A comparison of different lysis buffers to assess allele dropout from single cells for preimplantation genetic diagnosis

Single cell polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD) requires high efficiency and accuracy. Allele dropout (ADO), the random amplification failure of one of the two parental alleles, remains the most significant problem in PCR-based PGD testing since it can result in serious misdiagnosis for compound heterozygous or autosomal dominant conditions. A number of different strategies (including the use of lysis buffers to break down the cell and make the DNA accessible) have been employed to combat ADO with varying degrees of success, yet there is still no consensus among PGD centres over which lysis buffer should be used (ESHRE PGD Consortium, 1999 ). To address this issue, PCR amplification of three genes (CFTR, LAMA3 and PKP1) at different chromosomal loci was investigated. Single lymphocytes from individuals heterozygous for mutations within each of the three genes were collected and lysed in either alkaline lysis buffer (ALB) or proteinase K/SDS lysis buffer (PK). PCR amplification efficiencies were comparable between alkaline lysis and proteinase K lysis for PCR products spanning each of the three mutated loci (ΔF508 in CFTR 90% vs 88%; R650X in LAMA3 82% vs 78%; and Y71X in PKP1 91% vs 87%). While there was no appreciable difference between ADO rates between the two lysis buffers for the LAMA3 PCR product (25% vs 26%), there were significant differences in ADO rates between ALB and PK for the CFTR PCR product (0% vs 23%) and the PKP1 PCR product (8% vs 56%). Based on these results, we are currently using ALB in preference to PK/SDS buffer for the lysis of cells in clinical PGD. Copyright © 2001 John Wiley & Sons, Ltd.     

Modelling nitrogen fluxes at the landscape scale

The distribution and impacts of different nitrogen pollutants are inextricably linked. To understand the problem fully, the interactions between the different pollutants need to be taken into account. This is particularly important when it comes to abatement techniques, since measures to reduce emissions of one nitrogen pollutant can often lead to an increase in another. This project represents a step towards greater understanding of these issues by linking together new and existing nitrogen flux models into a larger framework. The modelling framework has been constructed and some of the nitrogen flows between fields, farms and the atmosphere have been modelled for a UK study area for typical farm management scenarios.     

Perceptions of river managers of institutional constraints on floodplain restoration in the UK

The restoration of river environments has been of growing importance to river management and planning in the UK. The extension of ecological restoration to floodplains as well as river channels is more complex, partly because of the range of stakeholders and the diversity of relevant management institutions. This paper draws on a qualitative survey of river managers in the UK to identify institutional factors relevant to the success or failure of floodplain restoration projects.     

Does Including Soil Moisture Observations Improve Operational Streamflow Forecasts in Snow‐Dominated Watersheds?

Changing climate and growing water demand are increasing the need for robust streamflow forecasts. Historically, operational streamflow forecasts made by the Natural Resources Conservation Service have relied on precipitation and snow water equivalent observations from Snow Telemetry (SNOTEL) sites. We investigate whether also including SNOTEL soil moisture observations improve April‐July streamflow volume forecast accuracy at 0, 1, 2, and 3‐month lead times at 12 watersheds in Utah and California. We found statistically significant improvement in 0 and 3‐month lead time accuracy in 8 of 12 watersheds and 10 of 12 watersheds for 1 and 2‐month lead times. Surprisingly, these improvements were insensitive to soil moisture metrics derived from soil physical properties. Forecasts were made with volumetric water content (VWC) averaged from October 1 to the forecast date. By including VWC at the 0‐month lead time the forecasts explained 7.3% more variability and increased the streamflow volume accuracy by 8.4% on average compared to standard forecasts that already explained an average 77% of the variability. At 1 to 3‐month lead times, the inclusion of soil moisture explained 12.3‐26.3% more variability than the standard forecast on average. Our findings indicate including soil moisture observations increased statistical streamflow forecast accuracy and thus, could potentially improve water supply reliability in regions affected by changing snowpacks.     

