Characterization of the Phytochelatin Synthase of Schistosoma mansoni

Treatment for schistosomiasis, which is responsible for more than 280,000 deaths annually, depends exclusively on the use of praziquantel. Millions of people are treated annually with praziquantel and drug resistant parasites are likely to evolve. In order to identify novel drug targets the Schistosoma mansoni sequence databases were queried for proteins involved in glutathione metabolism. One potential target identified was phytochelatin synthase (PCS). Phytochelatins are oligopeptides synthesized enzymatically from glutathione by PCS that sequester toxic heavy metals in many organisms. However, humans do not have a PCS gene and do not synthesize phytochelatins. In this study we have characterized the PCS of S. mansoni (SmPCS). The conserved catalytic triad of cysteine-histidine-aspartate found in PCS proteins and cysteine proteases is also found in SmPCS, as are several cysteine residues thought to be involved in heavy metal binding and enzyme activation. The SmPCS open reading frame is considerably extended at both the N- and C-termini compared to PCS from other organisms. Multiple PCS transcripts are produced from the single encoded gene by alternative splicing, resulting in both mitochondrial and cytoplasmic protein variants. Expression of SmPCS in yeast increased cadmium tolerance from less than 50 µM to more than 1,000 µM. We confirmed the function of SmPCS by identifying PCs in yeast cell extracts using HPLC-mass spectrometry. SmPCS was found to be expressed in all mammalian stages of worm development investigated. Increases in SmPCS expression were seen in ex vivo worms cultured in the presence of iron, copper, cadmium, or zinc. Collectively, these results indicate that SmPCS plays an important role in schistosome response to heavy metals and that PCS is a potential drug target for schistosomiasis treatment. This is the first characterization of a PCS from a parasitic organism

Engineering and use of magnetosomes for medical applications

Les magnetosomes sont des nanoparticules d’oxyde de fer produites génétiquement par les bactéries magnétotactiques. De part, la pureté de leurs cristaux, leur taille contrôlée, leur solubilité et biocompatibilité liées à la membrane les entourant ainsi que la possibilité de les fonctionnaliser par voie génétique, les magnétosomes peuvent être exploités dans un grand nombre d’applications biotechnologiques, rivalisant avec des particules magnétiques produites par voie chimique. C’est dans ce contexte que mon travail de thèse vise à explorer l’utilisation des magnétosomes comme agent théranostique. Mes travaux se sont centré sur l’utilisation de magnétosomes étiquetés génétiquement avec le peptide RGD (magnétosomes@RGD) comme outils biogéniques pour le diagnostic et le thérapie du cancer. En effet, nous démontrons que la présence du peptide RGD, se liant au récepteur ανβ3, permet le ciblage des cellules cancéreuses et augmente l'internalisation de la bioparticule de fer dans la tumeur. Après validation in cellulo, ces expériences ont été conduites chez la souris porteuse de tumeurs de la prostate, de mélanome ou de glioblastome. L'accumulation spécifique dans les tumeurs a été démontrée en imagerie par résonnance magnétique. La présence d'une grande quantité de fer dans les cellules nous a également permis d'aborder le potentiel thérapeutique des magnetosomes@RGD. Nous avons développé pour cela une approche par photothermie ainsi que par effet de radiosensibilisation aux rayons X ou protons appliquée in cellulo puis in vivo. L’ensemble de ces travaux contribue à démontrer le potentiel théranostique des magnétosomes bactériens via une fonctionnalisation adaptée.Magnetosomes are iron oxide nanoparticles produced genetically by magnetotactic bacteria. Due to the purity of their crystals, their controlled size, their solubility and biocompatibility related to the surrounding membrane and the possibility of their genetic functionalization, magnetosomes can be exploited in a large number of biotechnological applications, competing with chemically produced magnetic particles. It is in this context that my thesis work aims to explore the use of magnetosomes as theranostic agents. My work has focused on the use of magnetosomes genetically labelled with the RGD peptide (magnetosomes@RGD) as biogenic tools for cancer diagnosis and therapy. Indeed, we demonstrate that the presence of the RGD peptide, which binds to the ανβ3 receptor, allows the targeting of cancer cells and increases the internalization of the iron bioparticle in the tumor. After validation in cellulo, these experiments were conducted in mice with prostate tumours, melanoma or glioblastoma. Specific accumulation in tumours has been demonstrated in magnetic resonance imaging. The presence of a large amount of iron in the cells also allowed us to address the therapeutic potential of magnetosomes@RGD. For this purpose, we have developed an approach based on photothermia as well as on radiosensitivity to X-rays or protons applied in cellulo and then in vivo. All this work contributes to demonstrating the theranostic potential of bacterial magnetosomes through appropriate functionalization

Magnetosomes: biogenic iron nanoparticles produced by environmental bacteria

International audienc

Geographically-Distributed Databases: A Big Data Technology for Production Analysis in the Oil & Gas Industry

International audienceThe paper discusses some reported shortcomings of state-of-the-start IT technologies currently employed in the data management of Oil & Gas production operations. Most current IT infrastructures connect historian databases, production databases and application servers. This creates complex issues of data consistency between these systems. In the discussion, a particular focus is put on the geographically-distributed nature of the network which suffers from low-bandwidth limitations and un-reliabilities, e.g. due to satellite communication links.Taking the production engineers’ viewpoint, an example of production allocation using Data Validation and Reconciliation (DVR) serves to stress the malicious impacts of the described architecture. Production allocation represents one of the various monitoring and analysis tasks that are performed, on a daily basis, at the centralized level of data management systems. A quantitative study shows that the problem of mis-synchronization of databases is of great practical importance.We propose solutions to improve the robustness to communication outages. To improve data consistency across sites in a decentralized manner, the paper exposes the key concepts of distributed storage, message-based communication, and clustering. More generally, the paper proposes to shine a light on the potential relevance of several recent advances in the scientific field of “big-data” to the world of Oil & Gas upstream industry. These off-the-shelf technologies must be specifically tailored to geographically-distributed networks. The specificities are detailed, the necessary development work is outlined, and the potential qualitative benefits are estimated. A possible implementation is sketched

