Effects of long-term irrigation with treated wastewater. Part I: Evolution of soil physico-chemical properties

The long-term impact of irrigation on a Mediterranean sandy soil irrigated with Treated wastewater (TWW) since 1980 was evaluated. The main soil properties (CEC, pH, size distribution, exchangeable cations and chloride, hydraulic conductivity) as well as the organic matter and Cu, Cr and Pb speciation in an irrigated soil and a non-irrigated control soil at various soil depths were monitored and compared during a 2 years experiment. In this first part, the evolution of the physico-chemical soil properties was described. The irrigation with TWW was beneficial with regard to water and nutrient supplying. All the exchangeable cations other than K(+) were higher in the irrigated soil than in the reference one. A part of the exchangeable cations was not fixed on the exchange complex but stored as labile salts or in concentrated soil solution. Despite the very sandy soil texture, both saturated and unsaturated hydraulic conductivity exhibited a significant diminution in the irrigated soil, but remained high enough to allow water percolation during rainy periods and subsequent leaching of accumulated salts, preventing soil salinization. In the irrigated soil, exchangeable sodium percentage (ESP) exhibited high values (20% on average) and the soil organic C was lower than in the reference. No significant effect was noticed on soil mineralogical composition due to irrigation. (C) 2010 Published by Elsevier Ltd.

Influence of physico-chemical material characteristics on staphylococcal biofilm formation--a qualitative and quantitative in vitro analysis of five different calcium phosphate bone grafts.

Various compositions of synthetic calcium phosphates (CaP) have been proposed and their use has considerably increased over the past decades. Besides differences in physico-chemical properties, resorption and osseointegration, artificial CaP bone graft might differ in their resistance against biofilm formation. We investigated standardised cylinders of 5 different CaP bone grafts (cyclOS, chronOS (both β-TCP (tricalcium phosphate)), dicalcium phosphate (DCP), calcium-deficient hydroxyapatite (CDHA) and α-TCP). Various physico-chemical characterisations e.g., geometrical density, porosity, and specific surface area were investigated. Biofilm formation was carried out in tryptic soy broth (TSB) and human serum (SE) using Staphylococcus aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984). The amount of biofilm was analysed by an established protocol using sonication and microcalorimetry. Physico-chemical characterisation showed marked differences concerning macro- and micropore size, specific surface area and porosity accessible to bacteria between the 5 scaffolds. Biofilm formation was found on all scaffolds and was comparable for α-TCP, chronOS, CDHA and DCP at corresponding time points when the scaffolds were incubated with the same germ and/or growth media, but much lower for cyclOS. This is peculiar because cyclOS had an intermediate porosity, mean pore size, specific surface area, and porosity accessible to bacteria. Our results suggest that biofilm formation is not influenced by a single physico-chemical parameter alone but is a multi-step process influenced by several factors in parallel. Transfer from in vitro data to clinical situations is difficult; thus, advocating the use of cyclOS scaffolds over the four other CaP bone grafts in clinical situations with a high risk of infection cannot be clearly supported based on our data.

Validation and long-term evaluation of a modified on-line chiral analytical method for therapeutic drug monitoring of (R,S)-methadone in clinical samples.

Matrix effects, which represent an important issue in liquid chromatography coupled to mass spectrometry or tandem mass spectrometry detection, should be closely assessed during method development. In the case of quantitative analysis, the use of stable isotope-labelled internal standard with physico-chemical properties and ionization behaviour similar to the analyte is recommended. In this paper, an example of the choice of a co-eluting deuterated internal standard to compensate for short-term and long-term matrix effect in the case of chiral (R,S)-methadone plasma quantification is reported. The method was fully validated over a concentration range of 5-800 ng/mL for each methadone enantiomer with satisfactory relative bias (-1.0 to 1.0%), repeatability (0.9-4.9%) and intermediate precision (1.4-12.0%). From the results obtained during validation, a control chart process during 52 series of routine analysis was established using both intermediate precision standard deviation and FDA acceptance criteria. The results of routine quality control samples were generally included in the +/-15% variability around the target value and mainly in the two standard deviation interval illustrating the long-term stability of the method. The intermediate precision variability estimated in method validation was found to be coherent with the routine use of the method. During this period, 257 trough concentration and 54 peak concentration plasma samples of patients undergoing (R,S)-methadone treatment were successfully analysed for routine therapeutic drug monitoring.

