Quantification of DNA damage by PFGE: development of an analytical approach to correct for the background distribution

Purpose: To analyse the currently existing methods to infer the extent of cellular DNA damage induced by ionizing radiation when the pulsed field gel electrophoresis (PFGE) technique is used.

Development of an Arcellacean (testate lobose amoebae) based transfer function for sedimentary phosphorous in lakes.

Arcellacea (testate lobose amoebae) communities were assessed from 73 sediment-water interface samples collected from 33 lakes in urban and rural settings within the Greater Toronto Area (GTA), Ontario, Canada, as well as from forested control areas in the Lake Simcoe area, Algonquin Park and eastern Ontario. The results were used to: (1) develop a statistically rigorous arcellacean-based training set for sedimentary phosphorus (Olsen P (OP)) loading; and (2) derive a transfer function to reconstruct OP levels during the post-European settlement era (AD1870s onward) using a chronologically well-constrained core from Haynes Lake on the environmentally sensitive Oak Ridges Moraine, within the GTA. Ordination analysis indicated that OP most influenced arcellacean assemblages, explaining 6.5% (p < 0.005) of total variance. An improved training set where the influence of other important environmental variables (e.g. total organic carbon, total nitrogen, Mg) was reduced, comprised 40 samples from 31 lakes, and was used to construct a transfer function for lacustrine arcellaceans for sedimentary phosphorus (Olsen P) using tolerance downweighted weighted averaging (WA-Tol) with inverse deshrinking (RMSEPjack-77pp; r2jack = 0.68). The inferred reconstruction indicates that OP levels remained near pre-settlement background levels from settlement in the late AD 1970s through to the early AD 1970s. Since OP runoff from both forests and pasture is minimal, early agricultural land use within the lake catchment was as most likely pasture and/or was used to grow perennial crops such as Timothy-grass for hay. A significant increase in inferred OP concentration beginning ~ AD 1972 may have been related to a change in crops (e.g. corn production) in the catchment resulting in more runoff, and the introduction of chemical fertilizers. A dramatic decline in OP after ~ AD 1985 probably corresponds to a reduction in chemical fertilizer use related to advances in agronomy, which permitted a more precise control over required fertilizer application. Another significant increase in OP levels after ~ AD 1995 may have been related to the construction of a large golf course upslope and immediately to the north of Haynes Lake in AD 1993, where significant fertilizer use is required to maintain the fairways. These results demonstrate that arcellaceans have great potential for reconstructing lake water geochemistry and will complement other proxies (e.g. diatoms) in paleolimnological research.

The role women play in sustainable agriculture and rural development in Northern Ireland.

Reference: ECA: WPW/02/CASE NORTHERN IRELAND W/X0000/e

Development of a solid-phase extraction coupling chemiluminescent enzyme immunoassay for determination of organophosphorus pesticides in environmental water samples

Solid-phase extraction (SPE) and direct competitive chemiluminescence enzyme immunoassay (dcCL-EIA) were combined for the detection of organophosphorus pesticides (OPs) in environmental water samples. dcCL-EIA based on horseradish peroxidase labeled with a broad-specificity monoclonal antibody against OPs was developed, and the effects of several physicochemical parameters on dcCL-EIA performance were studied. SPE was used for the pretreatment of water samples to remove interfering substances and to concentrate the OP analytes. The coupling of SPE and dcCL-EIA can detect seven OPs (parathion, coumaphos, phoxim, quinalphos, triazophos, dichlofenthion, and azinphos-ethyl) with the limit of quantitation below 0.1 ng/mL. The recoveries of OPs from spiked water samples ranged from 62.5% to 131.7% by SPE-dcCL-EIA and 69.5% to 112.3% by SPE-HPLC-MS/MS. The screening of OP residues in real-world environmental water samples by the developed SPE-dcCL-EIA and their confirmatory analysis using SPE-HPLC-MS/MS demonstrated that the assay is ideally suited as a monitoring method for OP residues prior to chromatographic analysis.

Development of a Broad-Specificity Monoclonal Antibody-Based Immunoaffinity Chromatography Cleanup for Organophosphorus Pesticide Determination in Environmental Samples

An immunoaffinity chromatographic (IAC) method for the selective extraction and concentration of 13 organophosphorus pesticides (OPs, including coumaphos, parathion, phoxim, quinalphos, dichlofenthion, triazophos, azinphos-ethyl, phosalone, isochlorthion, parathion-methyl, cyanophos, disulfoton, and phorate) prior to analysis by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. The IAC column was prepared by covalently immobilizing a monoclonal antibody with broad specificity for OPs on CNBr-activated Sephrose 4B. The column capacity ranged from 884 to 2641 ng/mL of gel. The optimum elution solvent was 0.01 M phosphate-buffered saline containing 80% methanol. The breakthrough volume of the IAC column was found to be 400 mL. Recoveries of OPs from spiked environmental samples by IAC cleanup and HPLC-MS/MS analysis ranged from 60.2 to 107.1%, with a relative standard deviation below 11.1%. The limit of quantitation for 13 OPs ranged from 0.01 to 0.13 ng/mL (ng/g). The application of IAC cleanup coupled to HPLC-MS/MS in real environmental samples demonstrated the potential of this method for the determination of OP residues in environmental samples at trace levels.

Testate amoebae (thecamoebians) as water quality indicators in lakes: development of a modern distributional dataset from the Greater Toronto Area

Thecamoebians were examined from 123 surface sediment samples collected from 45 lakes in the Greater Toronto Area (GTA) and the surrounding region to i) elucidate the controls on faunal distribution in modern lake environments; and ii) to consider the utility of thecamoebians in quantitative studies of water quality change. This area was chosen because it includes a high density of lakes that are threatened by urban development and where water quality has deteriorated locally as a result of contaminant inputs, particularly nutrients. Canonical Correspondence analysis (CCA) and a series of partial CCAs were used to examine species-environment relationships. Twenty-four environmental variables were considered, including water properties (e.g. pH, DO, conductivity), substrate characteristics, nutrient loading, and environmentally available metals. The thecamoebian assemblages showed a strong association with Olsen's Phosphorus, reflecting the eutrophic status of many of the lakes, and locally to elevated conductivity measurements, which appear to reflect road salt inputs associated with winter de-icing operations. A transfer function was developed for Olsen P using this training set based on weighted averaging with inverse deshrinking (WA Inv). The model was applied to infer past changes in Phosphorus enrichment in core samples from several lakes, including eutrophic Haynes Lake within the GTA. Thecamoebian-inferred changes in sedimentary Phosphorus from a 210Pb dated core from Haynes Lake are related to i) widespread introduction of chemical fertilizers to agricultural land in the post WWII era; ii) a steep decline in Phosphorous with a change in agricultural practices in the late 1970s; and iii) the construction of a golf course in close proximity to the lake in the early 1990s. This preliminary study confirms that thecamoebians have considerable potential as indicators of eutrophication in lakes and can provide an estimate of baseline conditions.

Age- and Light-Dependent Development of Localised Retinal Atrophy in CCL2(-/-)CX3CR1(GFP/GFP) Mice

Previous studies have shown that CCL2/CX3CR1 deficient mice on C57BL/6N background (with rd8 mutation) have an early onset (6 weeks) of spontaneous retinal degeneration. In this study, we generated CCL2(-/-)CX3CR1(GFP/GFP) mice on the C57BL/6J background. Retinal degeneration was not detected in CCL2(-/-)CX3CR1(GFP/GFP) mice younger than 6 months. Patches of whitish/yellowish fundus lesions were observed in 17~60% of 12-month, and 30~100% of 18-month CCL2(-/-)CX3CR1(GFP/GFP) mice. Fluorescein angiography revealed no choroidal neovascularisation in these mice. Patches of retinal pigment epithelium (RPE) and photoreceptor damage were detected in 30% and 50% of 12- and 18-month CCL2(-/-)CX3CR1(GFP/GFP) mice respectively, but not in wild-type mice. All CCL2(-/-)CX3CR1(GFP/GFP) mice exposed to extra-light (~800lux, 6 h/day, 6 months) developed patches of retinal atrophy, and only 20-25% of WT mice which underwent the same light treatment developed atrophic lesions. In addition, synaptophysin expression was detected in the outer nucler layer (ONL) of area related to photoreceptor loss in CCL2(-/-)CX3CR1(GFP/GFP) mice. Markedly increased rhodopsin but reduced cone arrestin expression was observed in retinal outer layers in aged CCL2(-/-)CX3CR1(GFP/GFP) mice. GABA expression was reduced in the inner retina of aged CCL2(-/-)CX3CR1(GFP/GFP) mice. Significantly increased Müller glial and microglial activation was observed in CCL2(-/-)CX3CR1(GFP/GFP) mice compared to age-matched WT mice. Macrophages from CCL2(-/-)CX3CR1(GFP/GFP) mice were less phagocytic, but expressed higher levels of iNOS, IL-1ß, IL-12 and TNF-a under hypoxia conditions. Our results suggest that the deletions of CCL2 and CX3CR1 predispose mice to age- and light-mediated retinal damage. The CCL2/CX3CR1 deficient mouse may thus serve as a model for age-related atrophic degeneration of the RPE, including the dry type of macular degeneration, geographic atrophy.

Development and Validation of a Lateral Flow Immunoassay for the Rapid Screening of Okadaic Acid and All Dinophysis Toxins from Shellfish Extracts

A single-step lateral flow immunoassay was developed and validated to detect okadaic acid (OA) and dinophysis toxins (DTXs), which cause diarrhetic shellfish poisoning. The performance characteristics of the test were investigated, in comparison to reference methods (liquid chromatography tandem mass spectrometry and/or bioassay), using both spiked and naturally contaminated shellfish. A portable reader was used to generate a qualitative result, indicating the absence or presence of OA-group toxins, at concentrations relevant to the maximum permitted level (MPL). Sample homogenates could be screened in 20 min (including extraction and assay time) for the presence of free toxins (OA, DTX1, DTX2). DTX3 detection could be included with the addition of a hydrolysis procedure. No matrix effects were observed from the species evaluated (mussels, scallops, oysters, and clams). Results from naturally contaminated samples (n = 72) indicated no false compliant results and no false noncompliant results at <50% MPL. Thus, the development of a new low-cost but highly effective tool for monitoring a range of important phycotoxins has been demonstrated.