The three lipocalins of egg-white: only Ex-FABP inhibits siderophore-dependent iron sequestration by Salmonella Enteritidis

Salmonella Enteritidis is the most prevalent food-borne pathogen associated with egg-related outbreaks in the European Union. During egg colonization, S. Enteritidis must resist the powerful anti-bacterial activities of egg white (EW) and overcome ovotransferrin-imposed iron-restriction (the most important anti-bacterial mechanism of EW). Many pathogens respond to iron restriction by secreting iron-chelating chemicals called siderophores but EW contains a siderophore-sequestering “lipocalin” protein (Ex-FABP) that is predicted to limit the usefulness of siderophores in EW. S. Enteritidis produces two siderophores: enterobactin, which is strongly bound by Ex-FABP; and the di-glucosylated enterobactin-derivative, salmochelin (a so-called “stealth” siderophore), which is not recognized by Ex-FABP. Thus, production of salmochelin may allow S. Enteritidis to escape Ex-FABP-mediated growth inhibition under iron restriction although it is unclear whether its EW concentration is sufficient to inhibit pathogens. Further, two other lipocalins (Cal-γ and α-1-ovoglycoprotein) are found in EW but their siderophore sequestration potential remains unexplored. In addition, the effect of EW lipocalins on the major EW pathogen, S. Enteritidis, has yet to be reported. We overexpressed and purified the three lipocalins of EW and investigated their ability to interact with the siderophores of S. Enteritidis, as well as their EW concentrations. The results show that Ex-FABP is present in EW at concentrations (5.1 μM) sufficient to inhibit growth of a salmochelin-deficient S. Enteritidis mutant under iron restriction but has little impact on the salmochelin-producing wildtype. Neither Cal-γ nor α-1-ovoglycoprotein bind salmochelin or enterobactin, nor do they inhibit iron-restricted growth of S. Enteritidis. However, both are present in EW at significant concentrations (5.6 and 233 μM, respectively) indicating that α-1-ovoglycoprotein is the 4th most abundant protein in EW, with Cal-γ and Ex-FABP at 11th and 12th most abundant. Further, we confirm the preference (16-fold) of Ex-FABP for the ferrated form (Kd of 5.3 nM) of enterobactin over the iron-free form (Kd of 86.2 nM), and its lack of affinity for salmochelin. In conclusion, our findings show that salmochelin production by S. Enteritidis enables this key egg-associated pathogen to overcome the enterobactin-sequestration activity of Ex-FABP when this lipocalin is provided at levels found in EW

Occurrence of 30 trace elements in foods from a multi-centre Sub-Saharan Africa Total Diet Study: Focus on Al, As, Cd, Hg, and Pb

International audienceThis paper reports occurrence data related to 30 trace elements in food composite samples from a multi-regional Sub-Saharan Africa Total Diet Study. Herein, 2700 samples grouped in 225 food composite samples corresponding to 13 food groups: cereals, tubers, legumes, vegetables, fruits, nuts/seeds, meat, eggs, fish, milk/dairy, oil/fats, and beverages from eight locations in four countries, namely Benin (Littoral/Borgou), Cameroon (Duala/North), Mali (Bamako/Sikasso), and Nigeria (Lagos/Kano) were prepared as consumed, pooled, and analysed using a validated method based on inductively coupled plasma-mass spectrometry. The occurrence data for Al, As, Cd, Hg, and Pb as regulated by the Codex Alimentarius are discussed herein. Although the levels of As, Cd, Hg, and Pb were above the limit of quantification, they were below the maximum limits set by the Codex in most samples analysed. A distinct feature was observed for cereals and tubers, as they were mostly contaminated with Al and Pb. A pilot study regarding the impact of using artisanal cookware (made from recycled aluminium) on the contamination of food samples was performed. Relevant contamination with Al and Pb when cooking tomato samples from Cameroon and Nigeria using artisanal aluminium cookware was compared to that when cooked using stainless-steel

Quantitative study of lipase secretion, extracellular lipolysis, and lipid storage in the yeast Yarrowia lipolytica grown in the presence of olive oil: analogies with lipolysis in humans