Isolation and characterization of carcinoembryonic antigen (CEA) extracted from normal human colon mucosa.

Carcinoembryonic antigen (CEA) was identified in perchloric acid (PCA)_extract from normal colon mucosa by 2 immunological criteria: a line of identity in double diffusion and a parallel inhibition curve in radioimmunoassay (RIA), both with reference colon carcinoma-CEA (CEA-Tu). The average concentration of CEA in normal colon mucosa (CEA-No) was 35 times lower than in primary large bowel carcinomas and 230 times lower than in metastatic colon or rectum carcinomas. CEA-No was purified from PCA extracts of normal colon mucosa by Sephadex G-200 filtration and immunoadsorbent columns. Purified CEA-No had quatitatively the same inhibition activity in RIA as the British Standard CEA coded 73/601. Purified CEA-No was labelled with 125I. The percentage of binding of labelled CEA-No to a specific goat anti-CEA-Tu antiserum was similar to that of CEA-Tu. Labelled CEA-No could be used as radioactive tracer in RIA as well as labelled CEA-Tu. The physico-chemical properties of purified CEA-Tu as demonstrated by Sepharose 6 B filtration, SDS Polyacrylamide gel analysis and cesium chloride density gradient, were found to be almost identical to those of reference CEA-Tu. Preliminary results showed that CEA-No and CEA-Tu contained the same types of carbohydrates in similar proportions. A rabbit antiserum against CEA-No was obtained which demonstrated the same specificity as conventional anti-CEA-Tu antisera.

Nanomedicines for active targeting: physico-chemical characterization of paclitaxel-loaded anti-HER2 immunonanoparticles and in vitro functional studies on target cells.

Paclitaxel (Tx)-loaded anti-HER2 immunonanoparticles (NPs-Tx-HER) were prepared by the covalent coupling of humanized monoclonal anti-HER2 antibodies (trastuzumab, Herceptin) to Tx-loaded poly (dl-lactic acid) nanoparticles (NPs-Tx) for the active targeting of tumor cells that overexpress HER2 receptors. The physico-chemical properties of NPs-Tx-HER were compared to unloaded immunonanoparticles (NPs-HER) to assess the influence of the drug on anti-HER2 coupling to the NP surface. The immunoreactivity of sulfo-MBS activated anti-HER2 mAbs and the in vitro efficacy of NPs-Tx-HER were tested on SKOV-3 ovarian cancer cells that overexpress HER2 antigens. Tx-loaded nanoparticles (NPs-Tx) obtained by a salting-out method had a size of 171+/-22 nm (P.I.=0.1) and an encapsulation efficiency of about of 78+/-10%, which corresponded to a drug loading of 7.8+/-0.8% (w/w). NPs-Tx were then thiolated and conjugated to activated anti-HER2 mAbs to obtain immunonanoparticles of 237+/-43 nm (P.I.=0.2). The influence of the activation step on the immunoreactivity of the mAbs was tested on SKOV-3 cells using 125I-radiolabeled mAbs, and the activity of the anti-HER2 mAbs was minimally affected after sulfo-MBS functionalization. Approximately 270 molecules of anti-HER2 mAbs were bound per nanoparticle. NPs-Tx-HER exhibited a zeta potential of 0.2+/-0.1 mV. The physico-chemical properties of the Tx-loaded immunonanoparticles were very similar to unloaded immunonanoparticles, suggesting that the encapsulation of the drug did not influence the coupling of the mAbs to the NPs. No drug loss was observed during the preparation process. DSC analysis showed that encapsulated Tx is in an amorphous or disordered-crystalline phase. These results suggest that Tx is entrapped in the polymeric matrix and not adsorbed to the surface of the NPs. In vitro studies on SKOV-3 ovarian cancer cells demonstrated the greater cytotoxic effect of NPs-Tx-HER compared to other Tx formulations. The results showed that at 1 ng Tx/ml, the viability of cells incubated with drug encapsulated in NP-Tx-HER was lower (77.32+/-5.48%) than the viability of cells incubated in NPs-Tx (97.4+/-12%), immunonanoparticles coated with Mabthera, as irrelevant mAb (NPs-Tx-RIT) (93.8+/-12%) or free drug (92.3+/-9.3%).