Acid sulfate soils and human health—A Millennium Ecosystem Assessment

Acid sulfate soils have been described as the “nastiest soils on earth” because of their strong acidity, increased mobility of potentially toxic elements and limited bioavailability of nutrients. They only cover a small area of the world's total problem soils, but often have significant adverse effects on agriculture, aquaculture and the environment on a local scale. Their location often coincides with high population density areas along the coasts of many developing countries. As a result, their negative impacts on ecosystems can have serious implications to those least equipped for coping with the low crop yields and reduced water quality that can result from acid sulfate soil disturbance. The Millennium Ecosystem Assessment called on by the United Nations in 2000 emphasised the importance of ecosystems for human health and well-being. These include the service they provide as sources of food and water, through the control of pollution and disease, as well as for the cultural services ecosystems provide. While the problems related to agriculture, aquaculture and the environment have been the focus of many acid sulfate soil management efforts, the connection to human health has largely been ignored. This paper presents the potential health issues of acid sulfate soils, in relation to the ecosystem services identified in the Millennium Ecosystem Assessment. It is recognised that significant implications on food security and livelihood can result, as well as on community cohesiveness and the spread of vector-borne disease. However, the connection between these outcomes and acid sulfate soils is often not obvious and it is therefore argued that the impact of such soils on human well-being needs to be recognised in order to raise awareness among the public and decision makers, to in turn facilitate proper management and avoid potential human ill-health.     

In vitro approaches to assess bioavailability and human gastrointestinal mobilization of food-borne polychlorinated biphenyls (PCBs)

This study reports on the potential for gastrointestinal (GI) mobilization and bioavailability of food-borne PCBs in humans. The development and validation of a GI simulator and operational protocols, developed in compliance with the requirements of German DIN 19738 risk assessment test procedure, are presented. Food, naturally contaminated with PCBs, was homogenized with simulated saliva fluid and shaken in the GI simulator with simulated gastric fluids (containing pepsin, mucine) for 2 h at 37°C. Afterwards, the simulated intestinal fluids (containing pepsin, mucine, trypsin, pancreatin, bile) were added and the mixture shaken for a further 6 h prior to centrifugation and filtration using Buchner funnels to separate the undigested GI residues from GI fluids. PCBs were recovered from GI residues and fluids by Soxhlet and liquid-liquid extraction respectively, cleaned up using silica-SFE, and analyzed by gas chromatography mass spectrometry detection (GC-MSD). Detailed studies with fish indicate variations in mobilization and bioavailability of Σ PCBs (28, 52, 101, 118, 153, 138 and 180). For example, the bioavailable fractions (fractions mobilized) in mackerel, salmon, crab and prawn were 0.77, 0.60, 0.54, and 0.72 respectively of the Σ PCBs initially present in these food samples. The bioavailable fraction was dependent on the physicochemical characteristics of the PCBs. In mackerel bioavailable fractions for individual PCB congeners ranged from 0.47–0.82, from 0.30–0.70 in salmon, 0.44–0.64 in crab and in prawn from 0.47–0.77. Future studies will focus on understanding better, the variability in bioavailable fractions to be expected for different foodstuffs, in addition to tissue culture techniques using human gut cell lines to investigate a simultaneous mobilization and absorption of food-borne PCBs.     

Predicting molybdenum toxicity to higher plants: Estimation of toxicity threshold values

Four plant species (oilseed rape, Brassica napus L.; red clover, Trifolium pratense L.; ryegrass, Lolium perenne L.; and tomato, Lycopersicon esculentum L.) were tested on ten soils varying widely in soil properties to assess molybdenum (Mo) toxicity. A larger range (66-fold-609-fold) of added Mo concentrations resulting in 50% inhibition of yield (ED₅₀) was found among soils than among plant species (2-fold-38-fold), which illustrated that the soils differed widely in the expression of Mo toxicity. Toxicity thresholds based on soil solution Mo narrowed the variation among soils compared to thresholds based on added Mo concentrations. We conclude that plant bioavailability of Mo in soil depends on Mo solubility, but this alone did not decrease the variability in observed toxicity enough to be used in risk assessment and that other soil properties influencing Mo toxicity to plants need to be considered.     

