Enrichment of fetal cells from maternal blood by high gradient magnetic cell sorting (double MACS) for PCR-based genetic analysis

For simple and effective isolation of fetal cells from peripheral maternal blood, we combined depletion of maternal cells and enrichment of fetal cells by high-gradient magnetic cell separation (MACS). First CD45⁺ and CD14⁺ cells were depleted from maternal peripheral blood mononuclear cells by MACS. From the depleted fraction, CD71⁺ erythroid cells were enriched up to 80 per cent by MACS. This ‘double-MACS’ procedure yielded an average depletion rate of 780-fold and an average enrichment rate of 500-fold, with approximate recovery rates of 40–55 per cent. For paternity testing, cells from unseparated blood and the various fractions were analysed for polymorphism of the HLA-DQ-A1 locus and D1S80 locus by the polymerase chain reaction (PCR). In CD45⁻/CD71⁺ sorted cells from maternal blood, but not in unfractionated cells from maternal blood or CD45⁻/CD14⁻ cells, paternal alleles could be detected. In the CD45⁻/CD71⁺ fraction, the relative frequency of paternal alleles compared with maternal alleles ranged from 1 in 20 to 1 in 200 (determined by titration and depending on the quality of separation and biological variation). In 7 out of 11 cases, between weeks 12 and 25 of gestation, we could identify paternal alleles by PCR, either HLA-DQ-A1 or D1S80. This double-MACS procedure is simple, fast, efficient, and reliable for non-invasive prenatal diagnosis.     

False-negative prenatal exclusion of Wiskott-Aldrich syndrome by measurement of fetal platelet count and size

The study of the fetal platelet count and size can, according to the literature, be used for the prenatal diagnosis of the Wiskott-Aldrich syndrome (WAS). So far, no affected fetuses have been identified by this method. All pregnancies in which this method had been applied to resulted, as correctly predicted, in the birth of normal children. Here we report on a familial case of WAS where the haematological parameters failed to reveal the affected second child. Hence we assume that the platelet count and size of platelets remain normal in fetuses with WAS to the gestational age of 22 weeks and cannot be used for prenatal diagnosis.     

Soil functional responses to excess nitrogen inputs at global scale

There is little evidence that nitrogen (N) cycling in the highly weathered, low-phosphorus (P), acidic soils found in Southern Hemisphere continents will differ greatly from that in North America and Europe. Evidence from the 'south' shows: the similarity in forms and temporal patterns in losses of N from different land uses; that the C:N ratios of the forest floor/litter layer from different continents are strongly predictive of a range of processes on a global scale; that generalizations based on Northern Hemisphere experience of the impact of N additions to 'P-limited' ecosystems are likely to fail for southern ecosystems where anatomical and physiological adaptation of native plants to low-P soils makes questionable the concept of 'P-limitation'; that the greatest threats in the 'south' are probably changes in land use that may greatly increase N inputs and turnover; that localized increases in N inputs produce similar effects to those seen in the 'north'.     

Investigations of nitrogen fluxes and pools on a limestone site in the Alps

In the North Tyrolean Limestone Alps a site was investigated over a four-year period (1998–2001) in order to assess the nitrogen saturation status, the nitrogen budget (quantification of the net uptake of nitrogen by the canopy and of the nitrogen mineralization, nitrogen uptake from roots and N₂O emission rates, proof of the origin of nitrate in the soil water with stable isotope analyses), and the effects of the actual nitrogen input on ground water quality. The main goals were to quantify the nitrogen input rate, the nitrogen pools in above-ground and below-ground compartments, nitrogen turnover processes in the soil as well as the output into the groundwater and into the atmosphere. The findings are based on continuous and discontinuous field measurements as well as on model results.While nitrogen input exceeded the Critical Loads of the WHO (1995), nitrogen deficiency and nutrient imbalances were verified by needle analyses. The atmospheric input of inorganic nitrogen was higher than the nitrogen output in 50 cm soil depth. A tracer experiment with¹⁵N helped to prove that not more than half of the applied nitrate could be discharged. This allows the conclusion that nitrogen is stored in the system and that the site cannot yet be said to be saturated with nitrogen. The same result was also obtained by modelling. In addition, it was proved that the nitrogen discharge did not stem from deposition but from processes within the system.     

Nitrate dynamics in an alpine forest site (Mühleggerköpfl) O and N stable isotope analysis in natural water samples

Stable isotope analysis of¹⁵N/¹⁴N and¹⁸O/¹⁶O - nitrate was used to investigate the nitrate dynamics and potential groundwater pollution in an Alpine forest stand in Tyrol/Austria. The dynamics of δ¹⁵−N_nitrate values were followed in a forest ecosystem. The stable isotopic values of the throughfall are comparable with other studies. The completely decoupled dynamics of the δ¹⁵−N_nitrate of the precipitation and the surface water was observed. High variations in δ¹⁵-N - nitrate values in rainfall indicate that nitrate of different sources is deposited at that site. A significant correlation between the δ¹⁵N_nitrate values of the surface water and soil water was obtained, while no significant correlation between the δ¹⁵N_nitrate values of any precipitation sample with the surface water could be found. This suggests that the main source of nitrate in soil water originates from microbiological activity such as nitrification reactions and less from nitrate input by deposition. The results of δ¹⁸O_nitrate measurements strongly supported the microbiological origin of nitrate in the surface and soil water. In an additional lysimeter experiment,¹⁵N - labelled nitrate was applied to study nitrate transport in soil. After 130 days and the collection of 300 L leachate, a total of 52% of the applied nitrate was detected in seepage water.

     

Modelling the responses of the Lagoon of Venice ecosystem to variations in physical forcings

A Finite Element Ecological Model for the Lagoon of Venice (VELFEEM) has been used to test the responses of the Lagoon of Venice ecosystem to variations in physical conditions.The model is obtained by coupling a finite element hydrodynamic model, that computes the velocity fields of water, an energetic model to compute the water temperature fields, and an ecological model that simulates the dynamic of phytoplankton, zooplankton, nutrients (ammonia, nitrate and phosphate) organic detritus (organic nitrogen, organic phosphorous and CBOD) and dissolved oxygen.The transport model is a two-dimensional barotropic finite element model which allows for a better resolution of the lagoon morphology.The ecological model has been developed by starting from the ecological module EUTRO of WASP (Water Analysis Simulation System released by US EPA), and by adapting it to the peculiarity of the Lagoon of Venice.A reference condition has been identified by running a 1-year simulation under climatologic condition. Then, the sensitivity to physical forcing (tide and wind) and to the input of macronutrients has been investigated, by comparing model predictions of spatial and temporal evolution of major state variables and of an aggregate index of Water Quality Trophic Index (TRIX).