Développement de méthodes d’analyse protéomique pour l’exploration translationnelle de la maladie de Wilson.

Wilson’s disease is a rare genetic disorder triggered by mutations in the ATP7B gene, which encodes a transport protein involved in copper transport and excretion, triggering toxic copper overloads in the liver and the brain. The lack of genotype-phenotype correlation and phenotype variability lead to clinical care difficulties, especially for the diagnosis and biological follow up of patients. In this study, we initiated the discovery and evaluation of biomarker candidates for the diagnosis of Wilson’s disease and for early prognosis towards neurological manifestation. With the availability of the Atp7b-/- mice model, we engaged a preclinical study leading to the qualification of a panel of 7 biomarker candidates. These results allowed us to raise the interest of the National Reference Center for Wilson’s disease (CNR) medical teams in Lyon and Paris and to engage a close collaboration to initiate clinical study. Using a first plasma cohort from Wilson’s disease patients, we assessed the translational and clinical value of the 7 biomarker candidates and engage discovery study on patients’ plasma samples. Furthermore, we also studied the molecular mechanisms involved in liver pathophysiology using the Atp7b-/- mice model using discovery proteomics. These investigations led to the identification of a new potential therapeutic target.La maladie de Wilson est une atteinte génétique rare associée à des mutations du gène codant pour l’ATP7B, protéine de transport et d’excrétion du cuivre dans l’organisme, entrainant une accumulation toxique de cuivre dans l’organisme au niveau du foie et du cerveau. La difficulté d’établir une corrélation génotype-phénotype et la grande hétérogénéité du tableau clinique conduisent à des difficultés de prise en charge, notamment concernant le diagnostic et le suivi biologique et thérapeutique des patients. Dans cette étude, nous avons orienté nos recherches vers la découverte et l’évaluation de candidats biomarqueurs de diagnostics et de pronostics précoce de l’évolution vers des formes neurologiques de la maladie. L’accessibilité au modèle préclinique murin Atp7b-/- nous a permis d’engager une étude préclinique permettant dans un premier temps d’optimiser la partie expérimentale pour l’identification la plus fiable de nouveaux candidats biomarqueurs plasmatiques. Cette étude a permis l’identification d’un panel de 7 candidats biomarqueurs. Ces résultats nous ont permis de susciter l’intérêt des équipes médicales du Centre National de Référence Wilson (CNR) à Lyon et à Paris et d’engager une étroite collaboration pour débuter l’étude clinique. L’obtention d’une première cohorte d’échantillons plasmatiques provenant de patients atteints de la maladie de Wilson a permis d’évaluer la valeur translationnelle et clinique des candidats biomarqueurs identifiés et de débuter l’étude clinique d’exploration du protéome plasmatique de patients atteints de la maladie de Wilson. En outre, la compréhension des mécanismes moléculaires associés au développement de la physiopathologie hépatique a été étudiée et a permis de mettre en évidence de nouvelles cibles pour, à terme, améliorer la prise en charge clinique des patients atteints de la maladie de Wilson

Développement de méthodes d’analyse protéomique pour l’exploration translationnelle de la maladie de Wilson.

Wilson’s disease is a rare genetic disorder triggered by mutations in the ATP7B gene, which encodes a transport protein involved in copper transport and excretion, triggering toxic copper overloads in the liver and the brain. The lack of genotype-phenotype correlation and phenotype variability lead to clinical care difficulties, especially for the diagnosis and biological follow up of patients. In this study, we initiated the discovery and evaluation of biomarker candidates for the diagnosis of Wilson’s disease and for early prognosis towards neurological manifestation. With the availability of the Atp7b-/- mice model, we engaged a preclinical study leading to the qualification of a panel of 7 biomarker candidates. These results allowed us to raise the interest of the National Reference Center for Wilson’s disease (CNR) medical teams in Lyon and Paris and to engage a close collaboration to initiate clinical study. Using a first plasma cohort from Wilson’s disease patients, we assessed the translational and clinical value of the 7 biomarker candidates and engage discovery study on patients’ plasma samples. Furthermore, we also studied the molecular mechanisms involved in liver pathophysiology using the Atp7b-/- mice model using discovery proteomics. These investigations led to the identification of a new potential therapeutic target.La maladie de Wilson est une atteinte génétique rare associée à des mutations du gène codant pour l’ATP7B, protéine de transport et d’excrétion du cuivre dans l’organisme, entrainant une accumulation toxique de cuivre dans l’organisme au niveau du foie et du cerveau. La difficulté d’établir une corrélation génotype-phénotype et la grande hétérogénéité du tableau clinique conduisent à des difficultés de prise en charge, notamment concernant le diagnostic et le suivi biologique et thérapeutique des patients. Dans cette étude, nous avons orienté nos recherches vers la découverte et l’évaluation de candidats biomarqueurs de diagnostics et de pronostics précoce de l’évolution vers des formes neurologiques de la maladie. L’accessibilité au modèle préclinique murin Atp7b-/- nous a permis d’engager une étude préclinique permettant dans un premier temps d’optimiser la partie expérimentale pour l’identification la plus fiable de nouveaux candidats biomarqueurs plasmatiques. Cette étude a permis l’identification d’un panel de 7 candidats biomarqueurs. Ces résultats nous ont permis de susciter l’intérêt des équipes médicales du Centre National de Référence Wilson (CNR) à Lyon et à Paris et d’engager une étroite collaboration pour débuter l’étude clinique. L’obtention d’une première cohorte d’échantillons plasmatiques provenant de patients atteints de la maladie de Wilson a permis d’évaluer la valeur translationnelle et clinique des candidats biomarqueurs identifiés et de débuter l’étude clinique d’exploration du protéome plasmatique de patients atteints de la maladie de Wilson. En outre, la compréhension des mécanismes moléculaires associés au développement de la physiopathologie hépatique a été étudiée et a permis de mettre en évidence de nouvelles cibles pour, à terme, améliorer la prise en charge clinique des patients atteints de la maladie de Wilson

Development of proteomic analysis methods for translational exploration of Wilson's disease

La maladie de Wilson est une atteinte génétique rare associée à des mutations du gène codant pour l’ATP7B, protéine de transport et d’excrétion du cuivre dans l’organisme, entrainant une accumulation toxique de cuivre dans l’organisme au niveau du foie et du cerveau. La difficulté d’établir une corrélation génotype-phénotype et la grande hétérogénéité du tableau clinique conduisent à des difficultés de prise en charge, notamment concernant le diagnostic et le suivi biologique et thérapeutique des patients. Dans cette étude, nous avons orienté nos recherches vers la découverte et l’évaluation de candidats biomarqueurs de diagnostics et de pronostics précoce de l’évolution vers des formes neurologiques de la maladie. L’accessibilité au modèle préclinique murin Atp7b-/- nous a permis d’engager une étude préclinique permettant dans un premier temps d’optimiser la partie expérimentale pour l’identification la plus fiable de nouveaux candidats biomarqueurs plasmatiques. Cette étude a permis l’identification d’un panel de 7 candidats biomarqueurs. Ces résultats nous ont permis de susciter l’intérêt des équipes médicales du Centre National de Référence Wilson (CNR) à Lyon et à Paris et d’engager une étroite collaboration pour débuter l’étude clinique. L’obtention d’une première cohorte d’échantillons plasmatiques provenant de patients atteints de la maladie de Wilson a permis d’évaluer la valeur translationnelle et clinique des candidats biomarqueurs identifiés et de débuter l’étude clinique d’exploration du protéome plasmatique de patients atteints de la maladie de Wilson. En outre, la compréhension des mécanismes moléculaires associés au développement de la physiopathologie hépatique a été étudiée et a permis de mettre en évidence de nouvelles cibles pour, à terme, améliorer la prise en charge clinique des patients atteints de la maladie de Wilson.Wilson’s disease is a rare genetic disorder triggered by mutations in the ATP7B gene, which encodes a transport protein involved in copper transport and excretion, triggering toxic copper overloads in the liver and the brain. The lack of genotype-phenotype correlation and phenotype variability lead to clinical care difficulties, especially for the diagnosis and biological follow up of patients. In this study, we initiated the discovery and evaluation of biomarker candidates for the diagnosis of Wilson’s disease and for early prognosis towards neurological manifestation. With the availability of the Atp7b-/- mice model, we engaged a preclinical study leading to the qualification of a panel of 7 biomarker candidates. These results allowed us to raise the interest of the National Reference Center for Wilson’s disease (CNR) medical teams in Lyon and Paris and to engage a close collaboration to initiate clinical study. Using a first plasma cohort from Wilson’s disease patients, we assessed the translational and clinical value of the 7 biomarker candidates and engage discovery study on patients’ plasma samples. Furthermore, we also studied the molecular mechanisms involved in liver pathophysiology using the Atp7b-/- mice model using discovery proteomics. These investigations led to the identification of a new potential therapeutic target

Synthesis of photoactive polymer colloids by polymerization in aqueous dispersed media to product singlet oxygen

International audienceAPME 2019: The 13thInternational Conference on Advanced Polymers via Macromolecular Engineering, 15-18April 2019, Stellenbosch, SASYNTHESIS OF PHOTOACTIVE POLYMER COLLOIDS BY POLYMERIZATION IN AQUEOUS DISPERSEDMEDIA TO PRODUCE SINGLET OXYGENCharlène Boussiron, Luca Petrizza, Mickaël Le Bechec, Sylvie Lacombe, Maud Save*CNRS/ Univ Pau & Pays Adour/ E2S UPPA, IPREM, Institut des Sciences Analytiqueset de Physicochimie pour l'Environnement et les Matériaux (IPREM), UMR5254, 64000, Pau, France.* email: [email protected] design of photoactive polymer substrates producing singlet oxygen under visible light irradiation has great technological potential. Production of singlet oxygen (1O2), a selective reactive oxygen species (ROS) produced by irradiation under visible light of organic photosensitizers, has attracted increasing interest in the fields of fine chemistry,1photo-decontamination of air/water,2antimicrobial materials,3or Photodynamic Therapy (PDT).4Immobilization of photosensitizers on solid substrates improves their handling, recyclability, stability and facilitates purification steps to remove photocatalyst from reactants in fine chemistry. In that context, functional waterborne latex synthesized by the scalable emulsion polymerization process, are versatile to prepare photosensitizer-supported materials either as stable colloidal particles or as polymer film. We will present two strategies to design photosensitizer-grafted polymer colloids. First, film forming latex particle with a decorated shell will be synthesized by polymerization-induced self-assembly (PISA, Fig. 1 left).5Secondly, crosslinked particles will be synthesized by miniemulsion copolymerization to produce core-functionalized particles(PISA, Fig. 1 right). The organic photosensitizer chosen to synthesize the functional monomer is the Rose Bengal molecule

Synthesis of Film-Forming Photoactive Latex Particles by Emulsion Polymerization-Induced Self-Assembly to Produce Singlet Oxygen

International audienceThe design of photoactive polymer substrates producing singlet oxygen under visible light irradiation has great technological potential. Aqueous dispersion of novel photoactive core-shell particles was synthesized by surfactant-free reversible addition-fragmentation chain transfer (RAFT) emulsion polymerization of n-butyl acrylate. The surface of the nanoparticles is directly decorated thanks to the polymerization-induced self-assembly process using a hydrophilic macromolecular chain transfer agent (macro-CTA) functionalized with the organic photosensitizer. The macro-CTA was synthesized by statistical copolymerization of acrylic acid and 2-Rose Bengal ethyl acrylate (RBEA) at 80 °C mediated with 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid. Monitoring polymerization kinetics of RAFT polymerization highlights that increasing amount of RBEA induces retardation, still more pronounced when using the vinylbenzyl Rose Bengal comonomer. The present work provides insight into the quantum yield of singlet oxygen production in water (ΦΔ = 0.2-0.6) for the three types of synthesized polymers (hydrophilic polymer, latex particles, and polymer film). The photoactive core-shell latex particles enabled the easy preparation of photoactive polymer film by simple casting

Tuning photosensitized singlet oxygen production from microgels synthesized by polymerization in aqueous dispersed media

International audienceNovel sub-micronic photoactive polymer colloids grafted with Rose Bengal (RB) photosensitizer were designed to promote singlet oxygen production from a supported organic photosensitizer. Photooxygenation of fine chemicals under visible light irradiation is considered as a green process. To enhance the overall process sustainability, stable colloidal particles were synthesized by polymerization in aqueous dispersed media with the ability to be transferred into ethanol, recycled by a centrifugation step and reused with no significant decrease of the quantum yield of singlet oxygen production. The microgels were synthesized for the first time by miniemulsion copolymerization of vinyl acetate (VAc), N-vinyl caprolactam (VCL), polymerizable vinyl benzyl Rose Bengal (VBRB) monomers and divinyl adipate (DVA) crosslinker. The microgels were characterized by UV-visible spectroscopy and compared with the homologue non-crosslinked polymer in order to discriminate the effect of RB grafted onto the linear polymer from its grafting inside crosslinked microgels. The quantum yields of singlet oxygen production were almost null in water but interestingly in the range of 0.27–0.47 in ethanol. The singlet oxygen quantum yield of these polymer materials is tuned by the aggregation state of VBRB units, hence producing an ON/OFF photosensitizing colloidal system. The absorption and emission spectra of the VBRB containing microgels in water were characteristic of strongly aggregated VBRB, while no evidence of aggregation was observed from the spectra in ethanol. The highest singlet oxygen quantum yield of the linear polymer was correlated with a less aggregated state of RB units compared with the crosslinked microgels. The present RB-based microgels were 20% more resistant to photobleaching than free RB

From solutions to porous film: Influence of the media on fluorescent acridine end-capped polystyrene photophysic properties

