Développement d'outils microbiologiques et chimiques permettant d'identifier l'origine des pollutions fécales dans les eaux de baignades

La pollution organique issue des effluents d'élevage et des stations d'épuration urbaines conduit à un problème essentiel de santé publique lié à la contamination des eaux de surface où s'exercent des activités sensibles telles que la baignade. S'il est possible de déterminer les pollutions localisées liées à un dysfonctionnement des systèmes de traitement, il est beaucoup plus difficile d'identifier les pollutions organiques diffuses qui participent pourtant majoritairement à la dégradation de la qualité des eaux de surface. La problématique des pollutions diffuses est d'autant plus importante que la nouvelle réglementation européenne concernant les eaux de baignade (Directive 2006/7/CE) demande de constituer des profils de baignade qui nécessitent une identification et une hiérarchisation des sources de pollutions fécales. Le dénombrement de Escherichia coli et des entérocoques intestinaux stipulé par les textes réglementaires européens, représente actuellement le seul outil analytique permettant la mise en évidence d'une contamination fécale du milieu aquatique, sans toutefois différencier l'origine humaine ou animale de cette contamination. Il est donc nécessaire de développer de nouvelles méthodes de détection de la pollution fécale qui puissent non seulement mettre en évidence une contamination mais aussi en indiquer l'origine. C'est d'ailleurs dans cet objectif que s'est développé depuis quelques années, le concept de "Microbial Source Tracking" ("Traceurs de Sources Microbiennes") qui consiste à identifier à l'aide de marqueurs microbiologiques ou chimiques les sources de pollutions fécales. Dans ce contexte, six laboratoires de recherche se sont associés pour développer des techniques de traçage des contaminations fécales afin de proposer un outil opérationnel utilisable pour différencier les sources de pollution, de leur point d'émission jusqu'au milieu récepteur final que constituent les eaux de surface. Les marqueurs qui ont fait l'objet de cette étude sont des molécules chimiques naturelles (stéroïdes, caféine), des molécules de synthèse retrouvées dans les effluents de stations d'épuration ou des rapports de fluorescence de la matière organique ainsi que des micro-organismes (bactériophages, bactéries). A la suite des développements méthodologiques, plusieurs marqueurs ont été sélectionnés : - bactéries appartenant aux groupes bactériens dominants du tractus intestinal humain (Bifidobacterium adolescentis) et porcin (Lactobacillus amylovorus) ; - Bacteroidales spécifiques des humains, porcins et bovins (HF183, Pig-2-Bac, Rum-2-Bac); - génogroupes humains des bactériophages F ARN spécifiques; - rapports de stéroïdes : coprostanol/(24ethylcoprostanol+coprostanol) (R1) et sitostanol/coprostanol (R2); - caféine, benzophénone et tri(2-chloroethyl)phosphate (TCEP)

Relative Decay of Fecal Indicator Bacteria and Human-Associated Markers: A Microcosm Study Simulating Wastewater Input into Seawater and Freshwater

Fecal contaminations of inland and coastal waters induce risks to human health and economic losses. To improve water management, specific markers have been developed to differentiate between sources of contamination. This study investigates the relative decay of fecal indicator bacteria (FIB, Escherichia coli and enterococci) and six human-associated markers (two bacterial markers: Bacteroidales HF183 (HF183) and Bifidobacterium adolescentis (BifAd); one viral marker: genogroup II F-specific RNA bacteriophages (FRNAPH II); three chemical markers: caffeine and two fecal stanol ratios) in freshwater and seawater microcosms seeded with human wastewater. These experiments were performed in darkness, at 20 °C and under aerobic conditions. The modeling of the decay curves allows us (i) to compare FIB and markers and (ii) to classify markers according to their persistence in seawater (FRNAPH II < HF183, stanol ratios < BifAd, caffeine) and in freshwater (HF183, stanol ratios < FRNAPH II < BifAd < caffeine). Although those results depend on the experimental conditions, this study represents a necessary step to develop and validate an interdisciplinary toolbox for the investigation of the sources of fecal contaminations

Constitutive Notch2 signaling in neural stem cells promotes tumorigenic features and astroglial lineage entry

Recent studies identified a highly tumorigenic subpopulation of glioma stem cells (GSCs) within malignant gliomas. GSCs are proposed to originate from transformed neural stem cells (NSCs). Several pathways active in NSCs, including the Notch pathway, were shown to promote proliferation and tumorigenesis in GSCs. Notch2 is highly expressed in glioblastoma multiforme (GBM), a highly malignant astrocytoma. It is therefore conceivable that increased Notch2 signaling in NSCs contributes to the formation of GBM. Here, we demonstrate that mice constitutively expressing the activated intracellular domain of Notch2 in NSCs display a hyperplasia of the neurogenic niche and reduced neuronal lineage entry. Neurospheres derived from these mice show increased proliferation, survival and resistance to apoptosis. Moreover, they preferentially differentiate into astrocytes, which are the characteristic cellular population of astrocytoma. Likewise, we show that Notch2 signaling increases proliferation and resistance to apoptosis in human GBM cell lines. Gene expression profiling of GBM patient tumor samples reveals a positive correlation of Notch2 transcripts with gene transcripts controlling anti-apoptotic processes, stemness and astrocyte fate, and a negative correlation with gene transcripts controlling proapoptotic processes and oligodendrocyte fate. Our data show that Notch2 signaling in NSCs produces features of GSCs and induces astrocytic lineage entry, consistent with a possible role in astrocytoma formation

