary single factor experiments, the identified limiting extraction parameters (i.e., ultrasound frequency, extraction duration, and ethanol concentration) were further optimized using a full factorial design approach. The method was then validated following the recommendations of the association of analytical communities (AOAC) to ensure the precision and accuracy of the method used to quantify trans-rosmarinic acid. Highest trans-rosmarinic acid content was obtained using pure ethanol as extraction solvent following a 45-minute extraction in an ultrasound bath operating at an ultrasound frequency of 30 kHz. The antioxidant (in vitro radical scavenging activity) and antimicrobial (directed toward Staphylococcus aureus ACTT6538) activities were significantly correlated with the trans-rosmarinic acid concentration of the extract evidencing that these key biological activities were retained following the extraction using this validated method. Under these conditions, 110.8 mg/g DW of trans-rosmarinic acid were extracted from lyophilized P. scutellarioides leaves as starting material evidencing the great potential of this renewable material for cosmetic applications. Comparison to other classical extraction methods evidenced a clear benefit of the present USAE method both in terms of yield and extraction duration.
33 […]
DOUAT, Céline; BORNERIE, Mégane; ANTUNES, Stéphanie; GUICHARD, Gilles;
Hybrid Cell-Penetrating Foldamer with Superior Intracellular Delivery Properties and Serum Stability
Bioconjugate Chemistry, 2019, 30, 1133-1139
Sequence specific molecules with high folding ability (i.e., foldamers) can be used to precisely control the distribution and projection of side chains in space and have recently been introduced as tailored systems for delivering nucleic acids into cells. Designed oligourea sequences with an amphipathic distribution of Arg- and His-type residues were shown to form tight complexes with plasmid DNA, and to effectively promote the release of DNA from the endosomes. Herein, we report the synthesis of new cell-penetrating foldamer sequences in which the foldamer segment is conjugated via a reducible disulfide bond to a ligand that binds cell-surface expressed nucleoproteins with the idea that this system could facilitate both assemblies with nucleic acids and cell entry. This new system was evaluated for delivery of DNA in several cell lines and was found to compare favorably with all comparators tested (DOTAP and b-PEI as well as a number of known cell penetrating peptides) in various cell lines and particularly in culture medium containing up to 50% of serum. These results suggest that this dual molecular platform which is long lasting and noncytotoxic could be of practical use for in vivo applications.
34 […]
ZHANG, Jiarong; BATHANY, Katell; CHAUDIERE, Jean;
Oxidative Transformation of Leucocyanidin by Anthocyanidin Synthase from Vitis vinifera Leads Only to Quercetin
Journal of Agricultural and Food Chemistry, 2019, 67, 3595-3604
Anthocyanidin synthase from Vitis vinifera (VvANS) catalyzes the in vitro transformation of the natural isomer of leucocyanidin, 2R,3S,4S-cis-leucocyanidin, into 2R,4S-flavan-3,3,4-triol ([M + H](+), m/z 323) and quercetin. The C-3-hydroxylation product 2R,4S-flavan-3,3,4-triol is first produced and its C-3,C-4-dehydration product is in tautomeric equilibrium with (+)-dihydroquercetin. The latter undergoes a second VvANS-catalyzed C-3-hydroxylation leading to a 4-keto-2R-flavan-3,3-gem-diol which upon dehydration gives quercetin. The unnatural isomer of leucocyanidin, 2R,3S,4R-trans-leucocyanidin, is similarly transformed into quercetin upon C-3,C-4-dehydration, but unlike 3,4-cis-leucocyanidin, it also undergoes some C2,C-3-dehydration followed by an acid-catalyzed hydroxyl group extrusion at C-4 to give traces of cyanidin. Overall, the C-3,C-4-trans isomer of leucocyanidin is transformed into 2R,4R-flavan-3,3,4-triol (M + 1, m/z 323), (+)-DHQ, (-)-epiDHQ, quercetin, and traces of cyanidin. Our data bring the first direct observation of 3,4-cis-leucocyanidin- and 3,4-trans-leucocyanidin-derived 3,3-gem-diols, supporting the idea that the generic function of ANS is to catalyze the C-3-hydroxylation of its substrates. No cyanidin is produced with the natural cis isomer of leucocyanidin, and only traces with the unnatural trans isomer, which suggests that anthocyanidin synthase requires other substrate(s) for the in vivo formation of anthocyanidins.