In situ evaluation of single-cell lysis by cytosol extraction observation through fluorescence decay and dielectrophoretic trapping time

We present a new method for lysis of single cells in continuous flow, where cells are sequentially trapped, lysed and released in an automatic process. Using optimized frequencies, dielectrophoretic trapping allows exposing cells in a reproducible way to high electrical fields for long durations, thereby giving good control on the lysis parameters. In situ evaluation of cytosol extraction on single cells has been studied for Chinese hamster ovary (CHO) cells through out-diffusion of fluorescent molecules for different voltage amplitudes. A diffusion model is proposed to correlate this out-diffusion to the total area of the created pores, which is dependent on the potential drop across the cell membrane and enables evaluation of the total pore area in the membrane. The dielectrophoretic trapping is no longer effective after lysis because of the reduced conductivity inside the cells, leading to cell release. The trapping time is linked to the time required for cytosol extraction and can thus provide additional validation of the effective cytosol extraction for non-fluorescent cells. Furthermore, the application of one single voltage for both trapping and lysis provides a fully automatic process including cell trapping, lysis, and release, allowing operating the device in continuous flow without human intervention.

SwissSidechain: a molecular and structural database of non-natural sidechains.

Amino acids form the building blocks of all proteins. Naturally occurring amino acids are restricted to a few tens of sidechains, even when considering post-translational modifications and rare amino acids such as selenocysteine and pyrrolysine. However, the potential chemical diversity of amino acid sidechains is nearly infinite. Exploiting this diversity by using non-natural sidechains to expand the building blocks of proteins and peptides has recently found widespread applications in biochemistry, protein engineering and drug design. Despite these applications, there is currently no unified online bioinformatics resource for non-natural sidechains. With the SwissSidechain database (http://www.swisssidechain.ch), we offer a central and curated platform about non-natural sidechains for researchers in biochemistry, medicinal chemistry, protein engineering and molecular modeling. SwissSidechain provides biophysical, structural and molecular data for hundreds of commercially available non-natural amino acid sidechains, both in l- and d-configurations. The database can be easily browsed by sidechain names, families or physico-chemical properties. We also provide plugins to seamlessly insert non-natural sidechains into peptides and proteins using molecular visualization software, as well as topologies and parameters compatible with molecular mechanics software.

Functional diversification of duplicate genes through subcellular adaptation of encoded proteins.