Technical summary

Human interference with the climate system is occurring. Climate change poses risks for human and natural systems. The assessment of impacts, adaptation, and vulnerability in the Working Group II contribution to the IPCC's Fifth Assessment Report (WGII AR5) evaluates how patterns of risks and potential benefits are shifting due to climate change and how risks can be reduced through mitigation and adaptation. It recognizes that risks of climate change will vary across regions and populations, through space and time, dependent on myriad factors including the extent of mitigation and adaptation

Less than 5 Netrin-1 molecules initiate attraction but 200 Sema3A molecules are necessary for repulsion

Guidance molecules, such as Sema3A or Netrin-1, induce growth cone (GC) repulsion or attraction. In order to determine the speed of action and efficiency of these guidance cues we developed an experimental procedure to deliver controlled amounts of these molecules. Lipid vesicles encapsulating 10-10 4 molecules of Sema3A or Netrin-1 were manipulated with high spatial and temporal resolution by optical tweezers and their photolysis triggered by laser pulses. Guidance molecules released from the vesicles diffused and reached the GC membrane in a few seconds. Following their arrival, GCs retracted or grew in 20-120 s. By determining the number of guidance molecules trapped inside vesicles and estimating the fraction of guidance molecules reaching the GC, we show that the arrival of less than 5 Netrin-1 molecules on the GC membrane is sufficient to induce growth. In contrast, the arrival of about 200 Sema3A molecules is necessary to induce filopodia repulsion

Correlated MRI and Ultramicroscopy (MR-UM) of Brain Tumors Reveals Vast Heterogeneity of Tumor Infiltration and Neoangiogenesis in Preclinical Models and Human Disease

Diffuse tumor infiltration into the adjacent parenchyma is an effective dissemination mechanism of brain tumors. We have previously developed correlated high field magnetic resonance imaging and ultramicroscopy (MR-UM) to study neonangiogenesis in a glioma model. In the present study we used MR-UM to investigate tumor infiltration and neoangiogenesis in a translational approach. We compare infiltration and neoangiogenesis patterns in four brain tumor models and the human disease: whereas the U87MG glioma model resembles brain metastases with an encapsulated growth and extensive neoangiogenesis, S24 experimental gliomas mimic IDH1 wildtype glioblastomas, exhibiting infiltration into the adjacent parenchyma and along white matter tracts to the contralateral hemisphere. MR-UM resolves tumor infiltration and neoangiogenesis longitudinally based on the expression of fluorescent proteins, intravital dyes or endogenous contrasts. Our study demonstrates the huge morphological diversity of brain tumor models regarding their infiltrative and neoangiogenic capacities and further establishes MR-UM as a platform for translational neuroimaging

The Notch pathway in ovarian carcinomas and adenomas

Elements of the Notch pathway regulate differentiation; we investigated the expression of such elements in epithelial ovarian tumours. A total of 32 ovarian tumour samples (17 adenocarcinomas, three borderline tumours, 12 adenomas), two human ovarian cancer (A2780, OVCAR3), and one ovarian surface (IOSE 144) cell lines were analysed. The expression of Notch pathway elements was assessed by RT–PCR, real-time PCR (Notch 1), and by immunoblots (Notch 1 extracellular domain (EC), HES1). The proliferation and colony formation of A2780 cells were measured after stable transfection with activated Notch 1 (intracellular domain). Jagged 2, Delta-like-1, Manic Fringe, and TSL1 were expressed more frequently in adenocarcinomas whereas Deltex, Mastermind, and Radical Fringe were more frequent in adenomas. Quantitative PCR revealed decreased Notch 1 mRNA in ovarian adenocarcinomas compared with adenomas. The expression of Notch 1-EC protein was similar in benign and malignant tumours. HES1 protein was strongly expressed in 18/19 ovarian cancers and borderline tumours but not in adenomas. Transfecting A2780 cells with active Notch 1-IC resulted in a proliferative and colony formation advantage compared to mock transfected cells. Thus, Notch pathway elements are expressed in ovarian epithelial tumours and some of them are differentially expressed between adenomas and carcinomas. The Notch pathway could be a target for the development of therapies for ovarian cancer