35 […]
CULLIN, Christophe; BATHANY, Katell;
Production, purification and characterization of an elastin-like polypeptide containing the Ile-Lys-Val-Ala-Val (IKVAV) peptide for tissue engineering applications
Journal of biotechnology, 2019, 298, 35-44
Elastin-like polypeptides (ELPs) are biocompatible-engineered polypeptides, with promising interest in tissue engineering due to their intrinsic biological and physical properties, and their ease of production. The IKVAV (Ile-Lys-Val-Ala-Val) laminin-1 sequence has been shown to sustain neuron attachment and growth. In this study, the IKVAV adhesion sequence, or a scrambled VKAIV sequence, were incorporated by genetic engineering in the structure of an ELP, expressed in Escherichia coli and purified. The transition temperatures of the ELP-IKVAV and ELP-VKAIV were determined to be 23°C. Although the phase transition was fully reversible for ELP-VKAIV, we observed an irreversible aggregation for ELP-IKVAV. The corresponding aggregates shared some characteristics with amyloid-like polypeptides. The two ELPs were then reacted with functionalized polyethylene glycol (PEG) to form hydrogels. These hydrogels were characterized for rheological properties, tested with cultures of rat primary sensory neurons, and implanted subcutaneously in mice for 4 weeks. Sensory neurons cultured on high IKVAV concentration hydrogels (20%) formed longer neurite than those of neurons grown on hydrogels containing the scrambled IKVAV sequence. Finally, in vivo evaluation showed the absence of detectable inflammation. In conclusion, this functionalized ELP-IKVAV biomaterial shows interesting properties for tissue engineering requiring neurotization. Copyright © 2019 Elsevier B.V. All rights reserved.
36 […]
ALVES, Isabel; LECOMTE, Sophie;
Study of G-Protein Coupled Receptor Signaling in Membrane Environment by Plasmon Waveguide Resonance
Accounts of Chemical Research, 2019, 52, 1059-1067
Here we describe an experimental technique, termed plasmon waveguide resonance (PWR) spectroscopy that enables the characterization of molecular interactions occurring at the level of anisotropic thin films as lipid membranes and therein inserted or interacting molecules. PWR allows one to characterize such molecular interactions at different levels: (1) acquire binding curves and calculate dissociation constants; (2) obtain kinetic information; (3) obtain information about associated anisotropy changes and changes in membrane thickness; (4) obtain insight about lateral homogeneity (formation of domains). Points 1, 2, and 4 can be directly obtained from the data. Point 3 requires spectral fitting procedures so that the different optical parameters characterizing thin films as proteolipid membranes, namely refractive index and extinction coefficient for both p- (TM component of light that is parallel to the incident light) and s- (TE component of light that is perpendicular to the incident light) polarizations and thickness, can be determined. When applied to membrane proteins as the G-protein coupled receptor (GPCR) family, both ligand-induced conformational changes of the receptor can be followed as well as interactions with effectors (e.g., G-proteins). Additionally, by either altering the lipid composition in cellular membranes or specifically controlling its composition in the case of lipid model membranes with reconstituted proteins, the role of the lipid environment in receptor activation and signaling can be determined. Additionally, the eventual partition of receptors in different lipid microdomains (e.g., lipid rafts) can be followed. Such information can be obtained ex cellulo with mammalian cell membrane fragments expressing the protein of interest and/or in vitro with lipid model systems where the protein under investigation has been reconstituted. Moreover, PWR can also be applied to directly follow the reconstitution of membrane proteins in lipid model membranes. The measurements are performed directly (no labeling of molecular partners), in real time and with very high sensitivity. Here we will discuss different aspects of GPCR activation and signaling where PWR brought important information in parallel with other approaches. The utility of PWR is not limited to GPCRs but can be applied to any membrane protein. PWR is also an excellent tool to characterize the interaction of membrane active molecules (as cell penetrating, antimicrobial, viral and amyloid peptides) with lipids. A brief section is dedicated to such applications, with particular emphasis on amyloid peptides. To finalize, as PWR is a homemade technology, ongoing instrument developments aiming at breaking current experimental limitations are briefly discussed, namely, the coupling of PWR with electrochemical measurements and the expansion of measurements from the visible to the infrared region.