International audienceLipase secretion, extracellular lipolysis, and fatty acid uptake were quantified in the yeast Yarrowia lipolytica grown in the presence of olive oil and/or glucose. Specific lipase assays, Western blot analysis, and ELISA indicated that most of the lipase activity measured in Y. lipolytica cultures resulted from the YLLIP2 lipase. Lipase production was triggered by olive oil and, during the first hours of culture, most of the lipase activity and YLLIP2 immunodetection remained associated with the yeast cells. YLLIP2 was then released in the culture medium before it was totally degraded by proteases. Olive oil triglycerides were largely degraded when the lipase was still attached to the cell wall. The fate of lipolysis products in the culture medium and inside the yeast cell, as well as lipid storage, was investigated simultaneously by quantitative TLC-FID and GC analysis. The intracellular levels of free fatty acids (FFA) and triglycerides increased transiently and were dependent on the carbon sources. A maximum fat storage of 37.8% w/w of yeast dry mass was observed with olive oil alone. A transient accumulation of saturated FFA was observed whereas intracellular triglycerides became enriched in unsaturated fatty acids. So far, yeasts have been mainly used for studying the intracellular synthesis, storage, and mobilization of neutral lipids. The present study shows that yeasts are also interesting models for studying extracellular lipolysis and fat uptake by the cell. The quantitative data obtained here allow for the first time to establish interesting analogies with gastrointestinal and vascular lipolysis in humans

Diffusion of amoxicillin into heart valves from infective endocarditis patients

International audienceOBJECTIVES: Amoxicillin is the drug of choice in the management of streptococcal and enterococcal infective endocarditis (IE) but little is known regarding amoxicillin diffusion into infected heart valves. Herein, we assessed amoxicillin valvular distribution and related pharmacokinetic/pharmacodynamic (PK/PD) target attainment in IE patients undergoing heart valve surgery. PATIENTS AND METHODS: In this 2-year prospective study, patients with IE treated by continuous infusion of amoxicillin and undergoing a surgical valve replacement were included. Both amoxicillin plasma and tissue concentrations were measured the day of surgery. Amoxicillin concentration in plasma and crushed heart valves were measured by a validated liquid chromatography method coupled with ultra-violet and tandem mass spectrometry, respectively. MIC and MBC of amoxicillin were determined for all available isolates. The rate of achievement of PK/PD efficacy parameters were assessed. RESULTS: Twenty-two heart valves were removed from 20 patients. Bacterial aetiology was streptococcal (n = 17) and enterococcal (n = 3). Amoxicillin mean daily dose was 12 ± 3 g/24 h, mean plasma concentration was 29 ± 21 mg/L (n = 15), mean tissue concentration was 23 ± 15 mg/L (n = 22). Median diffusion rate was 62%. Patients reached a plasma concentration target >4XCMI (n = 13). Tissue concentrations were bactericidal for all streptococcal IE but not for enterococcal IE. CONCLUSIONS: Amoxicillin intravalvular measurements in IE treated patients showed significant penetration into the infectious site. These data are reassuring that in situ bactericidal concentrations can be largely achieved in the management of streptococcal IE and support the need for combination antibiotic therapy for enterococcal IE

Duck pluripotent stem cells and their susceptibility to influenza virus

Tremendous efforts have been made to derive embryonic stem cells (ESC) from various species, primarily in mammals. Developing these cell types with self-renewal and differentiation capacities in avian species presents a double interest: 1) fundamental, to study early development and to decipher the mechanisms involved in the maintenance of pluripotency in vitro. 2) biotechnological, to analyze viral replication and to produce specific vaccines instead of using embryonated eggs. So far, only chicken ESC lines and one proprietary duck ESC line have been established and all of them were derived in the presence of serum. The small number of available stem cell lines limits the possibility of comparing avian species among themselves and with mammalian species. Their derivation in media containing serum decreases their potential use for pharmaceutical or veterinary purposes. We have therefore chosen to derive, in chemically defined media, new cell lines from stage X Pekin duck embryos. We have established different independent isolates which can be established in long term culture. The cells maintain the transcriptional signature such as OCT4, NANOG, TERT and ALPL gene transcription and physiological markers of ESC (telomerase activity, Alkaline phosphatase activity) and plasticity of differentiation. These populations then share most of the characteristics expected of pluripotent stem cells (PSCs). Therefore, these new duck PSCs can provide a working basis to analyze the mechanisms of maintenance of pluripotency in avian species by comparing them with already existing chicken cells.Moreover, it has been shown that certain endogenous retroviral elements, usually silent in most cell types, can be transcriptionally reactivated in pluripotent cells. This property remains a major drawback for their use as a substrate for the production of human and veterinary vaccines. We therefore analyzed the activity of reverse transcriptase in the supernatant of different chicken and duck cell lines. We have shown that the new duck ES lines exhibit a very low RT activity, 3 log below the activities measured in the chicken ES cell lines. We then tested the susceptibility of these duck ES cells to influenza viruses. We have shown that duck ES cells, but not duck embryonic fibroblasts, can replicate both human or avian influenza viruses as much as chicken ES cells. These Duck stem cells can therefore be a perfect substrate for producing vaccines under greater safety conditions than those with chicken embryos

Duck pluripotent stem cells and their susceptibility to influenza virus

Tremendous efforts have been made to derive embryonic stem cells (ESC) from various species, primarily in mammals. Developing these cell types with self-renewal and differentiation capacities in avian species presents a double interest: 1) fundamental, to study early development and to decipher the mechanisms involved in the maintenance of pluripotency in vitro. 2) biotechnological, to analyze viral replication and to produce specific vaccines instead of using embryonated eggs. So far, only chicken ESC lines and one proprietary duck ESC line have been established and all of them were derived in the presence of serum. The small number of available stem cell lines limits the possibility of comparing avian species among themselves and with mammalian species. Their derivation in media containing serum decreases their potential use for pharmaceutical or veterinary purposes. We have therefore chosen to derive, in chemically defined media, new cell lines from stage X Pekin duck embryos. We have established different independent isolates which can be established in long term culture. The cells maintain the transcriptional signature such as OCT4, NANOG, TERT and ALPL gene transcription and physiological markers of ESC (telomerase activity, Alkaline phosphatase activity) and plasticity of differentiation. These populations then share most of the characteristics expected of pluripotent stem cells (PSCs). Therefore, these new duck PSCs can provide a working basis to analyze the mechanisms of maintenance of pluripotency in avian species by comparing them with already existing chicken cells.Moreover, it has been shown that certain endogenous retroviral elements, usually silent in most cell types, can be transcriptionally reactivated in pluripotent cells. This property remains a major drawback for their use as a substrate for the production of human and veterinary vaccines. We therefore analyzed the activity of reverse transcriptase in the supernatant of different chicken and duck cell lines. We have shown that the new duck ES lines exhibit a very low RT activity, 3 log below the activities measured in the chicken ES cell lines. We then tested the susceptibility of these duck ES cells to influenza viruses. We have shown that duck ES cells, but not duck embryonic fibroblasts, can replicate both human or avian influenza viruses as much as chicken ES cells. These Duck stem cells can therefore be a perfect substrate for producing vaccines under greater safety conditions than those with chicken embryos

RAPPORT FINAL « PROJET SERVICE NUMERIQUE CULTUREL INNOVANT 2012»

Partenaires : Jean-Louis Dauga, expert en patrimoine maritime du Ministère de la Culture Morel Mapping WorkShop MgDesign Budget total du projet : 30000 Subvention : 16000 1 BILAN-Rappel des objectifs :-La Bretagne est la première région française pour le nombre des bateaux protégés au titre des monuments historiques : 27 bateaux classés y sont en effet rattachés, soit près du quart du total national. Parmi les derniers classements, signalons «Patron François Morin», ancien canot de sauvetage d'Ouessant, «Danycan», sloop bermudien de plan Cornu, «La Janine», ancien caseyeur camaretois, «Minahouet II », ancien pilote de Gironde.... qui sont venus rejoindre, en 2010, les autres unités, bateaux de travail et de plaisance déjà protégés. Les propriétaires sont tant privés que publics, et souvent gérés au quotidien par des associations.-La restauration de ces monuments historiques est placée sous le contrôle scientifique de l'État, au travers des missions données par la DRAC aux experts nationaux, et par l'action de la conservation régionale des monuments historiques. Un conservateur du patrimoine et un architecte des bâtiments de France sont en effet, entre autres missions, en charge de cet aspect particulier de notre patrimoine. Des subventions sont régulièrement accordées aux propriétaires qui doivent faire restaurer leur patrimoine : en 2010, ce sont 359 610 euros qui ont ainsi été accordés par l'Etat. Les collectivités (Région Bretagne, départements...) sont également très présentes dans ce domaine.-L'autre versant de l'aide de l'État se concentre sur l'aspect scientifique et technique avec la mise en place de dossiers de restauration après chaque intervention afin de constituer un véritable « dossier d'oeuvre » pour chaque bateau, comportant plans, relevés des restaurations effectuées, description des travaux, photos, etc… qui servira à documenter le bateau pour les interventions futures.-Cependant, cet aspect se heurte à plusieurs difficultés : le coût de ces opérations et l'établissement d'une méthodologie qui permettrait d'acquérir les données lors, par exemple, d'un grand carénage, et de pouvoir d'une part les partager (avec le maître d'oeuvre, les experts mandatés par le Ministère de la Culture, le propriétaire, le public etc) et d'autre part de les conserver afin qu'un véritable suivi sanitaire puisse être établi au fil des années.-Le projet-En 2012 le Ministère de la Culture et de la Communication lance un appel d'offre pour la création de " Services Numériques Culturels Innovants " pour lequel le laboratoire GERSA de l'Ecole Nationale Supérieure d'Architecture de Nantes (ENSA Nantes), gagne le concours. Les chercheurs de cette équipe ont, en effet, développé des outils méthodologiques reconnus concernant les relevés patrimoniaux et leur mise en forme.-La proposition a été de constituer une base de données pour les concepteurs et les propriétaires de bateaux classées comme monuments historiques, afin de préserver le patrimoine, d'enrichir les connaissances et d'offrir un accès public aux informations, tout cela en temps réel. Cela suppose de faire avancer en parallèle deux axes majeurs : le premier concerne la modélisation numérique des bateaux. Le modèle doit être parfaitement fidèle à l'état existant puisqu'il sert non seulement à décrire le bateau mais également à surveiller son état sanitaire. C'est en quelque sorte, une photographie 3D qui servira de référence pour les actions à venir

Archives Virtuelle En Ligne - AVEL: Enjeux et techniques pour le relevé des bateaux classés Monuments Historiques

International audienceIn 2012, the Ministry of Culture and Communication launchs a project for the creation of "Innovative Digital Cultural Services" for which the GERSA laboratory of the Nantes Superior School of Architecture (ENSA Nantes) Competition. The researchers of this team have indeed developed recognized methodological tools concerning heritage surveys and their shaping.The proposal was to create a database for designers and owners of vessels classified as historical monuments, in order to preserve heritage, enrich knowledge and provide public access to information, all in real time. This entails advancing two major axes in parallel: the first concerns the numerical modeling of ships. The model must be perfectly faithful to the existing condition since it serves not only to describe the boat but also to monitor its health status. In a way, it is a 3D photograph that will serve as a reference for future actions.En 2012 le Ministère de la Culture et de la Communication lance un appel d’offre pour la création de “Services Numériques Culturels Innovants” pour lequel le laboratoire GERSA de l’Ecole Nationale Supérieure d’Architecture de Nantes (ENSA Nantes), gagne le concours. Les chercheurs de cette équipe ont, en effet, développé des outils méthodologiques reconnus concernant les relevés patrimoniaux et leur mise en forme.La proposition a été de constituer une base de données pour les concepteurs et les propriétaires de bateaux classées comme monuments historiques, afin de préserver le patrimoine, d’enrichir les connaissances et d’offrir un accès public aux informations, tout cela en temps réel. Cela suppose de faire avancer en parallèle deux axes majeurs : le premier concerne la modélisation numérique des bateaux. Le modèle doit être parfaitement fidèle à l’état existant puisqu’il sert non seulement à décrire le bateau mais également à surveiller son état sanitaire. C’est en quelque sorte, une photographie 3D qui servira de référence pour les actions à venir

