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.     

Nest-building by crested tit Parus cristatus males: an analysis of costs and benefits

In a population of crested tits in Belgium, one out of three breeding males participated in nest-building. Since variation in the amount of nest-building among 11 building males was low, analysis of male nest-building allowed a logistic regression design. The probability of building increased with male condition and for late nests. The effect of male condition decreased with date, and date was the only factor predicting nest-building for repeat broods. The effect of male condition on nest-building suggests that building entails important costs for males. Based on correlative data, we argue that building by males shortens the interval between the start of nest-building and the onset of egg-laying by about 5 days. Since clutch size, incubation and nestling time did not differ between early and late broods, male nest-building apparently affects fledging date. Since early fledgers (early dispersers) are more likely to settle in future winter flocks, building behaviour by crested tit males should be favoured by natural selection. So far, male nest-building in hole-breeding birds might have been overlooked due to the widespread use of artificial nest-boxes in behavioural studies.     

Review of present European environmental policies and legislation involving cultural heritage

Cultural heritage is being addressed by a number of charters and conventions and it is clear that its consideration within decision-making processes is progressively becoming a real challenge, both for developers and public authorities. Against this background, this paper reviews the environmental assessment framework developed by the European Community, as this should increasingly influence decision making about cultural heritage in an urban setting. The legislative framework for access to environmental information is also reviewed because of its relevance to the decision-making process.The Directives on Environmental Impact Assessment and Strategic Environmental Assessment both require a consideration of cultural heritage in decision making. This requirement can go some way to addressing the paradox in the European Community's position whereby the community wishes to conserve and enhance its own cultural identity whilst, at the same time, cultural heritage is usually defined at a local level. The present paper suggests that, given their flexibility, the two European Community directives on environmental assessment constitute a promising opportunity to address this seeming paradox, but that there is a significant gap between legal obligations and the methodological tools to meet them.     

OBSERVATIONS ON PHYTOPLANKTON PRODUCTIVITY IN RELATION TO HYDROGRAPHY IN THE NORTHERN ADRIATIC

This work, carried out within the framework of the PRISMA II project, aims at evaluating the effects of the Po River flow on primary productivity, measured by C with in situ sample incubations. A total of four cruises was carried out in late winter and early summer (1996-1998) along transects from the coast offshore. In both seasons, the highest primary productivity was found in the most oligohaline stations. The effects of the Po River inputs were therefore clearly evident in the westernmost area, both in high productivity values and in the shape of productivity profiles. In late winter, extensive blooms of Skeletonema costatum and Pseudo-nitzschia delicatissima resulted in productivity values that were sometimes higher than in early summer. In early summer 1996, greatly reduced river flow clearly influenced productivity which was much lower than in 1997.     

Effects of calibration on L-THIA GIS runoff and pollutant estimation

Urbanization can result in alteration of a watershed's hydrologic response and water quality. To simulate hydrologic and water quality impacts of land use changes, the Long-Term Hydrologic Impact Assessment (L-THIA) system has been used. The L-THIA system estimates pollutant loading based on direct runoff quantity and land use based pollutant coefficients. The accurate estimation of direct runoff is important in assessing water quality impacts of land use changes. An automated program was developed to calibrate the L-THIA model using the millions of curve number (CN) combinations associated with land uses and hydrologic soil groups. L-THIA calibration for the Little Eagle Creek (LEC) watershed near Indianapolis, Indiana was performed using land use data for 1991 and daily rainfall data for six months of 1991 (January 1-June 30) to minimize errors associated with use of different temporal land use data and rainfall data. For the calibration period, the Nash-Sutcliffe coefficient was 0.60 for estimated and observed direct runoff. The calibrated CN values were used for validation of the model for the same year (July 1-December 31), and the Nash-Sutcliffe coefficient was 0.60 for estimated and observed direct runoff. The Nash-Sutcliffe coefficient was 0.52 for January 1, 1991 to December 31, 1991 using uncalibrated CN values. As shown in this study, the use of better input parameters for the L-THIA model can improve accuracy. The effects on direct runoff and pollutant estimation of the calibrated CN values in the L-THIA model were investigated for the LEC. Following calibration, the estimated average annual direct runoff for the LEC watershed increased by 34%, total nitrogen by 24%, total phosphorus by 22%, and total lead by 43%. This study demonstrates that the L-THIA model should be calibrated and validated prior to application in a particular watershed to more accurately assess the effects of land use changes on hydrology and water quality.