37 […]
LOQUET, Antoine;
Tetrameric Abeta40 and Abeta42 beta-Barrel Structures by Extensive Atomistic Simulations. I. In a Bilayer Mimicking a Neuronal Membrane
The journal of physical chemistry. B, 2019, 123, 3643-3648
The amyloid-beta (Abeta) 42 oligomers are much more toxic than Abeta40 oligomers in Alzheimer's disease. Numerous experiments indicate that toxicity could involve the formation of pores in membranes, but experimental high-resolution structure determination of these pore-forming Abeta oligomers has been impeded by aggregate heterogeneity. Using extensive atomistic simulations, low-resolution data obtained in lipid bilayers, and other theoretical factors, we proposed atomic structures of Abeta40 and Abeta42 beta-barrels in a bilayer mimicking a neuronal membrane. The 3D model, which consists of tetramer subunits, two distinct beta-hairpin motifs and an asymmetric arrangement of eight antiparallel beta-strands, is drastically destabilized for Abeta40 compared to its Abeta42 counterpart. Our computational modeling has several implications in Alzheimer's disease, sheds light on the amyloid pore hypothesis, and explains the higher deleterious property of Abeta42.
38 […]
LAMON, Gaëlle; LOQUET, Antoine;
The extracellular matrix protects Bacillus subtilis colonies from Pseudomonas invasion and modulates plant co-colonization
Nature Communications, 2019, 10,
Bacteria of the genera Pseudomonas and Bacillus can promote plant growth and protect plants from pathogens. However, the interactions between these plant-beneficial bacteria are understudied. Here, we explore the interaction between Bacillus subtilis 3610 and Pseudomonas chlororaphis PCL1606. We show that the extracellular matrix protects B. subtilis colonies from infiltration by P. chlororaphis. The absence of extracellular matrix results in increased fluidity and loss of structure of the B. subtilis colony. The P. chlororaphis type VI secretion system (T6SS) is activated upon contact with B. subtilis cells, and stimulates B. subtilis sporulation. Furthermore, we find that B. subtilis sporulation observed prior to direct contact with P. chlororaphis is mediated by histidine kinases KinA and KinB. Finally, we demonstrate the importance of the extracellular matrix and the T6SS in modulating the coexistence of the two species on melon plant leaves and seeds.
39 […]
HABENSTEIN, Birgit; LOQUET, Antoine;
Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of Pseudomonas aeruginosa
Frontiers in Microbiology, 2019, 10,
The type three secretion system (T3SS) is a macromolecular protein nano-syringe used by different bacterial pathogens to inject effectors into host cells. The extracellular part of the syringe is a needle-like filament formed by the polymerization of a 9-kDa protein whose structure and proper localization on the bacterial surface are key determinants for efficient toxin injection. Here, we combined in vivo, in vitro, and in silico approaches to characterize the Pseudomonas aeruginosa T3SS needle and its major component PscF. Using a combination of mutagenesis, phenotypic analyses, immunofluorescence, proteolysis, mass spectrometry, atomic force microscopy, electron microscopy, and molecular modeling, we propose a model of the P aeruginosa needle that exposes the N-terminal region of each PscF monomer toward the outside of the filament, while the core of the fiber is formed by the C-terminal helix. Among mutations introduced into the needle protein PscF, D76A, and P47A/Q54A caused a defect in the assembly of the needle on the bacterial surface, although the double mutant was still cytotoxic on macrophages in a T3SS-dependent manner and formed filamentous structures in vitro. These results suggest that the T3SS needle of P aeruginosa displays an architecture that is similar to that of other bacterial needles studied to date and highlight the fact that small, targeted perturbations in needle assembly can inhibit T3SS function. Therefore, the T3SS needle represents an excellent drug target for small molecules acting as virulence blockers that could disrupt pathogenesis of a broad range of bacteria.
40 […]
MOLINARI, Michael;
Quantitative characterization of single-cell adhesion properties by atomic force microscopy using protein-functionalized microbeads
Journal of Molecular Recognition, 2019, 32,
A method was developed to characterize the adhesion properties of single cells by using protein-functionalized atomic force microscopy (AFM) probes. The quantification by force spectroscopy of the mean detachment force between cells and a gelatin-functionalized colloidal tip reveals differences in cell adhesion properties that are not within reach of a traditional bulk technique, the washing assay. In this latter method, experiments yield semiquantitative and average adhesion properties of a large population of cells. They are also limited to stringent conditions and cannot highlight disparities in adhesion in the subset of adherent cells. In contrast, this AFM-based method allows for a reproducible and quantitative investigation of the adhesive properties of individual cells in common cell culture conditions and allows for the detection of adhesive subpopulations of cells. These characteristics meet the critical requirements of many fields, such as the study of cancer cell migratory abilities.
