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.     

THE INFLUENCE OF SETTINGS AND ENVIRONMENTAL EVENTS ON METAMOTIVATIONAL STATE AND AROUSAL: FURTHER TESTS OF REVERSAL THEORY ON ASIAN SAMPLES

The two studies reported further test reversal theory hypotheses concerning human psychological responses to different types of settings and environmental events on an Asian sample. In study 1, individuals in four settings representing different combinations of metamotivational state and arousal level (telic and paratelic/low arousal; telic and paratelic/high arousal) completed the telic state measure (TSM; Svebak & Murgatroyd, 1985). Significant differences between participants' mean scores across the four settings were obtained for TSM items serious–playful, planning–spontaneous, felt arousal, preferred arousal, effort and arousal discrepancy. In study 2 different environmental events in the same setting (changes in reading tasks), used in a crossover design, were used to induce reversals in individuals' operative metamotivational states. The results obtained from TSM scores pretask 1, between tasks and posttask 2. indicated significant time-of-testing interaction effects on the TSM serious–playful, planning–spontaneous and effort items. In addition, a main effect for TSM felt arousal was found. These results showed that reversals in metamotivational states were induced by the environmental event and that felt arousal scores increased across the three administrations of the TSM.     

Human induced salinisation and the use of quantitative methods

Sustainable development of the earth's limited water and land resources is of paramount importance because of rising world population and existing conflicting demands for these resources. Enormous capital investment has been made in developing these resources, but now there is irrefutable evidence that such developments have led to major resource degradation. This includes problems of salinisation and damages to ecosystems. The countries predominantly affected by human induced salinisation are located in arid and semi-arid regions of the world and include Australia, China, Egypt, India, Pakistan, USSR, and USA. This paper describes the processes of salinisation, its impacts and the use of quantitative methods in salinity investigation and management. Australia is used as a case study of typical salinity problems and as a demonstration of the fruitful application of quantitative methods. The paper concludes that quantitative methods such as surface water and groundwater models are powerful design, management and predictive tools in salinity investigation. However, application of some models, such as those for unsaturated flow and transport and groundwater solute transport, are not widespread due to uncertainties in describing the complexity of the processes and the lack of hydrodispersive data.     

Mangrove Mapping Using Landsat Imagery and Aerial Photographs: Kemaman District, Terengganu, Malaysia

Classification and distribution of mangrove vegetation are vital information for the proper development of a mangrove management plan. In this study, classification for the mangroves of the district of Kemaman were done using both 1 : 5000 aerial photographs and Landsat TM imageries. The coverage by aerial photographs is limited to the coastal and estuarine areas only. Thus, for areas further upstream of the aerial photo coverage, Landsat TM imageries were used. Analysis of aerial photographs and remote sensing images revealed that the mangroves of Kemaman could be classified into 14 different classes of vegetation. All the 14 classes were identified from areas covered by the aerial photographs. For areas covered by the Landsat images only 7 classes of vegetation were identified. The accuracy for aerial photograph and Landsat images are 91.2% and 87.8%, respectively. It can be concluded that although both techniques are useful in determining the mangrove vegetation classes, the large 1 : 5000 aerial photographs are more accurate and provided more detailed information comparatively.     

Assessment of environmental changes in the Orinoco River delta

Major anthropogenic driven changes in the hydrologic and sedimentation patterns of the Orinoco River have had an impact on environmental conditions in the delta. The abrupt water flow reduction from 3,600 to 200 m³ s^–1 in one of its major distributaries resulting from dam construction forced its transformation from a fresh-water body into a tidal channel with an increase in salinity level (as far as 100 km upstream) and with well-mixed water at the mouth and estuarine connection to the Paria Gulf. Three different sectors along this distributary can be identified (indicated by the Na/Cl ratio in the water). As a result, noticeable changes have occurred in the mangrove community which moved about 60 km further upstream. The changes have also promoted the formation of new islands of sediment progradation at the mouth of this distributary, where successional colonization and species replacement by different species of grasses and mangroves take place. Electronic Publication     

Gas chromatographic methods for monitoring of wastewater chlorophenol degradation in anaerobic reactors

Wastewater samples from an anaerobic reactor were extracted with hexane and derivatized with diazomethane (method 1) and with acetic anidride (method 2). Gas chromatography with electron-capture detection (ECD) was employed for separating the parent compound and intermediates trichlorophenols (TCP) and dichlorophenols (DCP) which originated from the penta chlorophenol (PCP) degradation process. The relations between concentrations of PCP, TCP and DCP areas were linear in the range of concentrations of 0.2 to 8 mg/L and 0.025 mg/L to 5 mg/L for methods 1 and 2, respectively. The repeatability of the extraction methods was satisfactory, with variation coefficients lower than 11%. For method 1, at the fortification level of 0.2 mg/L, recovery of PCP, TCP, and DCP was 112%, 74% and 45%, respectively. For method 2, the corresponding recovery values at the fortification level of 0.1 mg/L were 91%, 93% and 103%, respectively. Storage of the frozen samples did not alter their PCP determination properties. The chromatographic methods adapted for chlorophenol determination in wastewater were suitable with relatively simple manipulation techniques. The obtained results were reproducible and allowed identification of intermediates formed during the PCP degradation process.     

Inputs of chemicals from recreational activities into the Norwegian coastal zone

Wastewater treatment works effluent is often considered to be one of the most important point sources of a wide range of anthropogenic contaminants to aquatic systems, however, this paper discusses other potential sources. With the aim of establishing the important sources of UV filters, insect repellent and biocides to the aquatic environment, samples were collected from sites with direct (bathing areas and marinas) and indirect (sites receiving wastewater effluent) human influences. Sunscreens containing UV filters are used in large volumes during the summer months and often applied shortly before a person enters the water for swimming activities. The results presented here demonstrate that washing directly from the skin is an important point source of 4 UV filters to the Oslofjord. The insect repellent, diethyl toluamide, was also measured and it was concluded that washing from the skin was not such an important point source into the fjord. Concentrations of the biocide Irgarol 1051 were also measured and were elevated in the small boat marina and surrounding enclosed area. This work demonstrates that man recreational water-based activities are a diffuse source of some contaminants into coastal and fjord environments and this study provides an initial assessment of the levels being released.