Quantification of ionophores in aged poultry litter using liquid chromatography tandem mass spectrometry

Veterinary anticoccidials, biochemically known as ionophores, are widely used in poultry feed at therapeutic levels to treat Coccidiosis and at sub-therapeutic levels for growth- promotion. Commonly used ionophores in the US poultry industry are monensin, salinomycin, lasalocid and narasin. There is an increasing concern regarding the persistence of these anticoccidials in the environment. However, little attention has been directed to methods development for quantitatively measuring ionophores in complex environmental matrices such as poultry litters that are land applied. Here, we describe a rapid and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method developed for simultaneous quantification of monensin, lasalocid, salinomycin, and narasin in aged poultry litter samples. Results show significant level of monensin (97.8 ± 3.2?μg kg(-1)), lasalocid (19.2 ± 6.6?μg kg(-1)), salinomycin (70 ± 2.7?μg kg(-1)) and narasin (57.3 ± 2.6?μg kg(-1)) in poultry litter stored for over three years at < 5°C. Our findings indicate that even after several years of unmanaged storage of poultry litter, ionophores may continue to persist in this matrix, raising the possibility of prolonged release into the environment.     

Cadmium content of wheat grain from a long-term field experiment with sewage sludge

Grain Cd concentrations were determined in the wheat (Triticum aestivum L.) cultivars Soissons, Brigadier, and Hereward grown in 1994,1996, and 1999, respectively, in soils of a long-term field experiment to which sewage sludges contaminated with Zn, Cu, Ni, or Cr had previously been added. Soil pore water soluble Cd and free Cd2+ increased linearly with increasing total soil Cd (R2=0.82 and 0.84, respectively; P<0.001). Similarly, soil pore water free Cd2+ increased linearly with increasing soil pore water soluble Cd (R2=0.98; P<0.001). There was no evidence of a plateau in soil pore water Cd concentrations with increasing soil Cd concentrations. Grain Cd concentrations were significantly correlated with total soil Cd (P<0.001), soil pore water Cd (P<0.001), and free Cd2+ (P<0.001). A slight curvilinear relationship between grain Cd and soil Cd was apparent, but there was no plateau, even at the maximum soil Cd concentration of about 2.7 mg kg(-1). The relationship between soil pore water Cd and grain Cd was linear for all three cultivars. The slopes were in the order 1994 > 1996 > 1999, with more Cd being taken up into the grain by Soissons grown in 1994, and least by Hereward grown in 1999. For Soissons, Cd concentration in the grain greater than the EU limit (0.24 mg kg(-1) dry wt.) occurred at soil Cd less than the current UK limit of 3 mg kg(-1) for soils receiving sewage sludge. In contrast, for Brigadier and Hereward, grain Cd concentrations were near to and less than the EU limit, respectively, at soil Cd concentrations of 3 mg kg(-1).     

The dynamics of arsenic in four paddy fields in the Bengal delta

Irrigation with arsenic contaminated groundwater in the Bengal Delta may lead to As accumulation in the soil and rice grain. The dynamics of As concentration and speciation in paddy fields during dry season (boro) rice cultivation were investigated at 4 sites in Bangladesh and West Bengal, India. Three sites which were irrigated with high As groundwater had elevated As concentrations in the soils, showing a significant gradient from the irrigation inlet across the field. Arsenic concentration and speciation in soil pore water varied temporally and spatially; higher As concentrations were associated with an increasing percentage of arsenite, indicating a reductive mobilization. Concentrations of As in rice grain varied by 2-7 fold within individual fields and were poorly related with the soil As concentration. A field site employing alternating flooded-dry irrigation produced the lowest range of grain As concentration, suggesting a lower soil As availability caused by periodic aerobic conditions.