41 […]
HENRY, Sarah; FEUILLIE, Cecile; CULLIN, Christophe; LECOMTE, Sophie; MOLINARI, Michael;
High speed atomic force microscopy to investigate the interactions between toxic A(1-42) peptides and model membranes in real time: impact of the membrane composition
Nanoscale, 2019, 11, 7229-7238
Due to an aging population, neurodegenerative diseases have become a major health issue, the most common being Alzheimer's disease. The mechanisms leading to neuronal loss still remain unclear but recent studies suggest that soluble A oligomers have deleterious effects on neuronal membranes. Here, high-speed atomic force microscopy was used to assess the effect of oligomeric species of a variant of A(1-42) amyloid peptide on model membranes with various lipid compositions. Results showed that the peptide does not interact with membranes composed of phosphatidylcholine and sphingomyelin. Ganglioside GM1, but not cholesterol, is required for the peptide to interact with the membrane. Interestingly, when they are both present, a fast disruption of the membrane was observed. It suggests that the presence of ganglioside GM1 and cholesterol in membranes promotes the interaction of the oligomeric A(1-42) peptide with the membrane. This interaction leads to the membrane's destruction in a few seconds. This study highlights the power of high-speed atomic force microscopy to explore lipid-protein interactions with high spatio-temporal resolution.
42 […]
CATARGI, Bogdan;
Sealing hyaluronic acid microgels with oppositely-charged polypeptides: A simple strategy for packaging hydrophilic drugs with on-demand release
Journal of Colloid and Interface Science, 2019, 535, 16-27
A simple route to deliver on demand hydrosoluble molecules such as peptides, packaged in biocompatible and biodegradable microgels, is presented. Hyaluronic acid hydrogel particles with a controlled structure are prepared using a microfluidic approach. Their porosity and their rigidity can be tuned by changing the crosslinking density. These negatively-charged polyelectrolytes interact strongly with positively-charged linear peptides such as poly-L-lysine (PLL). Their interactions induce microgel deswelling and inhibit microgel enzymatic degradability by hyaluronidase. While small PLL penetrate the whole volume of the microgel, PLL larger than the mesh size of the network remain confined at its periphery. They make a complexed layer with reduced pore size, which insulates the microgel inner core from the outer medium. Consequently, enzymatic degradation of the matrix is fully inhibited and non affinity hydrophilic species can be trapped in the core. Indeed, negatively-charged or small neutral peptides, without interactions with the network, usually diffuse freely across the network. By simple addition of large PLL, they are packaged in the core and can be released on demand, upon introduction of an enzyme that degrades selectively the capping agent. Single polyelectrolyte layer appears as a simple generic method to coat hydrogel-based materials of various scales for encapsulation and controlled delivery of hydrosoluble molecules. (C) 2018 Elsevier Inc. All rights reserved.
43 […]
Remote Monitoring of Diabetes: A Cloud-Connected Digital System for Individuals With Diabetes and Their Health Care Providers
Journal of diabetes science and technology, 2019, 0, 1932296819834054-1932296819834054
Benefits of telemedicine have been proven in the field of diabetes. Among a number of technical solutions, Diabeo has been studied in both type 1 and type 2 diabetes with intensive insulin therapy. This digital therapeutic system contains a self-monitoring glucose logbook and offers automated insulin dose recommendations thanks to a fully customizable algorithm. In addition, the cloud-based dedicated software also has features to facilitate remote monitoring, including a platform for diabetes nurses who perform coaching and treatment adjustment. A detailed description of this telemedicine system is provided, as well as results of completed clinical studies. In particular, TeleDiab 1's positive results on HbA1c in type 1 diabetes are detailed. We conclude with a discussion of the role of this telemedicine system within the landscape of mobile apps for diabetes.
