Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative Luciferase-Vpr packaging-based assay

The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity

Socioeconomic Influence on Caries Susceptibility in Juvenile Individuals with Limited Dental Care: Example from an Early Middle Age Population (Great Moravia, 9th-10th Centuries A.D., Czech Republic)

International audienc

Cell Entry and Trafficking of Human Adenovirus Bound to Blood Factor X Is Determined by the Fiber Serotype and Not Hexon:Heparan Sulfate Interaction

Human adenovirus serotype 5 (HAdV5)-based vectors administered intravenously accumulate in the liver as the result of their direct binding to blood coagulation factor X (FX) and subsequent interaction of the FX-HAdV5 complex with heparan sulfate proteoglycan (HSPG) at the surface of liver cells. Intriguingly, the serotype 35 fiber-pseudotyped vector HAdV5F35 has liver transduction efficiencies 4-logs lower than HAdV5, even though both vectors carry the same hexon capsomeres. In order to reconcile this apparent paradox, we investigated the possible role of other viral capsid proteins on the FX/HSPG-mediated cellular uptake of HAdV5-based vectors. Using CAR- and CD46-negative CHO cells varying in HSPG expression, we confirmed that FX bound to serotype 5 hexon protein and to HAdV5 and HAdV5F35 virions via its Gla-domain, and enhanced the binding of both vectors to surface-immobilized hypersulfated heparin and cellular HSPG. Using penton mutants, we found that the positive effect of FX on HAdV5 binding to HSPG and cell transduction did not depend on the penton base RGD and fiber shaft KKTK motifs. However, we found that FX had no enhancing effect on the HAdV5F35-mediated cell transduction, but a negative effect which did not involve the cell attachment or endocytic step, but the intracellular trafficking and nuclear import of the FX-HAdV5F35 complex. By cellular imaging, HAdV5F35 particles were observed to accumulate in the late endosomal compartment, and were released in significant amounts into the extracellular medium via exocytosis. We showed that the stability of serotype 5 hexon∶FX interaction was higher at low pH compared to neutral pH, which could account for the retention of FX-HAdV5F35 complexes in the late endosomes. Our results suggested that, despite the high affinity interaction of hexon capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the dominant determinant of the internalization and trafficking pathway of HAdV5-based vectors

Protuberant Electrode Structures for New Retinal Implants

Focal stimulation of neural tissue is a challenge in retinal prosthetic devices in which the aim is to improve the spatial resolution of the stimulation and therefore increase the electrode density of the prosthetic devices. Our work intends to create a new implant able to enhance the focalisation of the stimulation signal through protuberant electrodes. These electrodes are micro fabricated on a soft polyimide substrate using classical metal electrodeposition techniques. Before proceeding with fabrication a FEM model of the electrode’s current density was done to select the best-performing structures and geometries in terms of local stimulation. Based on these models, several prototypes were fabricated and implanted in vivo into a rat’s eye to verify the adaptation to the retina tissue

Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells

Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins

Rapid Typing of Transmissible Spongiform Encephalopathy Strains with Differential ELISA

A strain-typing ELISA distinguishes bovine spongiform encephalopathy from other scrapie strains in small ruminants

Different viral genes modulate virulence in model mammal hosts and Culex pipiens vector competence in Mediterranean basin lineage 1 West Nile virus strains

West Nile virus (WNV) is a single-stranded positive-sense RNA virus (+ssRNA) belonging to the genus Orthoflavivirus. Its enzootic cycle involves mosquito vectors, mainly Culex, and wild birds as reservoir hosts, while mammals, such as humans and equids, are incidental dead-end hosts. It was first discovered in 1934 in Uganda, and since 1999 has been responsible for frequent outbreaks in humans, horses and wild birds, mostly in America and in Europe. Virus spread, as well as outbreak severity, can be influenced by many ecological factors, such as reservoir host availability, biodiversity, movements and competence, mosquito abundance, distribution and vector competence, by environmental factors such as temperature, land use and precipitation, as well as by virus genetic factors influencing virulence or transmission. Former studies have investigated WNV factors of virulence, but few have compared viral genetic determinants of pathogenicity in different host species, and even fewer have considered the genetic drivers of virus invasiveness and excretion in Culex vector. In this study, we characterized WNV genetic factors implicated in the difference in virulence observed in two lineage 1 WNV strains from the Mediterranean Basin, the first isolated during a significant outbreak reported in Israel in 1998, and the second from a milder outbreak in Italy in 2008. We used an innovative and powerful reverse genetic tool, e.g., ISA (infectious subgenomic amplicons) to generate chimeras between Israel 1998 and Italy 2008 strains, focusing on non-structural (NS) proteins and the 3′UTR non-coding region. We analyzed the replication of these chimeras and their progenitors in mammals, in BALB/cByJ mice, and vector competence in Culex (Cx.) pipiens mosquitoes. Results obtained in BALB/cByJ mice suggest a role of the NS2B/NS3/NS4B/NS5 genomic region in viral attenuation in mammals, while NS4B/NS5/3′UTR regions are important in Cx. pipiens infection and possibly in vector competence

