Chemical mixtures and fluorescence in situ hybridization analysis of natural microbial community in the Tiber river.

The Water Framework Directive (WFD) regulates freshwater and coastal water quality assessment in Europe. Chemical and ecological water quality status is based on measurements of chemical pollutants in water and biota together with other indicators such as temperature, nutrients, species compositions (phytoplankton, microalgae, benthos and fish) and hydromorphological conditions. However, in the current strategy a link between the chemical and the ecological status is missing. In the present WFD, no microbiological indicators are foreseen for integrating the different anthropogenic pressures, including mixtures of chemicals, nutrients and temperature changes, to provide a holistic view of the freshwater ecosystem water quality. The main aim of this work was to evaluate if natural microbial populations can be valuable indicators of multiple stressors (e.g. chemical pollutants, temperature, nutrients etc.) to guide preventive and remediation actions by water authorities. A preliminary survey was conducted to identify four sites reflecting a contamination gradient from the source to the mouth of a river suitable to the objectives of the European Marie Curie project, MicroCoKit. The River Tiber (Italy) was selected as a pilot case study to investigate the correlation between bacteria taxa and the chemical status of the river. The main physicochemical parameters, inorganic elements, organic pollutants and natural microbial community composition were assessed at four selected sites corresponding to pristine, agricultural, industrial and urban areas for three consecutive years. The overall chemical results indicated a correspondence between different groups of contaminants and the main contamination sources at the selected sampling points. Phylogenetic analysis of the microbial community analyzed by Fluorescence In Situ Hybridization method (FISH) revealed differences among the four sampling sites which could reflect an adaptive bacterial response to the different anthropogenic pressures.

Rationally modified estrogen receptor protein as a bio-recognition element for the detection of EDC pollutants: strategies and opportunities.

The estrogen receptor protein (ER) can bind a vast number of organic pollutants widely spread in the environment and collectively known as Endocrine Disrupting Chemicals, EDCs. Its broad selectivity makes it an ideal bio-recognition element for the detection of EDCs. Here we describe the strategy and rationale for the design of ER based biosensors and assays that generate a signal in the presence of EDCs. The opportunity to use either natural or rationally modified ER molecules is discussed. The latter approach was successfully applied in the EU-FP7 project RADAR, with the aim to develop a novel biosensor for the detection of organic pollutants both in the environment and in commercial water products.

Rational modification of estrogen receptor by combination of computational and experimental analysis.

In this manuscript, we modulate the binding properties of estrogen receptor protein by rationally modifying the amino acid composition of its ligand binding domain. By combining sequence alignment and structural analysis of known estrogen receptor-ligand complexes with computational analysis, we were able to predict estrogen receptor mutants with altered binding properties. These predictions were experimentally confirmed by producing single point variants with up to an order of magnitude increased binding affinity towards some estrogen disrupting chemicals and reaching an half maximal inhibitory concentration (IC50) value of 2 nM for the 17α-ethinylestradiol ligand. Due to increased affinity and stability, utilizing such mutated estrogen receptor instead of the wild type as bio-recognition element would be beneficial in an assay or biosensor.