44 […]
CATARGI, Bogdan;
Glycemic Variability Is a Powerful Independent Predictive Factor of Midterm Major Adverse Cardiac Events in Patients With Diabetes With Acute Coronary Syndrome
Diabetes Care, 2019, 42, 674-681
OBJECTIVE Acute glucose fluctuations are associated with hypoglycemia and are emerging risk factors for cardiovascular outcomes. However, the relationship between glycemic variability (GV) and the occurrence of midterm major cardiovascular events (MACE) in patients with diabetes remains unclear. This study investigated the prognostic value of GV in patients with diabetes and acute coronary syndrome (ACS). RESEARCH DESIGN AND METHODS This study included consecutive patients with diabetes and ACS between January 2015 and November 2016. GV was assessed using SD during initial hospitalization. MACE, including new-onset myocardial infarction, acute heart failure, and cardiac death, were recorded. The predictive effects of GV on patient outcomes were analyzed with respect to baseline characteristics and cardiac status. RESULTS A total of 327 patients with diabetes and ACS were enrolled. MACE occurred in 89 patients (27.2%) during a mean follow-up of 16.9 months. During follow-up, 24 patients (7.3%) died of cardiac causes, 35 (10.7%) had new-onset myocardial infarction, and 30 (9.2%) were hospitalized for acute heart failure. Multivariable logistic regression analysis showed that GV >2.70 mmol/L, a Synergy between PCI with Taxus and Cardiac Surgery (SYNTAX) score >34, and reduced left ventricular ejection fraction of <40% were independent predictors of MACE, with odds ratios (ORs) of 2.21 (95% CI 1.64-2.98; P < 0.001), 1.88 (1.26-2.82; P = 0.002), and 1.71 (1.14-2.54; P = 0.009), respectively, whereas a Global Registry of Acute Coronary Events (GRACE) risk score >140 was not (OR 1.07 [0.77-1.49]; P = 0.69). CONCLUSIONS A GV cutoff value of >2.70 mmol/L was the strongest independent predictive factor for midterm MACE in patients with diabetes and ACS.
45 […]
GUICHARD, Gilles; FREMAUX, Juliette;
, 2019, 0,
(EN) The present description provides compositions and methods for producing therapeutic oligomeric compounds. In another aspect the description provides methods for administering the oligomeric compounds for the treatment and prevention of disease in a mammal. In particular, the invention relates to medicaments comprising various novel oligomeric compounds and pharmaceutically acceptable salts thereof. The compounds of the invention may optionally be administered with at least one of a pharmaceutically acceptable excipient, additional pharmacologically active agent or a combination thereof.
46 […]
PLAWINSKI, Laurent; DURRIEU, Marie-Christine;
Label-free multi-parametric imaging of single cells: dual picosecond optoacoustic microscopy
Journal of biophotonics, 2019, 0, e201900045-e201900045
Advances in microscopy with new visualization possibilities often bring dramatic progress to our understanding of the intriguing cellular machinery. Picosecond optoacoustic micro-spectroscopy is an optical technique based on ultrafast pump-probe generation and detection of hypersound on time durations of picoseconds and length scales of nanometers. It is experiencing a renaissance as a versatile imaging tool for cell biology research after a plethora of applications in solid-state physics. In this emerging context, this work reports on a dual-probe architecture to carry out real-time parallel detection of the hypersound propagation inside a cell that is cultured on a metallic substrate, and of the hypersound reflection at the metal/cell adhesion interface. Using this optoacoustic modality, several biophysical properties of the cell can be measured in a noncontact and label-free manner. Its abilities are demonstrated with the multiple imaging of a mitotic macrophage-like cell in a single run experiment.
47 […]
GOIBIER, Lucie; PILLEMENT, Christophe; MONTEIL, Julien; FAURE, Chrystel; LEAL-CALDERON, Fernando;
Emulsification of non-aqueous foams stabilized by fat crystals: Towards novel air-in-oil-in-water food colloids
Food Chemistry, 2019, 293, 49-56
We designed Air-in-Oil-in-Water (A/O/W) emulsions. First, Air-in-Oil foams were fabricated by whipping anhydrous milk fat. The maximum overrun was obtained at 20 degrees C. The foams contained 30-35 vol% air and were stabilized solely by fat crystals. To refine the bubble size, foams were further sheared in a Couette's cell. The average bubble size reached a value as small as 6.5 mu m at a shear rate of 5250 s(-1). The nonaqueous foams were then dispersed in a viscous aqueous phase containing sodium caseinate to obtain A/O/W emulsions. The shear rate was varied from 1000 to 7500 s(-1), allowing to obtain Air-in-Oil globules whose average diameter ranged from 15 to 60 mu m. To avoid globule creaming, the aqueous phase was gelled by incorporating hydroxyethyl cellulose. Homogeneous emulsions were obtained with fat globules containing around 22 vol% of residual air. The systems were kinetically stable for at least 3 weeks at 4 degrees C.
