Etude des interactions protéine-polymères dans des conditions relevant de la formulation de vecteurs particulaires

Dans le cadre de cette étude, les propriétés interfaciales de polymères d'intérêt thérapeutique ont été caractérisées. Des films de poly(a-hydroxy acide)s (PAHA), le PLA50 et le PLA37,5GA25, et des poly(éthylène glycol)s, en particulier le PEG2000 et le PEG4000, formés par étalement ou par adsorption, séparément , sous forme de mélanges ou de copolymère dibloc, ont été étudiés à des interfaces fluide/liquide, en présence ou non d'une protéine, le lysozyme du blanc d'oeuf de poule. Pour ce faire, différentes techniques de tensiométrie (balance de Langmuir, tensiomètre à goutte), l'AFM, la réflectivité des rayons X et l'application d'un modèle rhéologique de type Maxwell ont été utilisés. Il a été montré que selon la nature des polymères, les segments de PEG restent enchevêtrés avec ceux de PAHA dans le plan de l'interface ou désorbent. Par ailleurs, les interactions entre PEG et lysozyme ont été caractérisées par différentes méthodes spectrophotométriques en volume, tout comme l'a été l'influence de la force ionique sur la stabilité conformationnelle de la protéine. Il a été montré que l'empêchement de l'adsorption interfaciale du lysozyme est dépendant de la densité de segments de PEG ancrés à l'interface. L'ensemble de ces résultats a permis de déterminer les interactions interfaciales existant entre les polymères impliqués dans la formulation de systèmes particulaires et d'expliquer expérimentalement les stratégies utilisées pour empêcher l'adsorption protéique.In this study, the interfacial properties of polymers of therapeutic interest were characterized. Some spreading or adsorbed films of poly(a-hydroxy acid)s (PAHA), the PLA50 and the PLA37.5GA25, and some poly(ethylene glycol)s, and among them, the PEG2,000 and the PEG4,000, were studied separately, or as a physical mixture, or as a dibloc copolymer, at some fluid/liquid interfaces, in presence or not of hen egg-white lysozyme. In this goal, different tensiometric techniques (the Langmuir trough, the pendant drop method), AFM, and X-ray reflectivity measurements were used, and a rheological Maxwell model was applied. According to the polymers, PEG segments remain interpenetrated with those of PAHA in the plane of the interface, or they desorb. Furthermore, results of the study on PEG-lysozyme bulk interactions by various spectrophotometric methods are reported. The influence of the ionic strength on the protein conformational stability was also tested. It was shown that the prevention of lysozyme adsorption depends on the PEG chain density anchored at the considered interface. From these different results, interfacial interactions between polymers implied in the formulation of therapeutic particles were determined, and the strategies used to prevent protein adsorption were clarified.ANGERS-BU Médecine-Pharmacie (490072105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Comparative analysis of response to selection with three insecticides in the dengue mosquito Aedes aegypti using mRNA sequencing

Background Mosquito control programmes using chemical insecticides are increasingly threatened by the development of resistance. Such resistance can be the consequence of changes in proteins targeted by insecticides (target site mediated resistance), increased insecticide biodegradation (metabolic resistance), altered transport, sequestration or other mechanisms. As opposed to target site resistance, other mechanisms are far from being fully understood. Indeed, insecticide selection often affects a large number of genes and various biological processes can hypothetically confer resistance. In this context, the aim of the present study was to use RNA sequencing (RNA-seq) for comparing transcription level and polymorphism variations associated with adaptation to chemical insecticides in the mosquito Aedes aegypti. Biological materials consisted of a parental susceptible strain together with three child strains selected across multiple generations with three insecticides from different classes: the pyrethroid permethrin, the neonicotinoid imidacloprid and the carbamate propoxur. Results After ten generations, insecticide-selected strains showed elevated resistance levels to the insecticides used for selection. RNA-seq data allowed detecting over 13,000 transcripts, of which 413 were differentially transcribed in insecticide-selected strains as compared to the susceptible strain. Among them, a significant enrichment of transcripts encoding cuticle proteins, transporters and enzymes was observed. Polymorphism analysis revealed over 2500 SNPs showing > 50% allele frequency variations in insecticide-selected strains as compared to the susceptible strain, affecting over 1000 transcripts. Comparing gene transcription and polymorphism patterns revealed marked differences among strains. While imidacloprid selection was linked to the over transcription of many genes, permethrin selection was rather linked to polymorphism variations. Focusing on detoxification enzymes revealed that permethrin selection strongly affected the polymorphism of several transcripts encoding cytochrome P450 monooxygenases likely involved in insecticide biodegradation. Conclusions The present study confirmed the power of RNA-seq for identifying concomitantly quantitative and qualitative transcriptome changes associated with insecticide resistance in mosquitoes. Our results suggest that transcriptome modifications can be selected rapidly by insecticides and affect multiple biological functions. Previously neglected by molecular screenings, polymorphism variations of detoxification enzymes may play an important role in the adaptive response of mosquitoes to insecticides