International audienceNitroxide-mediated radical polymerization (NMP) was used to synthesize polystyrene (PS) in the presence of N-tert-butyl-N-(1-diethylphosphono-2,2-dimethylpropyl)nitroxide (SG1) as a control agent and the BlocBuilder® alkoxyamine as an initiator. The α-telechelic carboxylic acid PS was further functionalized by the fluorescent 9-aminoacridine moiety (9AA) to synthesize α-functional fluorescent polystyrene (PS-9AA). The 9-aminoacridine end-capped PS was characterized by size exclusion chromatography, two dimensional heteronuclear multiple-bond correlation nuclear magnetic resonance and fluorescence spectroscopy. The PS-9AA α-telechelic polymer was used to prepare fluorescent continuous and porous polymer films via the Breath Figure (BF) method. The fluorescence properties of PS-9AA in solution were compared to the corresponding one in the continuous and porous polymer films

Patient-reported outcomes with direct-acting antiviral treatment for hepatitis C in West and Central Africa (TAC ANRS 12311 trial)

Background & Aims: Patient-reported outcomes (PROs) are poorly documented for patients with chronic hepatitis C on direct-acting antiviral (DAA) treatment in low-to-middle-income countries. We documented PROs during and after DAA treatment in participants of the TAC ANRS 12311 trial (West and Central Africa). Methods: Trial participants received a 12-week regimen containing either sofosbuvir plus ribavirin (HCV genotype 2, n = 40), or sofosbuvir plus ledipasvir (HCV genotypes 1 and 4, n = 80). Health-related quality of life (SF-12), fatigue (Piper Fatigue scale), and self-reported symptoms (35-symptom list) were assessed at enrolment (Week (W) 0), during treatment (W2, W4, W8 and W12) and after treatment (W24 and W36). These PROs were compared between W0 and W36 (Wilcoxon signed-rank or McNemar tests). Mixed-effects linear regression models helped identify correlates of physical and mental quality of life component summaries (PCS and MCS) in a longitudinal analysis. Results: Most PROs were significantly improved 24 weeks after treatment end (W36), without significant differences between treatment groups. For the post-treatment period, multivariable analysis showed significant increases in PCS for patients with cirrhosis and in MCS for patients in the sofosbuvir plus ribavirin group. A higher number of self-reported symptoms at W0 was associated with lower PCS and MCS, older age and cirrhosis with lower PCS, and male sex and HCV cure with higher PCS. Conclusions: Sofosbuvir-based DAA therapy was associated with a significant improvement in PROs 6 months after treatment end in patients with chronic HCV infection from Central and West Africa. These findings may guide HCV treatment providers in low-to-middle-income countries to deliver pre-treatment information concerning the benefits of DAAs beyond viral eradication. ClinicalTrials.gov Identifier: NCT02405013. Impact and implications: Perceptions and experiences (i.e. “patient-reported outcomes”) of patients with chronic hepatitis C receiving direct-acting antivirals (DAAs) are poorly documented in the African setting. This study shows significant improvements in health-related quality of life, fatigue, and self-reported symptoms 24 weeks after the end of a 12-week sofosbuvir-based DAA regimen in 120 patients from Central and West Africa. These findings substantially add to the body of knowledge about DAA therapy in the African setting. Treatment providers should be encouraged to inform patients of the benefits of DAAs beyond viral eradication, to increase treatment adherence and retention in care

Efficient Photooxygenation Process of Biosourced α-Terpinene by Combining Controlled LED-Driven Flow Photochemistry and Rose Bengal-Anchored Polymer Colloids

International audienceThis work studies the reactivity of poly(N-vinylcaprolactam-co-vinyl acetate-co-vinylbenzyl Rose Bengal) microgels (VBRB@MG) as heterogeneous photosensitizers in a continuous-flow process for sustainable singlet oxygen sensitized photooxygenation of a bio-based molecule. Experiments were carried out in a LED-driven spiral-shaped microreactor in which slurry Taylor flows were generated, allowing accurate control of irradiation, light absorption and gas-liquid flow conditions. The benchmark photooxygenation of -terpinene was implemented in ethanol to provide a green solvent using air as a safe supply of oxygen. Swollen RB-grafted colloids formed an efficient substrate for converting -terpinene into ascaridole, providing up to high conversion with high selectivity under continuous-flow conditions, and within short residence times of a few minutes. The supported RB exhibited a reactivity similar to that of the free RB. The reactivity of the supported photosensitizer was maintained for several cycles with a reproducible level after 8 months of storage. Under experimental conditions favouring photobleaching of RB, the photobleaching level of RB was lower with the VBRB@MG colloids than with free RB, suggesting that grafting RB molecules onto the colloid can prevent their photodegradation. KEYWORDS Flow photochemistry. Singlet oxygen. Photoactive polymer colloids. Green conditions. Rose Bengal. Alpha-terpinene

Proteomic characterization of human exhaled breath condensate

International audienc