BACKGROUND: Gene duplication is the primary source of new genes with novel or altered functions. It is known that duplicates may obtain these new functional roles by evolving divergent expression patterns and/or protein functions after the duplication event. Here, using yeast (Saccharomyces cerevisiae) as a model organism, we investigate a previously little considered mode for the functional diversification of duplicate genes: subcellular adaptation of encoded proteins. RESULTS: We show that for 24-37% of duplicate gene pairs derived from the S. cerevisiae whole-genome duplication event, the two members of the pair encode proteins that localize to distinct subcellular compartments. The propensity of yeast duplicate genes to evolve new localization patterns depends to a large extent on the biological function of their progenitor genes. Proteins involved in processes with a wider subcellular distribution (for example, catabolism) frequently evolved new protein localization patterns after duplication, whereas duplicate proteins limited to a smaller number of organelles (for example, highly expressed biosynthesis/housekeeping proteins with a slow rate of evolution) rarely relocate within the cell. Paralogous proteins evolved divergent localization patterns by partitioning of ancestral localizations ('sublocalization'), but probably more frequently by relocalization to new compartments ('neolocalization'). We show that such subcellular reprogramming may occur through selectively driven substitutions in protein targeting sequences. Notably, our data also reveal that relocated proteins functionally adapted to their new subcellular environments and evolved new functional roles through changes of their physico-chemical properties, expression levels, and interaction partners. CONCLUSION: We conclude that protein subcellular adaptation represents a common mechanism for the functional diversification of duplicate genes.

The role of gene duplication in the origin and evolution of new biological functions

Abstract : Gene duplication is an essential source of material for the origin of genetic novelty and the evolution of lineage- or species-specific phenotypic traits. The reverse transcription of source gene mRNA followed by the genomic insertion of the resulting cDNA - retroposition - has provided the human genome with a significant number of gene copies during the last ~63 million years (MYA) of primate evolution. We estimated that at least 1 new functional gene (retrogene) per MYA emerged by retroposition in the primate lineage leading to humans. Using a combination of comparative sequencing and evolutionary simulations, we obtained strong evidence of functionality for 7 primate specific retrogenes. Most of these genes are specifically expressed in testis suggesting that retroposition has contributed with genetic raw material necessary for the evolution ofmale-specific functions in primates. We characterized CDC14Bretro (identified in the previous survey) that originated from the retroposition of a cell cycle gene - CDC14B - in the common ancestor of humans and apes. We demonstrate that CDC14Bretro experienced a period of intense positive selection in the African ape ancestor. By virtue of the amino acid substitutions that occurred during this period CDC 14Bretro adapted to a new subcellular compartment in African apes. Further analyses indicate that this subcellular shift reflects the evolution of anew functional role of CDC 14Bretro. Prompted by this result, we used yeast (Saccharomyces cerevisiae) to investigate on a global scale the extent of functional diversification of duplicate genes through the subcellular adaptation of their encoded proteins. We found that duplicate proteins frequently evolved new cellular localization patterns, either by partitioning of ancestral localizations ("sublocalization"), or more frequently by relocalization to previously unoccupied compartments ("neolocalization"). Interestingly, proteins involved in processes with a wider subcellular distribution more frequently evolved new localization patterns suggesting that subcellular localization changes are dependent on progenitor gene functions. Relocated proteins adapted to their new subcellular environments and evolved new functional roles through changes of their physio-chemical properties, expression levels, and interaction partners. Our work suggests an important role of subcellular adaptation for the emergence of new gene functions.

Pre-analytical and methodological challenges in red blood cell microparticle proteomics.

Microparticles are phospholipid vesicles shed mostly in biological fluids, such as blood or urine, by various types of cells, such as red blood cells (RBCs), platelets, lymphocytes, endothelial cells. These microparticles contain a subset of the proteome of their parent cell, and their ready availability in biological fluid has raised strong interest in their study, as they might be markers of cell damage. However, their small size as well as their particular physico-chemical properties makes them hard to detect, size, count and study by proteome analysis. In this review, we report the pre-analytical and methodological caveats that we have faced in our own research about red blood cell microparticles in the context of transfusion science, as well as examples from the literature on the proteomics of various kinds of microparticles.

Constitutively active mutants of the beta1-adrenergic receptor.

We provide the first evidence that point mutations can constitutively activate the beta(1)-adrenergic receptor (AR). Leucine 322 of the beta(1)-AR in the C-terminal portion of its third intracellular loop was replaced with seven amino acids (I, T, E, F, C, A and K) differing in their physico-chemical properties. The beta(1)-AR mutants expressed in HEK-293 cells displayed various levels of constitutive activity which could be partially inhibited by some beta-blockers. The results of this study might have interesting implications for future studies aiming at elucidating the activation process of the beta(1)-AR as well as the mechanism of action of beta-blockers.