Photochromism and Dual-Color Fluorescence in a Polyoxometalate-Benzospiropyran Molecular Switch

The photophysical properties of a Keggin-type polyoxometalate (POM) covalently bounded to a benzospiropyran (BSPR) unit have been investigated. These studies reveal that both closed and open forms are emissive with distinct spectral features (lambda(em) (closed form) - 530 nm, lambda(em) (open form) = 670 nm) and that the fluorescence of the BSPR unit of the hybrid is considerably enhanced compared to BSPR parent compounds. While the fluorescence excitation energy of the BSPR reference compounds (370 nm) is close to the intense absorption responsible of the photochromic character (350 nm), the fluorescence excitation of the hybrid is shifted to lower energy (400 nm), improving the population of the emissive state. Combined NOESY NMR and theoretical calculations of the closed form of the hybrid give an intimate understanding of the conformation adopted by the hybrid and show that the nitroaryl moieties of the BSPR is folded toward the POM, which should affect the electronic properties of the BSPR

Hierarchical Self-Assembly of Polyoxometalate-Based Organo Palladium(II) Metallomacrocycles via Electrostatic Interactions

International audienceThe design and synthesis of a supramolecular square composed of polyoxometalate-based hybrid donors and ethylenediamine palladium(II) nodes are reported. The structure of the metallomacrocycle scaffold was inferred by diffusion NMR, small-angle X-ray scattering (SAXS), and molecular modeling. The metallomacrocycle scaffold that contains negatively and positively charged subunits can further self-assemble owing to a competition between the solvation energy of the discrete species and intermolecular electrostatic interactions. When the dissociating character of the solvent was lowered or when in the presence of a protic solvent, different types of multiscale organizations (vesicles and pseudo-1D structures) were selectively formed and were characterized by SAXS and transmission electron microscopy

NF-κB-Mediated Modulation of Inducible Nitric Oxide Synthase Activity Controls Induction of the Epstein-Barr Virus Productive Cycle by Transforming Growth Factor Beta 1▿†

Transforming growth factor beta 1 (TGF-β1) signal transduction has been implicated in many second-messenger pathways, including the NF-κB pathway. We provide evidence of a novel TGF-β1-mediated pathway that leads to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, which in turn induces expression of an Epstein-Barr virus (EBV) protein, ZEBRA, that is responsible for the induction of the viral lytic cycle. This pathway includes two unexpected steps, both of which are required to control ERK 1/2 phosphorylation: first, a quick and transient activation of NF-κB, and second, downregulation of inducible nitric oxide synthase (iNOS) activity that requires the participation of NF-κB activity. Although necessary, NF-κB alone is not sufficient to produce downregulation of iNOS, suggesting that another uncharacterized event(s) is involved in this pathway. Dissection of the steps involved in the switch from the EBV latent cycle to the lytic cycle will be important to understand how virus-host relationships modulate the innate immune system

uAUG creating variants in the 5’UTR of ENG causing Hereditary Hemorrhagic Telangiectasia

International audienceHereditary Hemorrhagic Telangiectasia (HHT) is a rare, autosomal dominant, vascular disorder. About 80% of cases are caused by pathogenic variants in ACVRL1 (also known as ALK1) and ENG, with the remaining cases being unexplained. We identified two variants, c.-79C>T and c.-68G>A, in the 5’UTR of ENG in two unrelated patients. They create upstream AUGs at the origin of upstream overlapping open reading frames (uoORFs) ending at the same stop codon. To assess the pathogenicity of these variants, we performed functional assays based on the expression of wild-type and mutant constructs in human cells and evaluated their effect on ALK1 activity in a BMP-response element assay. This assay is mandatory for molecular diagnosis and has been so far only applied to coding ENG variants. These variants were associated with a decrease of protein levels in HeLa and HUVEC cells and a decreased ability to activate ALK1. We applied the same experiments on three additional uoORF-creating variants (c.-142A>T, c.-127C>T and c.-10C>T) located in the 5’UTR of ENG and previously reported in HHT patients. We found that all the analyzed variants alter protein levels and function. Additional experiments relying on an artificial deletion in our mutated constructs show that identified uAUGs could initiate the translation indicating that the associated effect is translation-dependent. Overall, we have identified two 5’UTR ENG variations in HHT patients and shed new light on the role of upstream ORFs on ENG regulation. Our findings contribute to the amelioration of molecular diagnosis in HHT