Duck pluripotent stem cells replicate efficiently influenza viruses

Pluripotent stem cells (PSCs) have revolutionized in vitro approaches for disease modeling and regenerative medicine. Nowadays, avian stem cells represent also a tremendous alternative to primary cells or eggs for the study of viruses and for the production of vaccines. They offer the possibility of developing stable production methods while preventing safety risks and egg shortages due to the increasing number of epizootics in the poultry farms. However, so far, only chicken embryonic stem cells (ESC) lines and one proprietary duck ESC line have been established and all of them were derived in the presence of serum. Therefore, we sought to establish novel duck ESCs in chemically defined media and analyzed their permissiveness to influenza viruses to provide a novel substrate for viral replications.Methods:Derivation and characterization of duck ESCs: Pekin duck blastodermal cells from freshly laid eggs were cultured in chemically defined media and different independent isolates were established for long term culture. They were characterized by biochemical assays (Telomerase and alkaline phosphatase activities). Their transcriptomes were analyzed by RT-qCR and RNAseq, and their plasticity was evaluated by in vitro differentiation and by chimaera formation.Influenza infections:A/Lyon/969/2009 H1N1 (H1N1pdm09), A/Puerto Rico/8/1934 (H1N1), A/Switzerland/ 9715293/2013 (H3N2), B/Phuket/3073/2013 and A/Finch/England/2051/1991 (H5N2) (low pathogenic avian influenza virus) were propagated in the MDCK cells at 37°C in Eagle’s minimum essential medium (EMEM Lonza) supplemented with 1 µg/mL TPCK-treated trypsin (Sigma-Aldrich, St. Louis, MO, USA). The first HPAIV (High Pathogenicity Avian Influenza Virus) H5N8 was generated by reverse genetic using the eight segments from the A/mallard duck/France/171201g/2017 (H5N8) as described by Bessiere et al, 2022. H5N1/2021 and H5N8/2020 were isolated from field samples (feathers). All those three HPAIV were amplified on 9 to 11 days embryonated eggs. The allantoic liquid was harvested after 48 hours of infection, aliquoted and frozen at -70°C. The infectious viral titers were determined either by 50% tissue culture infectious dose (TCID50) in the MDCK cells from 4 replicates using the Reed and Muench method.Results :Independent isolates were established from duck embryos in chemical defined media without serum. We demonstrated that these cells exhibit a typical transcriptional signature of ESCs with high OCT4 and NANOG gene expressions but also express high level of germinal markers such as DDX4 and DAZL genes. These cells display some physiological markers of ESC, especially cellular plasticity demonstrated by in vitro differentiation and by their ability to colonize developing embryo and form chimaeras. These populations then share most of the characteristics expected of pluripotent stem cells and can provide a working basis to analyze the mechanisms of maintenance of pluripotency in avian species by comparing them with already existing chicken cells.Once established we first checked the expression level of endogenous retroviral elements (ERV), which are usually silent in most cell types, but which can be transcriptionally reactivated in pluripotent cells. Therefore, we analyzed the activity of reverse transcriptase activity in the supernatant of those new duck ESCs by qPERT assay. We demonstrated that those cells exhibit a much lower RT activity than their chicken ESCs homologs and could therefore be viewed as novel valuable substrates for viral studies. We then infected both chicken and duck ESCs with 6 different influenza viruses (H1N1/pr8, H1N1/pdm2009, H5N2, H5N1/2021, H5N8/2017 and H5N8/2020) and evaluated the cells permissiveness either by measuring the Influenza infectious particles in the supernatant by TCID50 on MDCK cells or by measuring the Influenza viral RNA load in supernatant by RT-qPCR using primers targeting the HA gene. We demonstrated that these new duck ESCs but not duck embryonic fibroblasts, can replicate very efficiently avian influenza viruses when compared to chicken ESCs and other reference cell lines. Conclusions and Perspectives:Therefore, all these properties encompassed in these new duck stem cell lines (long-term cultivation, permissiveness towards avian influenza viruses and low retroviral activity) make these cells a perfect substrate for evaluating new influenza isolates from the field as well as for producing vaccines under greater safety conditions when compared to chicken embryos

Avian pluripotent stem cells and their susceptibility to influenza virus

International audienceTremendous efforts have been made to derive pluripotent stem cells (PSC) from various species, primarily in mammals. Developing these cell types with self-renewal and differentiation capacities in avian species presents a double interest: 1) fundamental, to study early development and to decipher the mechanisms involved in the maintenance of pluripotency in vitro. 2) biotechnological, to analyze viral replication and to produce specific vaccines instead of using embryonated eggs. These cells can be directly derived from embryos and are then so-called embryonic stem cells (ESC) or by overexpressing some factors into somatic cells to reprogrammed them as induced pluripotent stem cells (iPSC).Only chicken ESC lines and one proprietary duck ESC line have been established and all of them were derived in the presence of serum. The small number of available stem cell lines limits the possibility of comparing avian species among themselves and with mammalian species. We have therefore chosen to reprogram chicken and duck fibroblast into pluripotent stem like cells (rPSC) and derive new duck embryonic stem cell lines from stage X Pekin duck embryos in chemically defined media. We have established different independent isolates which can be established in long term culture. We characterized these cells and we have shown that some isolates maintain the transcriptional signature such as OCT4, NANOG, TERT and ALPL gene transcription and physiological markers of ESC (telomerase activity, Alkaline phosphatase activity) and plasticity of differentiation. These populations then share most of the characteristics expected of pluripotent stem cells (PSCs). Therefore, these new avian PSCs can provide a working basis to analyze the mechanisms of maintenance of pluripotency in avian species by comparing them with already existing chicken cells.Moreover, it has been shown that certain endogenous retroviral elements, usually silent in most cell types, can be transcriptionally reactivated in pluripotent cells. This property remains a major drawback for their use as a substrate for the production of human and veterinary vaccines. We therefore analyzed the activity of reverse transcriptase in the supernatant of different chicken and duck cell lines. We have shown that the new duck ES lines exhibit a very low RT activity, 3 log below the activities measured in the chicken ES cell lines. We then tested the susceptibility of these duck ES cells to influenza viruses. We have shown that duck ES cells, but not duck embryonic fibroblasts, can replicate both human or avian influenza viruses as much as chicken ES cells. These Duck stem cells can therefore be a perfect substrate for producing vaccines under greater safety conditions than those with chicken embryos