Atmospheric deposition and migration of artificial radionuclides in Alpine soils (Val Piora, Switzerland) compared to the distribution of selected major and trace elements.

Artificial radionuclides ((137)Cs, (90)Sr, Pu, and (241)Am) are present in soils because of Nuclear Weapon Tests and accidents in nuclear facilities. Their distribution in soil depth varies according to soil characteristics, their own chemical properties, and their deposition history. For this project, we studied the atmospheric deposition of (137)Cs, (90)Sr, Pu, (241)Am, (210)Pb, and stable Pb. We compared the distribution of these elements in soil profiles from different soil types from an alpine Valley (Val Piora, Switzerland) with the distribution of selected major and trace elements in the same soils. Our goals were to explain the distribution of the radioisotopes as a function of soil parameters and to identify stable elements with analogous behaviors. We found that Pu and (241)Am are relatively immobile and accumulate in the topsoil. In all soils, (90)Sr is more mobile and shows some accumulations at depth into Fe-Al rich horizons. This behavior is also observed for Cu and Zn, indicating that these elements may be used as chemical analogues for the migration of (90)Sr into the soil.

Fate of the herbicide glyphosate and its metabolite AMPA in soils and their transfer to surface waters: A multi-scale approach in the Lavaux vineyards, western Switzerland

The use of herbicides in agriculture may lead to environmental problems, such as surface water pollution, with a potential risk for aquatic organisms. The herbicide glyphosate is the most used active ingredient in the world and in Switzerland. In the Lavaux vineyards it is nearly the only molecule applied. This work aimed at studying its fate in soils and its transfer to surface waters, using a multi-scale approach: from molecular (10-9 m) and microscopic scales (10-6 m), to macroscopic (m) and landscape ones (103 m). First of all, an analytical method was developed for the trace level quantification of this widely used herbicide and its main by-product, aminomethylphosphonic acid (AMPA). Due to their polar nature, their derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) was done prior to their concentration and purification by solid phase extraction. They were then analyzed by ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The method was tested in different aqueous matrices with spiking tests and validated for the matrix effect correction in relevant environmental samples. Calibration curves established between 10 and 1000ng/l showed r2 values above 0.989, mean recoveries varied between 86 and 133% and limits of detection and quantification of the method were as low as 5 and 10ng/l respectively. At the parcel scale, two parcels of the Lavaux vineyard area, located near the Lutrive River at 6km to the east of Lausanne, were monitored to assess to which extent glyphosate and AMPA were retained in the soil or exported to surface waters. They were equipped at their bottom with porous ceramic cups and runoff collectors, which allowed retrieving water samples for the growing seasons 2010 and 2011. Results revealed that the mobility of glyphosate and AMPA in the unsaturated zone was likely driven by the precipitation regime and the soil characteristics, such as slope, porosity structure and layer permeability discrepancy. Elevated glyphosate and AMPA concentrations were measured at 60 and 80 cm depth at parcel bottoms, suggesting their infiltration in the upper parts of the parcels and the presence of preferential flow in the studied parcels. Indeed, the succession of rainy days induced the gradual saturation of the soil porosity, leading to rapid infiltration through macropores, as well as surface runoff formation. Furthermore, the presence of more impervious weathered marls at 100 cm depth induced throughflows, the importance of which for the lateral transport of the herbicide molecules was determined by the slope steepness. Important rainfall events (>10 mm/day) were clearly exporting molecules from the soil top layer, as indicated by important concentrations in runoff samples. A mass balance showed that total loss (10-20%) mainly occurred through surface runoff (96%) and, to a minor extent, by throughflows in soils (4%), with subsequent exfiltration to surface waters. Observations made in the Lutrive River revealed interesting details of glyphosate and AMPA dynamics in urbanized landscapes, such as the Lavaux vineyards. Indeed, besides their physical and chemical properties, herbicide dynamics at the catchment level strongly depend on application rates, precipitation regime, land use and also on the presence of drains or constructed channels. Elevated concentrations, up to 4970 ng/l, observed just after the application, confirmed the diffuse export of these compounds from the vineyard area by surface runoff during main rain events. From April to September 2011, a total load of 7.1 kg was calculated, with 85% coming from vineyards and minor urban sources and 15% from arable crops. Small vineyard surfaces could generate high concentrations of herbicides and contribute considerably to the total load calculated at the outlet, due to their steep slopes (~10%). The extrapolated total amount transferred yearly from the Lavaux vineyards to the Lake of Geneva was of 190kg. At the molecular scale, the possible involvement of dissolved organic matter (DOM) in glyphosate and copper transport was studied using UV/Vis fluorescence spectroscopy. Combined with parallel factor (PARAFAC) analysis, this technique allowed characterizing DOM of soil and surface water samples from the studied vineyard area. Glyphosate concentrations were linked to the fulvic-like spectroscopic signature of DOM in soil water samples, as well as to copper, suggesting the formation of ternary complexes. In surface water samples, its concentrations were also correlated to copper ones, but not in a significant way to the fulvic-like signature. Quenching experiments with standards confirmed field tendencies in the laboratory, with a stronger decrease in fluorescence intensity for fulvic-like fluorophore than for more aromatic ones. Lastly, based on maximum concentrations measured in the river, an environmental risk for these compounds was assessed, using laboratory tests and ecotoxicity data from the literature. In our case and with the methodology applied, the risk towards aquatic species was found negligible (RF<1).

Glucocorticoids exert direct toxicity on microvasculature: analysis of cell death mechanisms.

Glucocorticoids (GCs) are routinely administered systemically or injected into the eye when treating numerous ocular diseases; however, their toxicity on the retinal microvasculature has not been previously investigated. In this article, the effects of hydrocortisone (Hydro), dexamethasone, dexamethasone-phosphate and triamcinolone acetonide (TA) were evaluated in vitro on human skin microcirculation cells and, bovine endothelial retinal cells, ex-vivo, on flat mounted rat retinas. The degree of GCs induced endothelial cell death varied according to the endothelial cell type and GCs chemical properties. GCs toxicity was higher in skin microvascular endothelial cells and for hydrophobic GC formulations. The mechanism of cell death differed between GCs, Hydro and TA activated the leukocyte elastase inhibitor/L-DNase II pathways but did not activate caspases. The mechanisms of cell death observed in cell cultures were similar to those observed in rat retinal explants. Taken together these results indicate that particular attention should be paid to the potential vascular side effects when administrating GCs clinically and in particular when developing sustained-release intraocular devices.

Local alignment refinement using structural assessment.

Homology modeling is the most commonly used technique to build a three-dimensional model for a protein sequence. It heavily relies on the quality of the sequence alignment between the protein to model and related proteins with a known three dimensional structure. Alignment quality can be assessed according to the physico-chemical properties of the three dimensional models it produces.In this work, we introduce fifteen predictors designed to evaluate the properties of the models obtained for various alignments. They consist of an energy value obtained from different force fields (CHARMM, ProsaII or ANOLEA) computed on residue selected around misaligned regions. These predictors were evaluated on ten challenging test cases. For each target, all possible ungapped alignments are generated and their corresponding models are computed and evaluated.The best predictor, retrieving the structural alignment for 9 out of 10 test cases, is based on the ANOLEA atomistic mean force potential and takes into account residues around misaligned secondary structure elements. The performance of the other predictors is significantly lower. This work shows that substantial improvement in local alignments can be obtained by careful assessment of the local structure of the resulting models.