48 […]
ATGIE, Claude; SAUVANT, Patrick; FAURE, Chrystel;
Encapsulation of epsilon-viniferin in onion-type multi-lamellar liposomes increases its solubility and its photo-stability and decreases its cytotoxicity on Caco-2 intestinal cells
Food & Function, 2019, 10, 2573-2582
epsilon-Viniferin, a resveratrol dimer, is a naturally occurring stilbene that has been studied so far for its potential beneficial effects on human health. Its low water solubility, its photo-sensitivity and its low bioavailability make its applications in the food industry complicated. To overcome these limitations, epsilon-viniferin was encapsulated in phospholipid-based multi-lamellar liposomes (MLLs) called spherulites or onions. In the best case, an encapsulation efficiency of 58 +/- 3% and a bioactive loading of 4.2 +/- 0.5% were reached. Encapsulation of epsilon-viniferin drastically increased its water solubility by more than 5 orders to reach 17.4 g L-1 and provided protection against its UV-induced isomerization. While epsilon-viniferin was shown to be significantly toxic to Caco-2 intestinal-like cells for concentrations higher than 25 M, once encapsulated in MLLs, those cells did not experience any mortality even for the highest tested stilbene concentration (100 M) as revealed by red neutral assay.
49 […]
LAMBERT, Olivier; BONNAFOUS, Pierre;
Complementary use of mass spectrometry and cryo-electron microscopy to assess the maturity of live attenuated dengue vaccine viruses
Vaccine, 2019, 37, 3580-3587
Dengue virus (DENV) infection is a global health threat with the potential to affect at least 3.6 billion people living in areas of risk. No specific curative treatments against dengue disease are available and vaccines are currently the only way to prevent the disease. The tetravalent dengue vaccine developed by Sanofi Pasteur has demonstrated significant efficacy in phase III studies and is now licensed in several countries for the prevention of disease in dengue-seropositives over 9 years of age. The vaccine is composed of four recombinant, live, attenuated vaccines (CYD 1-4) based on a yellow fever vaccine 17D (YFV 17D) backbone, each expressing the pre-membrane (prM) and envelope (E) genes of one of the four DENV serotypes. Virus maturity could impact the biological activity of the vaccine viruses. To address this question, the maturity of the four vaccine viruses used in phase III clinical studies was assessed by two complementary techniques: mass spectrometry (MS) and cryo-electron microscopy (cryoEM). MS assessed viral maturity at the molecular level by quantifying specifically the prM, and M proteins. CryoEM provided information at the particle level, allowing visualizing the different phenotypes of viral particles: spiky (immature), smooth/bumpy (mature), and mixed (partially mature). Results of the two assays used in this study show that all four CYD dengue vaccine viruses present in lots used in phase III efficacy trials, display in the majority a mature phenotype. (C) 2019 Elsevier Ltd. All rights reserved.
50 […]
GOLE, Bappaditya; KAUFFMANN, Brice; MAURIZOT, Victor; HUC, Ivan; FERRAND, Yann;
Light-Controlled Conformational Switch of an Aromatic Oligoamide Foldamer
Angewandte Chemie-International Edition, 2019, 58, 8063-8067
An aromatic oligoamide sequence composed of a light-responsive diazaanthracene-based aromatic beta-sheet flanked by two variable diameter helical segments was prepared. Structural investigations revealed that such oligomers adopt two distinct conformations: a canonical symmetrical conformation with the two helices stacked above and below the sheet, and an unanticipated unsymmetrical conformation in which one helix has flipped to directly stack with the first helix. Photoirradiation of the foldamer led to the quantitative, and thermally reversible, formation of a single photoproduct resulting from the [4+4] cycloaddition of two diazaanthracenes within the aromatic beta-sheet. NMR and crystallographic studies revealed a parallel arrangement of the diazaanthracene photoproduct and a complete conversion into a symmetrical conformation requiring a rearrangement of all unsymmetrical conformers. These results highlight the potential of foldamers, with structures more complex than isolated helices, for the design of photoswitches showing nontrivial nanometer scale shape changes.
51 […]
ANDRE, Christophe; GUICHARD, Gilles;
Interaction of a Model Peptide on Gram Negative and Gram Positive Bacterial Sliding Clamps
Acs Infectious Diseases, 2019, 5, 1022-1034
Bacterial sliding clamps control the access of DNA polymerases to the replication fork and are appealing targets for antibacterial drug development. It is therefore essential to decipher the polymerase-clamp binding mode across various bacterial species. Here, two residues of the E. coli clamp binding pocket, S-Ec(346) and M-Ec(362), and their cognate residues in M. tuberculosis and B. subtilis clamps, were mutated. The effects of these mutations on the interaction of a model peptide with these variant clamps were evaluated by thermodynamic, molecular dynamics, X-rays crystallography, and biochemical analyses. M-Ec(362) and corresponding residues in Gram positive clamps occupy a strategic position where a mobile residue is essential for an efficient peptide interaction. S-Ec(346) has a more subtle function that modulates the pocket folding dynamics, while the equivalent residue in B. subtilis is essential for polymerase activity and might therefore be a Gram positive-specific molecular marker. Finally, the peptide binds through an induced-fit process to Gram negative and positive pockets, but the complex stability varies according to a pocket-specific network of interactions.
52 […]
TOKARSKI, Caroline;
'Palaeoshellomics' reveals the use of freshwater mother-of-pearl in prehistory
Elife, 2019, 8,
The extensive use of mollusc shell as a versatile raw material is testament to its importance in prehistoric times. The consistent choice of certain species for different purposes, including the making of ornaments, is a direct representation of how humans viewed and exploited their environment. The necessary taxonomic information, however, is often impossible to obtain from objects that are small, heavily worked or degraded. Here we propose a novel biogeochemical approach to track the biological origin of prehistoric mollusc shell. We conducted an in-depth study of archaeological ornaments using microstructural, geochemical and biomolecular analyses, including 'palaeoshellomics', the first application of palaeoproteomics to mollusc shells (and indeed to any invertebrate calcified tissue). We reveal the consistent use of locally-sourced freshwater mother-of-pearl for the standardized manufacture of 'double-buttons'. This craft is found throughout Europe between 4200-3800 BCE, highlighting the ornament-makers' profound knowledge of the biogeosphere and the existence of cross-cultural traditions.
53 […]
TOKARSKI, Caroline;
Solvent-mediated extraction of fatty acids in bilayer oil paint models: a comparative analysis of solvent application methods
Heritage Science, 2019, 7,
The impact of solvent exposure on oil paintings and the differences between solvent application methods are long-standing topics in cleaning studies. Solvent exposure is ideally kept to a minimum, because solvent swelling can lead to the extraction and displacement of reactive paint components. In particular, important concerns are fatty acids displacement resulting in metal soap formation and embrittlement of paint due to solvent exposure. In this study, the extraction of a saturated fatty acid (SFA) marker and the formation of zinc soaps were monitored to measure the impact of solvent cleaning on tailored bilayer model systems for aged oil paint. Three methods of solvent application were compared: cotton swab, rigid gel and Evolon tissue (with different solvent loading). The samples were analysed by surface acoustic wave nebulization mass spectrometry (SAWN-MS) and thermally-assisted hydrolysis and methylation pyrolysis gas chromatography mass spectrometry (THM-Py-GC/MS) by comparing the calculated margaric:palmitic acid (C-17:C-16) ratio determined in the extracts (taken from the swab, gel or Evolon tissue). We conclude that both swab cleaning and squeezed Evolon tissue application result in comparable SFA extraction. The rigid gel and Evolon with controlled solvent-loading limit the amount of SFA extraction. The distribution of C-17 after solvent application was visualised using static Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) on cross sections, showing that C-17 redistribution took place in all cases where solvent was applied. Crystalline zinc soaps formation was not observed after 5 min of ethanol exposure in the embedded cross-sections with imaging ATR-FTIR, indicating that solvent exposure does not immediately trigger the formation of crystalline metal soaps. However, significant zinc soap formation was found after 30 min of ethanol exposure using Evolon tissue without controlled loading. This study contributes to a better understanding of the impact of different methods of solvent application on oil paintings and highlights important differences between these methods.