Accumulation and Dissolution of Magnetite Crystals in a Magnetically Responsive Ciliate

International audienceMagnetotactic bacteria (MTB) represent a group of microorganisms that are widespread in aquatic habitats and thrive at oxic-anoxic interfaces. They are able to scavenge high concentrations of iron thanks to the biomineralization of magnetic crystals in their unique organelles, the so-called magnetosome chains. Although their biodiversity has been intensively studied, their ecology and impact on iron cycling remain largely unexplored. Predation by protozoa was suggested as one of the ecological processes that could be involved in the release of iron back into the ecosystem. Magnetic protozoa were previously observed in aquatic environments, but their diversity and the fate of particulate iron during grazing are poorly documented. In this study, we report the morphological and molecular characterizations of a magnetically responsive MTB-grazing protozoan able to ingest high quantities of MTB. This protozoan is tentatively identified as $Uronema\ marinum$, a ciliate known to be a predator of bacteria. Using light and electron microscopy, we investigated in detail the vacuoles in which the lysis of phagocytized prokaryotes occurs. We carried out high-resolution observations of aligned magnetosome chains and ongoing dissolution of crystals. Particulate iron in the ciliate represented approximately 0.01% of its total volume. We show the ubiquity of this interaction in other types of environments and describe different grazing strategies. These data contribute to the mounting evidence that the interactions between MTB and protozoa might play a significant role in iron turnover in microaerophilic habitats

A bioluminescent arsenite biosensor designed for inline water analyzer

International audienceWhole-cell biosensors based on the reporter gene system can offer rapid detection of trace levels of organic or metallic compounds in water. They are well characterized in laboratory conditions, but their transfer into technological devices for the surveillance of water networks remains at a conceptual level. The development of a semi-autonomous inline water analyzer stumbles across the conservation of the bacterial biosensors over a period of time compatible with the autonomy requested by the end-user while maintaining a satisfactory sensitivity, specificity, and time response. We focused here on assessing the effect of lyophilization on two biosensors based on the reporter gene system and hosted in Escherichia coli. The reporter gene used here is the entire bacterial luciferase lux operon (luxCDABE) for an autonomous bioluminescence emission without the need to add any substrate. In the cell-survival biosensor that is used to determine the overall fitness of the bacteria when mixed with the water sample, lux expression is driven by a constitutive E. coli promoter P-rpoD. In the arsenite biosensor, the arsenite-inducible promoter P-ars involved in arsenite resistance in E. coli controls lux expression. Evaluation of the shelf life of these lyophilized biosensors kept at 4 degrees C over a year evidenced that about 40 % of the lyophilized cells can be revived in such storage conditions. The performances of the lyophilized biosensor after 7 months in storage are maintained, with a detection limit of 0.2 mu M arsenite for a response in about an hour with good reproducibility. These results pave the way to the use in tandem of both biosensors (one for general toxicity and one for arsenite contamination) as consumables of an autonomous analyzer in the field

Targeted thermal therapy with genetically engineered magnetite magnetosomes@RGD: Photothermia is far more efficient than magnetic hyperthermia

International audienc

In vitro evaluation of hexitol nucleic acid antisense oligonucleotides

status: publishe

a common highly conserved cadmium detoxification mechanism from bacteria to humans HEAVY METAL TOLERANCE CONFERRED BY THE ATP-BINDING CASSETTE (ABC)TRANSPORTER SpHMT1 REQUIRES GLUTATHIONE BUT NON METAL-CHELATING PHYTOCHELATIN PEPTIDES

International audienceCadmium poses a significant threat to human health due to its toxicity. In mammals and in bakers'yeast, cadmium is detoxified by ATP-binding cassette transporters after conjugation to glutathione. In fission yeast, phytochelatins constitute the co-substrate with cadmium for the transporter SpHMT1. In plants, a detoxification mechanism similar to the one in fission yeast is supposed, but the molecular nature of the transporter is still lacking. To investigate further the relationship between SpHMT1 and its co-substrate, we overexpressed the transporter in a Schizosaccharomyces pombe strain deleted for the phytochelatin synthase gene and heterologously in Saccharomyces cerevisiae and in Escherichia coli. In all organisms, overexpression of SpHMT1 conferred a markedly enhanced tolerance to cadmium but not to Sb(III), AgNO3, As(III), As(V), CuSO4, or HgCl2. Abolishment of the catalytic activity by expression of SpHMT1K623M mutant suppressed the cadmium tolerance phenotype independently of the presence of phytochelatins. Depletion of the glutathione pool inhibited the SpHMT1 activity but not that of AtHNIA4, a P-type ATPase, indicating that GSH is necessary for the SpHMT1-mediated cadmium resistance. In E. coli, SpHMT1 was targeted to the periplasmic membrane and led to an increased amount of cadmium in the periplasm. These results demonstrate that SpHMT1 confers cadmium tolerance in the absence of phytochelatins but depending on the presence of GSH and ATP. Our results challenge the dogma of the two separate cadmium detoxification pathways and demonstrate that a common highly conserved mechanism has been selected during the evolution from bacteria to humans. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc