Bioactive Seed Layer for Surface-Confined Self-Assembly of Peptides

International audienceThe design and control of molecular systems that self-assemble spontaneously and exclusively at or near an interface represents a real scientific challenge. We present here a new concept, an active seed layer that allows to overcome this challenge.It is based on enzyme-assisted self-assembly. An enzyme, alkaline phosphatase, which transforms an original peptide,Fmoc-FFY(PO 4 2À), into an efficient gelation agent by dephosphorylation, is embedded in a polyelectrolyte multilayer and constitutes the "reaction motor". A seed layer composed of a polyelectrolyte covalently modified by anchoring hydro-gelator peptides constitutes the top of the multilayer. This layer is the nucleation site for the Fmoc-FFY peptide self-assembly. When such a film is brought in contact with a Fmoc-FFY-(PO42-) solution, a nofiber network starts to form almost instantaneously which extents up to several micrometers into the solution after several hours. We demonstrate that the active seed layer allows convenient control over the self-assembly kinetics and the geometric features of the fiber network simply by changing its peptide density

Influence of substrate elasticity on chromatic plasticity of epithelial cells and on division of tumoral cells

Dans le domaine des biomatériaux, cette thèse s’intéresse à l’influence de l’élasticité du substrat sur la division et la plasticité de la chromatine de cellules épithéliales. La létalité des cellules est corrélée aux faibles rigidités des substrats. Cependant, quelques cellules tumorales SW480, incluant celles portant des anomalies de ségrégation des chromosomes, progressent en mitose. Ces anomalies seraient à l’origine de réarrangements chromosomiques, sources de nombreuses mutations. Les substrats mous conduisent à la formation d’hétérochromatine tandis que les substrats très mous induisent la nécrose des cellules PtK2. Sur ces substrats, l’euchromatine est maintenue après inhibition de HDAC, permettant aux cellules de résister à la nécrose, indépendamment de la compétence transcriptionnelle du noyau. Ces cellules s’étalent à nouveau après transfert sur un substrat rigide. Ces résultats suggèrent 1) une voie de signalisation entrante initiée par le substrat conduisant à la nécrose via la formation d’hétérochromatine 2) une voie de signalisation sortante initiée par l’euchromatine permettant la survie cellulaire.In the biomaterials field, this PhD work is about influence of substrate elasticity on cell division and chromatin plasticity of epithelial cells. Soft substrates cause massive death.However, some SW480 tumor cells, including those bearing chromosomal segregation abnormalities progress in mitosis. These abnormalities could result in more chromosomal rearrangements, increasing mutations. Soft substrates lead to heterochromatin remodelling and very soft substrates promote necrosis of PtK2 cells. On these substrates, euchromatin could be maintained after HDAC inhibition independently of the nuclear transcriptional competence.These cells spread again after tranfer on stiff substrates. These results suggest i) outside-insignalling cascade initiated at the soft substrate surface leading to heterochromatin remodelling and ultimately necrosis, ii) inside-out signaling cascade initiated from euchromatin allowing cell to overcome necrosis on soft substrate

Influence of substrate elasticity on chromatic plasticity of epithelial cells and on division of tumoral cells

Dans le domaine des biomatériaux, cette thèse s’intéresse à l’influence de l’élasticité du substrat sur la division et la plasticité de la chromatine de cellules épithéliales. La létalité des cellules est corrélée aux faibles rigidités des substrats. Cependant, quelques cellules tumorales SW480, incluant celles portant des anomalies de ségrégation des chromosomes, progressent en mitose. Ces anomalies seraient à l’origine de réarrangements chromosomiques, sources de nombreuses mutations. Les substrats mous conduisent à la formation d’hétérochromatine tandis que les substrats très mous induisent la nécrose des cellules PtK2. Sur ces substrats, l’euchromatine est maintenue après inhibition de HDAC, permettant aux cellules de résister à la nécrose, indépendamment de la compétence transcriptionnelle du noyau. Ces cellules s’étalent à nouveau après transfert sur un substrat rigide. Ces résultats suggèrent 1) une voie de signalisation entrante initiée par le substrat conduisant à la nécrose via la formation d’hétérochromatine 2) une voie de signalisation sortante initiée par l’euchromatine permettant la survie cellulaire.In the biomaterials field, this PhD work is about influence of substrate elasticity on cell division and chromatin plasticity of epithelial cells. Soft substrates cause massive death.However, some SW480 tumor cells, including those bearing chromosomal segregation abnormalities progress in mitosis. These abnormalities could result in more chromosomal rearrangements, increasing mutations. Soft substrates lead to heterochromatin remodelling and very soft substrates promote necrosis of PtK2 cells. On these substrates, euchromatin could be maintained after HDAC inhibition independently of the nuclear transcriptional competence.These cells spread again after tranfer on stiff substrates. These results suggest i) outside-insignalling cascade initiated at the soft substrate surface leading to heterochromatin remodelling and ultimately necrosis, ii) inside-out signaling cascade initiated from euchromatin allowing cell to overcome necrosis on soft substrate

Influence de l'élasticité du substrat sur la plasticité de la chromatine de cellules épithéliales et sur la division de cellules tumorales

In the biomaterials field, this PhD work is about influence of substrate elasticity on cell division and chromatin plasticity of epithelial cells. Soft substrates cause massive death.However, some SW480 tumor cells, including those bearing chromosomal segregation abnormalities progress in mitosis. These abnormalities could result in more chromosomal rearrangements, increasing mutations. Soft substrates lead to heterochromatin remodelling and very soft substrates promote necrosis of PtK2 cells. On these substrates, euchromatin could be maintained after HDAC inhibition independently of the nuclear transcriptional competence.These cells spread again after tranfer on stiff substrates. These results suggest i) outside-insignalling cascade initiated at the soft substrate surface leading to heterochromatin remodelling and ultimately necrosis, ii) inside-out signaling cascade initiated from euchromatin allowing cell to overcome necrosis on soft substrate.Dans le domaine des biomatériaux, cette thèse s’intéresse à l’influence de l’élasticité du substrat sur la division et la plasticité de la chromatine de cellules épithéliales. La létalité des cellules est corrélée aux faibles rigidités des substrats. Cependant, quelques cellules tumorales SW480, incluant celles portant des anomalies de ségrégation des chromosomes, progressent en mitose. Ces anomalies seraient à l’origine de réarrangements chromosomiques, sources de nombreuses mutations. Les substrats mous conduisent à la formation d’hétérochromatine tandis que les substrats très mous induisent la nécrose des cellules PtK2. Sur ces substrats, l’euchromatine est maintenue après inhibition de HDAC, permettant aux cellules de résister à la nécrose, indépendamment de la compétence transcriptionnelle du noyau. Ces cellules s’étalent à nouveau après transfert sur un substrat rigide. Ces résultats suggèrent 1) une voie de signalisation entrante initiée par le substrat conduisant à la nécrose via la formation d’hétérochromatine 2) une voie de signalisation sortante initiée par l’euchromatine permettant la survie cellulaire

Que devront bientôt affronter l’avocat et le juriste d’entreprise de propriété intellectuelle ?

International audienceCompte rendu des "Rendez-vous de 5h" organisés par le M2 PLA de l’Université Paris 2Les 30, 31 mai et 1er juin 2016 se sont tenus les traditionnels Rendez-vous de cinq heures, organisés par les étudiants du Master II Propriété littéraire, artistique et industrielle, sous la direction du Professeur Gautier. Autour du thème « Que devront bientôt affronter l’avocat et le juriste d’entreprise de propriété intellectuelle ? », à l’aune des réformes en propriété intellectuelle et en droit des contrats, de la « balance des intérêts », méthode de raisonnement de plus en plus courtisée par la Cour de cassation, et de l’extension des champs d’activité professionnelle des avocats, les débats ont ainsi porté sur les sujets suivants : « Affronter les lois nouvelles », « affronter les raisonnements juridiques nouveaux », et « affronter l’extension des champs d’activités professionnelles ». Les questions des étudiants et les interventions du public ont permis aux participants de faire valoir leurs différents points de vue, favorisant ainsi la discussion. À l’issue de chaque journée, les débats ont été conclus par un mot improvisé du Professeur Gautier

Insensitivity of dental pulp stem cells migration to substrate stiffness

Dental pulp stem cells (DPSCs) are a promising cell source for regeneration of dental pulp. Migration is a key event but influence of the microenvironment rigidity (5 kPa at the center of dental pulp to 20 GPa for the dentin) is largely unknown. Mechanical signals are transmitted from the extracellular matrix to the cytoskeleton, to the nuclei, and to the chromatin, potentially regulating gene expression. To identify the microenvironmental influence on migration, we analyzed motility on PDMS substrates with stiffness increasing from 1.5 kPa up to 2.5 MPa. We found that migration speed slightly increases as substrate stiffness decreases in correlation with decreasing focal adhesion size. Motility is relatively insensitive to substrate stiffness, even on a bi-rigidity PDMS substrate where DPSCs migrate without preferential direction. Migration is independent of both myosin II activity and YAP translocation after myosin II inhibition. Additionally, inhibition of Arp2/3 complex leads to significant speed decrease for all rigidities, suggesting contribution of the lamellipodia in the migration. Interestingly, the chromatin architecture remains stable after a 7-days exposure on the PDMS substrates for all rigidity. To design scaffold mimicking dental pulp environment, similar DPSCs migration for all rigidity, leaves field open to choose this mechanical parameter

Design of hybrid protein-coated magnetic core-mesoporous silica shell nanocomposites for MRI and drug release assessed in a 3D tumor cell model

International audienceIn this work, we describe the design and the use of a novel theranostic hybrid nanocomposite made of an iron oxide core and a mesoporous silica shell (IO@MS) of ca. 30 nm coated by human serum albumin (HSA) layer for magnetic resonance imaging and drug delivery applications. The porosity of IO@MS nanoparticles was loaded with an antitumoral drug, Doxorubicin (Dox) reaching a high drug loading capacity (DLC) of 34 w%. To entrap the drug, a tight HSA coating held via isobutyramide (IBAM) binders was deposited. We show that this protein nanoassembly entraps the drugs efficiently and behaves as an innovative enzyme-sensitive gatekeeper that is degraded upon protease action. Finally we assess the Dox release in a 3D cell model via confocal imaging and its cytotoxicity is shown by growth inhibition studies on liver cancer cell spheroid

The Contribution of Various In Vitro Methodologies to Comprehending the Filling Ability of Root Canal Pastes in Primary Teeth

A void-free obturation during root canal treatment on primary teeth is currently very difficult to attain. In this study, the pulpectomy filling abilities of Bio-C Pulpecto (Angelus, Basil, Londrina, Paraná, Brazil) and of zinc oxide eugenol, or "ZOE" (DenPro, Prevest, New York, NY, USA), were compared using several in vitro techniques. Therefore, 30 primary anterior teeth were used in the present in vitro study. Analysis of variance (ANOVA), including a multiple comparison procedure (Holm-Sidak method, Dunn's Method, or Tukey test), was used. On micro-CT, Bio-C Pulpecto exhibited higher void percentages than did ZOE (10.3 ± 3.8%, and 3.5 ± 1.3%), respectively (p < 0.05). With digital microscopy, higher total void percentages were found in the BC (13.2 ± 26.7%) group compared to the ZOE (2.7 ± 2.8%) group (p < 0.05). With the CLSM, mean tubular penetration depths were higher for Bio-C Pulpecto than for ZOE in all canal thirds (p < 0.05). SEM images demonstrated no tags into dentinal tubules in either group throughout the three thirds. Moreover, higher statistically significant flowability was found for Bio-C (2.657 ± 0.06 mm) compared to ZOE (1.8 ± 0.13 mm) (p < 0.05). The findings of this study indicate that neither ZOE nor Bio-C Pulpecto appears to meet the criteria for an ideal root canal filling paste for primary teeth. This study laid the groundwork for future research by determining how micro-CT, digital microscopy, SEM, and CLSM contribute to our understanding of the filling process of primary teeth. More thorough research on the mechanism of root canal obturation on primary teeth is required to achieve a long-term successful root canal therapy in young children

Unexpected Bactericidal Activity of Poly(arginine)/Hyaluronan Nanolayered Coatings

The number of nosocomial infections related to implants and medical devices increase alarmingly worldwide. New strategies based on the design of antimicrobial coatings are required to prevent such infections. Polyelectrolyte “multilayer” films constitute a powerful tool for nanoscale surface functionalization which allows addressing this issue. By investigating films built up with poly(arginine) (PAR) of various chain lengths (10, 30, 100, and 200 residues) and hyaluronic acid (HA), we demonstrate that exclusively films constructed with poly(arginine) composed of 30 residues (PAR30) acquire a strong antimicrobial activity against Gram-positive and Gram-negative pathogenic bacteria associated with infections of medical devices. This chain-size effect is extremely striking and is the first example reported where the length of the polyelectrolytes played a key-role in the functionality of the films. Moreover, this unexpected functionality of nanolayered polypeptide/polysaccharide PAR30/HA films occurs without adding any specific antimicrobial agent, such as antibiotics or antimicrobial peptides. PAR30/HA film inhibits bacteria through a contact-killing mechanism due to the presence of mobile PAR30 chains. These chains are assumed to diffuse toward the interface, where they interact with the bacteria with the consequence of killing them. This new coating with unique properties based on the association of a homopolypeptide of 30 residues with a polysaccharide constitutes a simple system to prevent implant-related infections with a reasonable production cost

D-Cateslytin: a new antifungal agent for the treatment of oral Candida albicans associated infections

International audienceThe excessive use of antifungal agents, compounded by the shortage of new drugs being introduced into the market, is causing the accumulation of multi-resistance phenotypes in many fungal strains. Consequently, new alternative molecules to conventional antifungal agents are urgently needed to prevent the emergence of fungal resistance. In this context, Cateslytin (Ctl), a natural peptide derived from the processing of Chromogranin A, has already been described as an effective antimicrobial agent against several pathogens including Candida albicans. In the present study, we compared the antimicrobial activity of two conformations of Ctl, L-Ctl and D-Ctl against Candida albicans. Our results show that both D-Ctl and L-Ctl were potent and safe antifungal agents. However, in contrast to L-Ctl, D-Ctl was not degraded by proteases secreted by Candida albicans and was also stable in saliva. Using video microscopy, we also demonstrated that D-Ctl can rapidly enter C. albicans, but is unable to spread within a yeast colony unless from a mother cell to a daughter cell during cellular division. Besides, we revealed that the antifungal activity of D-Ctl could be synergized by voriconazole, an antifungal of reference in the treatment of Candida albicans related infections. In conclusion, D-Ctl can be considered as an effective, safe and stable antifungal and could be used alone or in a combination therapy with voriconazole to treat Candida albicans related diseases including oral candidosis