New Magnetic compilation and interpretation of the Bay of Biscay and surrounding continental shelves

International audienc

Impact of Subthalamic Deep Brain Stimulation on Impulse Control Disorders in Parkinson's Disease: A Prospective Study

International audienceBackground Impact of subthalamic deep brain stimulation (DBS) on impulse control disorders (ICD) in Parkinson's disease (PD) remains controversial. Objectives The objectives of this study were to analyze the natural history of ICD between baseline and 1 year after subthalamic DBS in patients with PD and to identify predictive factors, taking into account the positions of the active contact and stimulation parameters. Methods We analyzed postoperative modifications of ICD based on the multicentric, prospective Predictive Factors and Subthalamic Stimulation in Parkinson's Disease cohort. ICD status and Ardouin Scale of Behaviour in PD were assessed at baseline and 1 year following subthalamic DBS. Location of active contacts within the 3 subthalamic nucleus functional territories was investigated. Results A total of 217 were patients included. Of the patients, 10.6% had ICD at baseline of which 95.6% improved at 1 year following subthalamic DBS; 3.6% of the patients experienced de novo ICD at 1 year following subthalamic DBS. Dopamine agonist dose reduction (from 309.8 to 109.3 mg) was the main driver of ICD regression (P= 0.05). Higher preoperative dyskinesias were associated with poorer ICD evolution (P= 0.04). Whereas baseline apathy was a risk factor of de novo ICD (P= 0.02), ICD improvement correlated with postoperative apathy (P= 0.004). Stimulation power and position of active contacts-mainly located within the sensorimotor part of the subthalamic nucleus-did not influence ICD. Conclusions This 1-year, postoperative follow-up study showed ICD regression and dopaminergic drug reduction with optimal position of the active contacts within the subthalamic nucleus. Whereas patients with PD with preoperative ICD were prone to postoperative apathy, we also showed that those with preoperative apathy had a higher risk to experience postoperative de novo ICD, further highlighting the meaningful influence of postoperative management of dopaminergic medication on outcome and the continuum between apathy and ICD. (c) 2020 International Parkinson and Movement Disorder Societ

Whole-body MR imaging in suspected physical child abuse: comparison with skeletal survey and bone scintigraphy findings from the PEDIMA prospective multicentre study

International audienceObjectives: To assess the contribution of whole-body magnetic resonance imaging (WBMRI) and bone scintigraphy (BS) in addition to skeletal survey (SS) in detecting traumatic bone lesions and soft-tissue injuries in suspected child abuse.Methods: In this prospective, multicentre, diagnostic accuracy study, children less than 3 years of age with suspected physical abuse were recruited. Each child underwent SS, BS and WBMRI. A blinded first review was performed in consensus by five paediatric radiologists and three nuclear medicine physicians. A second review investigated discrepancies reported between the modalities using a consensus result of all modalities as the reference standard. We calculated the sensitivity, specificity and corresponding 95% confidence interval for each imaging modality (SS, WBMRI and BS) and for the combinations [SS + WBMRI] and [SS + BS].Results: One hundred seventy children were included of which sixty-four had at least one lesion. In total, 146 lesions were included. The sensitivity and specificity of each examination were, respectively, as follows: 88.4% [95% CI, 82.0-93.1] and 99.7% [95% CI, 99.5-99.8] for the SS, 69.9% [95% CI, 61.7-77.2] and 99.5% [95% CI, 99.2-99.7] for WBMRI and 54.8% [95% CI, 46.4-63.0] and 99.7% [95% CI, 99.5-99.9] for BS. Sensitivity and specificity were, respectively, 95.9% [95% CI, 91.3-98.5] and 99.2% [95% CI, 98.9-99.4] for the combination SS + WBMRI and 95.2% [95% CI, 90.4-98.1] and 99.4% [95% CI, 99.2-99.6] for the combination SS + BS, with no statistically significant difference between them.Conclusion: SS was the most sensitive independent imaging modality; however, the additional combination of either WBMRI or BS examinations offered an increased accuracy.Key points: • SS in suspected infant abuse was the most sensitive independent imaging modality in this study, especially for detecting metaphyseal and rib lesions, and remains essential for evaluation. • The combination of either SS + BS or SS + WBMRI provides greater accuracy in diagnosing occult and equivocal bone injuries in the difficult setting of child abuse. • WBMRI is a free-radiation technique that allows additional diagnosis of soft-tissue and visceral injuries