54 […]
Human H4 tail stimulates HIV-1 integration through binding to the carboxy-terminal domain of integrase
Nucleic Acids Research, 2019, 47, 3607-3618
The integration of the retroviral genome into the chromatin of the infected cell is catalysed by the integrase (IN)center dot viral DNA complex (intasome). This process requires functional association between the integration complex and the nucleosomes. Direct intasome/histone contacts have been reported to modulate the interaction between the integration complex and the target DNA (tDNA). Both prototype foamy virus (PFV) and HIV-1 integrases can directly bind histone amino-terminal tails. We have further investigated this final association by studying the effect of isolated histone tails on HIV-1 integration. We show here that the binding of HIV-1 IN to a peptide derived from the H4 tail strongly stimulates integration catalysis in vitro. This stimulation was not observed with peptide tails from other variants or with alpha-retroviral (RAV) and spuma-retroviral PFV integrases. Biochemical analyses show that the peptide tail induces both an increase in the IN oligomerization state and affinity for the target DNA, which are associated with substantial structural rearrangements in the IN carboxy-terminal domain (CTD) observed by NMR. Our data indicate that the H4 peptide tail promotes the formation of active strand transfer complexes (STCs) and support an activation step of the incoming intasome at the contact of the histone tail.
55 […]
First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study
Analytical Chemistry, 2019, 91, 6953-6961
The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.
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MARTINEZ, Denis; LEGRAND, Anthony; LAMBERT, Olivier; BERBON, Mélanie; GRELARD, Axelle; LOQUET, Antoine; HABENSTEIN, Birgit;
Coiled-coil oligomerization controls localization of the plasma membrane REMORINs
Journal of Structural Biology, 2019, 206, 12-19
REMORINs are nanodomain-organized proteins located in the plasma membrane and involved in cellular responses in plants. The dynamic assembly of the membrane nanodomains represents an essential tool of the versatile membrane barriers to control and modulate cellular functions. Nevertheless, the assembly mechanisms and protein organization strategies of nanodomains are poorly understood and many structural aspects are difficult to visualize. Using an ensemble of biophysical approaches, including solid-state nuclear magnetic resonance, cryo-electron microscopy and in vivo confocal imaging, we provide first insights on the role and the structural mechanisms of REMORIN trimerization. Our results suggest that the formation of REMORIN coiledcoil trimers is essential for membrane recruitment and promotes REMORIN assembly in vitro into long filaments by trimer-trimer interactions that might participate in nanoclustering into membrane domains in vivo.
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BERBON, Mélanie; HABENSTEIN, Birgit; LOQUET, Antoine;
A polymorphic helix of a Salmonella needle protein relays signals defining distinct steps in type III secretion
PLoS biology, 2019, 17, e3000351-e3000351
Type III protein-secretion machines are essential for the interactions of many pathogenic or symbiotic bacterial species with their respective eukaryotic hosts. The core component of these machines is the injectisome, a multiprotein complex that mediates the selection of substrates, their passage through the bacterial envelope, and ultimately their delivery into eukaryotic target cells. The injectisome is composed of a large cytoplasmic complex or sorting platform, a multiring base embedded in the bacterial envelope, and a needle-like filament that protrudes several nanometers from the bacterial surface and is capped at its distal end by the tip complex. A characteristic feature of these machines is that their activity is stimulated by contact with target host cells. The sensing of target cells, thought to be mediated by the distal tip of the needle filament, generates an activating signal that must be transduced to the secretion machine by the needle filament. Here, through a multidisciplinary approach, including solid-state NMR (SSNMR) and cryo electron microscopy (cryo-EM) analyses, we have identified critical residues of the needle filament protein of a Salmonella Typhimurium type III secretion system that are involved in the regulation of the activity of the secretion machine. We found that mutations in the needle filament protein result in various specific phenotypes associated with different steps in the type III secretion process. More specifically, these studies reveal an important role for a polymorphic helix of the needle filament protein and the residues that line the lumen of its central channel in the control of type III secretion.
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informations Dernière mise à jour : 15/05/2019

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