INDIFFERENCIE : DROIT DE L'HOMME ET UNION EUROPEENNE

Conférences de l’IREDIES / IREDIES Conference Paper n° 2 /2019La présente étude est le fruit d’un travail collectif réalisé par les étudiants de la promotion 2018-2019 du Master 2 « Droits de l’homme et Union européenne » de l’Ecole de droit de la Sorbonne (Paris 1). Ce diplôme – qui a l’intérêt de disposer d’un séminaire entièrement consacré à l’étude de la Charte des droits fondamentaux de l’Union européenne – a été le lieu de réflexion de cette recherche menée avec enthousiasme et professionnalisme par tous les étudiants. Les travaux doctrinaux sur ce texte novateur sont en général centrés sur la manière dont la Cour de justice de l’Union l’interprète et l’applique. Or, il ne s’agit là que de la face visible de l’iceberg contentieux. Dans ce contexte, il est apparu essentiel de s’interroger sur la manière dont les juridictions internes avaient apprivoisé ce texte moderne (au regard de la variété des droits qu’il consacre) et complexe (au regard de son champ d’application).Les étudiants se sont répartis en trois groupes afin d’étudier la manière dont les juridictions internes (Conseil constitutionnel, Conseil d’Etat et Cour de cassation), mais également le législateur, se sont emparés de ce texte.Les résultats de cette étude, où l’outil statistique a constitué un support cardinal, démontrent que si la Charte des droits fondamentaux est connue du pouvoir législatif et des « acteurs » judiciaires, il n’est pas encore devenu une référence incontournable du processus législatif et de l’argumentaire des juges. L’antériorité de la Convention de sauvegarde des droits et libertés fondamentales comme les spécificités du champ d’application de la Charte sont deux éléments explicatifs d’un tel état de fait.Ce travail, mené avec sérieux et rigueur, méritait d’être rendu public au sein de la collection des Working papers de l’IREDIES

A phosphatase cascade by which rewarding stimuli control nucleosomal response

ArticleInternational audienceDopamine orchestrates motor behaviour and reward-driven learning. Perturbations of dopamine signalling have been implicated in several neurological and psychiatric disorders, and in drug addiction. The actions of dopamine are mediated in part by the regulation of gene expression in the striatum, through mechanisms that are not fully understood. Here we show that drugs of abuse, as well as food reinforcement learning, promote the nuclear accumulation of 32-kDa dopamine-regulated and cyclic-AMP-regulated phosphoprotein (DARPP-32). This accumulation is mediated through a signalling cascade involving dopamine D1 receptors, cAMP-dependent activation of protein phosphatase-2A, dephosphorylation of DARPP-32 at Ser 97 and inhibition of its nuclear export. The nuclear accumulation of DARPP-32, a potent inhibitor of protein phosphatase-1, increases the phosphorylation of histone H3, an important component of nucleosomal response. Mutation of Ser 97 profoundly alters behavioural effects of drugs of abuse and decreases motivation for food, underlining the functional importance of this signalling cascad

Comparative transcriptomics of drought responses in Populus: a meta-analysis of genome-wide expression profiling in mature leaves and root apices across two genotypes

<p>Abstract</p> <p>Background</p> <p>Comparative genomics has emerged as a promising means of unravelling the molecular networks underlying complex traits such as drought tolerance. Here we assess the genotype-dependent component of the drought-induced transcriptome response in two poplar genotypes differing in drought tolerance. Drought-induced responses were analysed in leaves and root apices and were compared with available transcriptome data from other <it>Populus </it>species.</p> <p>Results</p> <p>Using a multi-species designed microarray, a genomic DNA-based selection of probesets provided an unambiguous between-genotype comparison. Analyses of functional group enrichment enabled the extraction of processes physiologically relevant to drought response. The drought-driven changes in gene expression occurring in root apices were consistent across treatments and genotypes. For mature leaves, the transcriptome response varied weakly but in accordance with the duration of water deficit. A differential clustering algorithm revealed similar and divergent gene co-expression patterns among the two genotypes. Since moderate stress levels induced similar physiological responses in both genotypes, the genotype-dependent transcriptional responses could be considered as intrinsic divergences in genome functioning. Our meta-analysis detected several candidate genes and processes that are differentially regulated in root and leaf, potentially under developmental control, and preferentially involved in early and long-term responses to drought.</p> <p>Conclusions</p> <p>In poplar, the well-known drought-induced activation of sensing and signalling cascades was specific to the early response in leaves but was found to be general in root apices. Comparing our results to what is known in arabidopsis, we found that transcriptional remodelling included signalling and a response to energy deficit in roots in parallel with transcriptional indices of hampered assimilation in leaves, particularly in the drought-sensitive poplar genotype.</p