A rapid microwell fluorescence immunoassay for cellular protein detection

In this paper, we describe a simple, rapid, specific, sensitive, and reliable method, the FICP method (Fluorescence Immunoassay for Cellular Protein detection) which is readily applicable to the detection of proteins directly on cells cultured in 96-well plates. In order to illustrate this method, we report on the detection of two different proteins, the cell cycle proteins cyclin D1 and p21CIP1/WAF1, in untreated and 2-cyclopenten-1-one treated breast cancer cells. When the FICP method was compared with Western blot procedure, FICP was found to be superior for many characteristics. By using this method, we were able to quantify biological effects of a specific compound on protein levels in non-lysed cells and perform statistical analysis. Therefore, we believe this screening assay could be very useful for detecting poorly expressed proteins and for drug development

Preparation, characterization, and safety evaluation of poly(lactide-co-glycolide) nanoparticles for protein delivery into macrophages.

International audienceFollowing infection, HIV establishes reservoirs within tissues that are inaccessible to optimal levels of antiviral drugs or within cells where HIV lies latent, thus escaping the action of anti-HIV drugs. Macrophages are a persistent reservoir for HIV and may contribute to the rebound viremia observed after antiretroviral treatment is stopped. In this study, we further investigate the potential of poly(lactic-co-glycolic) acid (PLGA)-based nanocarriers as a new strategy to enhance penetration of therapeutic molecules into macrophages. We have prepared stable PLGA nanoparticles (NPs) and evaluated their capacity to transport an active molecule into the human monocyte/macrophage cell line THP-1 using bovine serum albumin (BSA) as a proof-of-concept compound. Intracellular localization of fluorescent BSA molecules encapsulated into PLGA NPs was monitored in live cells using confocal microscopy, and cellular uptake was quantified by flow cytometry. In vitro and in vivo toxicological studies were performed to further determine the safety profile of PLGA NPs including inflammatory effects. The size of the PLGA NPs carrying BSA (PLGA-BSA) in culture medium containing 10% serum was ~126 nm in diameter, and they were negatively charged at their surface (zeta potential =-5.6 mV). Our confocal microscopy studies and flow cytometry data showed that these PLGA-BSA NPs are rapidly and efficiently taken up by THP-1 monocyte-derived macrophages (MDMs) at low doses. We found that PLGA-BSA NPs increased cellular uptake and internalization of the protein in vitro. PLGA NPs were not cytotoxic for THP-1 MDM cells, did not modulate neutrophil apoptosis in vitro, and did not show inflammatory effect in vivo in the murine air pouch model of acute inflammation. In contrast to BSA alone, BSA encapsulated into PLGA NPs increased leukocyte infiltration in vivo, suggesting the in vivo enhanced delivery and protection of the protein by the polymer nanocarrier. We demonstrated that PLGA-based nanopolymer carriers are good candidates to efficiently and safely enhance the transport of active molecules into human MDMs. In addition, we further investigated their inflammatory profile and showed that PLGA NPs have low inflammatory effects in vitro and in vivo. Thus, PLGA nanocarriers are promising as a drug delivery strategy in macrophages for prevention and eradication of intracellular pathogens such as HIV and Mycobacterium tuberculosis

Complete Genome Sequence of Crohn's Disease-Associated Adherent-Invasive E. coli Strain LF82

International audienceBACKGROUND: Ileal lesions of Crohn's disease (CD) patients are abnormally colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells and macrophages. PRINCIPAL FINDINGS: We report here the complete genome sequence of E. coli LF82, the reference strain of adherent-invasive E. coli associated with ileal Crohn's disease. The LF82 genome of 4,881,487 bp total size contains a circular chromosome with a size of 4,773,108 bp and a plasmid of 108,379 bp. The analysis of predicted coding sequences (CDSs) within the LF82 flexible genome indicated that this genome is close to the avian pathogenic strain APEC_01, meningitis-associated strain S88 and urinary-isolated strain UTI89 with regards to flexible genome and single nucleotide polymorphisms in various virulence factors. Interestingly, we observed that strains LF82 and UTI89 adhered at a similar level to Intestine-407 cells and that like LF82, APEC_01 and UTI89 were highly invasive. However, A1EC strain LF82 had an intermediate killer phenotype compared to APEC-01 and UTI89 and the LF82 genome does not harbour most of specific virulence genes from ExPEC. LF82 genome has evolved from those of ExPEC B2 strains by the acquisition of Salmonella and Yersinia isolated or clustered genes or CDSs located on pLF82 plasmid and at various loci on the chromosome. CONCLUSION: LF82 genome analysis indicated that a number of genes, gene clusters and pathoadaptative mutations which have been acquired may play a role in virulence of AIEC strain LF82

Spatio-temporal variability in benthic microbial activity and particle flux in the Laurentian Trough

Spatial variability within a single oceanographic station (10-1000m radius) is poorly understood, potentially limiting detection of short-lived moderate fluctuations in organic matter input and the benthic response. Between-station and inter-annual comparisons are also confounded by unknown local spatial variations. The primary objective of this study was to document spatial variability in benthic microbial activity at a single station in the Laurentian Trough (350 m depth) during the early summer phytoplankton bloom period. We also evaluated the influence of local spatial variability on the detection of a benthic response to bloom-related increases in organic matter sedimentation. Box coring and deployment of multicylinder drifting sediment traps were carried out daily over a 20-day period. Two spatial scales were sampled within one boxcore, and a second boxcore provided data on between-core variability. Microbial processes (bacterial abundance, extracellular enzyme activity and 3H-thymidine incorporation into DNA) showed significant spatial variability at the 10-100 m and dm scales, and were correlated with the distribution of photosynthetic pigments and polychaete abundance in the sediments. Polychaetes also influenced microbial activity at finer spatial scales, in relation to tube location. Bacterial extracellular enzyme activity and thymidine incorporation into DNA behaved independently at all spatial scales studied, arguing for a decoupling of these two microbial processes. Significant variation in organic matter sedimentation between cylinders on drifting arrays is attributed to particle aggregation. This may contribute to spatial irregularity in organic matter supply to the seafloor. Spatial variability at the 10-100 m scale for all measures of microbial activity was of magnitude comparable to temporal signals over the course of the phytoplankton bloom. We suggest that local spatial variability and microbial responses to bloom events may be site specific, controlled by factors such as long term sedimentation regimes. These observations point out a need to develop new tools to better resolve the distribution of microbial activity on the seafloor

Les systèmes de retraite, le couple et les inégalités de genre à la retraite: Thèse de doctorat en Sciences économiques

This thesis seeks to contribute to our understanding of gender inequalities in retirement. Because of the gendered division of social roles within couples, women have less advantageous careers and hence unequal pension rights. Drawing from an empirical approach and the analysis of administrative data, this thesis investigates gender inequalities in retirement beyond the gender pension gap and assesses the role of conjugal pension rights (survivor benefits) in these inequalities.The first introductory section describes the French situation with regard to survivor benefits. The second part focuses on retirement duration as a component of gender inequalities. Chapter 2 proposes a measure of retirement duration as a widow(er). Chapter 3 analyzes the differences in retirement decisions between women and men. Retirement duration is very heterogeneous among retirees. Low-income widows spend more years in widowhood. Moreover, women and men do not make the same retirement decisions because men are more sensitive to financial incentives. Finally, the third part of this thesis evaluates two reforms of conjugal pension rights. Chapter 4 focuses on a French reform aiming to maintain survivors' monetary standard of living following their spouse's death. Chapter 5 looks into a Dutch reform in order to investigate the consequences of the elimination of survivor benefits schemes on working-age widows' labor supply. Survivor benefits do not currently maintain survivors' standard of living for low-income widows, while they overcompensate it for the others. Eliminating this scheme would increase the working-age widows' labor supply but would also increase the take-up of other social benefits.Cette thèse vise à améliorer les connaissances sur les inégalités de genre à la retraite. Les inégalités de pension entre les femmes et les hommes sont le reflet de l’acquisition différenciée de droits à la retraite pendant la carrière, conséquence de la division genrée des rôles sociaux au sein des couples. Grâce à une approche empirique et avec l’utilisation de données administratives, cette thèse interroge les inégalités de genre à la retraite au-delà des différences de pension moyenne et évalue le rôle des droits conjugaux à la retraite dans ces inégalités. Une première partie introductive décrit la situation française vis-à-vis des dispositifs de droits conjugaux à la retraite. Une deuxième partie propose de prendre en compte la durée de la retraite dans les inégalités de genre. Le chapitre 2 propose une mesure de la durée de retraite passée en situation de veuvage. Le chapitre 3 analyse les différences de décision de départ entre les femmes et les hommes. La durée passée à la retraite est très hétérogène parmi les individus. Les veuves dont les revenus sont les plus faibles passent plus de temps en situation de veuvage. Par ailleurs, les femmes et les hommes ne prennent pas les mêmes décisions de départ à la retraite car les hommes sont plus sensibles aux incitations financières. Enfin, la troisième partie de cette thèse propose des évaluations de réformes des droits conjugaux à la retraite. Le chapitre 4 évalue ex ante une réforme permettant de maintenir le niveau de vie monétaire des survivants au décès de leur conjoint en France. Le chapitre 5 évalue ex post une réforme néerlandaise pour comprendre les conséquences de la suppression de tels dispositifs sur les revenus des veuves en âge de travailler. Les droits conjugaux à la retraite ne remplissent actuellement pas leur objectif de maintien du niveau de vie suite au décès d’un conjoint pour les survivantes les moins aisées, tandis qu’ils le surcompensent pour d’autres. La suppression de ces dispositifs augmenterait l’offre de travail des veuves en âge de travailler mais amplifierait la proportion de bénéficiaires d’autres prestations sociales

Influence of the incorporation mode of sugar beet pulp in the finishing diet on the digestive tract and performances of geese reared for foie gras production

The aim of this work was to study the effects of incorporating sugar beet pulp (SBP) into the diet of geese in two feeding systems (complete pelleted feed or loose-mix feeding system) on crop development and performance. A total of 480 1-d-old male geese were divided into three groups whose diet differed from d 56 to 90: a complete pelleted diet containing 50% corn (control diet: AMEn 11.5 MJ/kg; CP 161 g/kg), and no SBP; a complete pelleted diet containing 50% corn and 10% SBP (SBPcp diet: AME(n): 11.5 MJ/kg; CP: 161 g/kg); and a mix in the same feeder (SBPlm diet) of 500 g/kg of protein-rich pellets containing 20% SBP (SBPprp: AME(n): 9.0 MJ/kg; CP: 250 g/kg) and 500 g/kg of whole corn (WC: AME(n): 14.0 MJ/kg; CP: 72 g/kg). Body traits, including crop volume, were measured at d 91. From d 91 to 106, 88 birds/group were overfed with a mixture contain-ing mainly corn and water before slaughter to measure fatty liver performance. Feed intake from d 56 to 90 was higher (+ 10%; P = 0.004) in the SBPcp group than the other two, but at d 90, the body weight (BW) of the birds was higher (+ 7%; P = 0.002) in the SBPlm group than the other two. At d 91, the volume of the crop was greater in the SBPcp group (80.4 mL/kg of BW, P 0.05) between the three groups. In conclusion, the use of sugar beet pulp in the diet of finishing geese helps the adaptation of the digestive tract to the overfeeding period, even in a loose-mix feeding system based on whole corn