Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease

Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8–14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligo-nucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F₅₀₈ deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F₅₀₈ primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR.     

Early amniocentesis at 11–14 weeks' gestation for the diagnosis of fetal chromosomal abnormality—a clinical evaluation

Early arnniocentesis between 11 and 14 weeks' gestation was offered to 110 women at risk of a chromosomally abnormal fetus due to maternal age. Four were found to be unsuitable for the procedure, and 106 early amniocenteses were performed. In 102 cases, clear amniotic fluid was obtained with a single tap. There were two dry taps and two bloodstained tapis; sampling was repeated in three of these cases before 15 weeks. In the fourth case, placental biopsy was performed at 16 weeks. Thus, we were able to obtain a satisfactory sample in all but three cases(2.8 percent). Karyotyping of cells harvested from the early amniotic fluid samples was successful in all the 105 cases. Cell culture from the initial samples revealed a normal karyotype in 99 cases, two balanced translocations, two tetraploid karyotypes, and two cases of pseudomosaicism. Of the 105 pregnancies successfully sampled, there have been two losses to date (1–8 per cent). Two further patients presented with premature rupture of membranes, both pregnancies having successful outcomes. Sixty-two babies have delivered to date, with four congenital anomalies. There were no respiratory problems. Twenty-nine pregnancies are continuing without known complications, and details are not yet available on the remaining 12. The results indicate that early arnniocentesis may replace the traditional test at 15–17 weeks.     

Biodiversity and industry ecosystem management

The term biodiversity describes the array of interacting, genetically distinct populations and species in a region, the communities they comprise, and the variety of ecosystems of which they are functioning parts. Ecosystem health, a closely related concept, is described in terms of a process identifying biological indicators, end points, and values. The decline of populations or species, an accelerating trend worldwide, can lead to simplification of ecosystem processes, thus threatening the stability and sustainability of ecosystem services directly relevant to human welfare in the chain of economic and ecological relationships. The challenge of addressing issues of such enormous scope and complexity has highlighted the limitations of ecology-as-science. Additionally, biosphere-scale conflicts seem to lie beyond the scope of conventional economics, leading to differences of opinion about the commodity value of biodiversity and of the services that intact ecosystems provide. In the face of these uncertainties, many scientists and economists have adopted principles that clearly assign burdens of proof to those who would promote the loss of biodiversity and that also establish near-trump (preeminent) status for ecological integrity. Electric utility facilities and operations impact biodiversity whenever construction, operation, or maintenance of generation, delivery, and support facilities alters landscapes and habitats and thereby impacts species. Although industry is accustomed to dealing with broad environmental concerns (such as global warming or acid rain), the biodiversity issue invokes hemisphere-wide, regional, local, and site-specific concerns all at the same time. Industry can proactively address these issues of scope and scale in two main ways: first, by aligning strategically with the broad research agenda put forth by informed scientists and institutions; and second, by supporting focused management processes whose results will contribute incrementally to the broader agenda of rebuilding or maintaining biodiversity.     

Reducing car-based journeys to work: What potential is there and whose responsibility is it?

To date the transport solutions seeking to reduce traffic impacts have tended to focus on city/town centres. There has, however, been a substantial increase in traffic levels both in and to suburban areas and the location of large employment sites in such areas has had a significant impact. The typical policy response to traffic in these areas has been the introduction of traffic calming measures and residents' parking but such policies do not address the fundamental issue of travel generation and its consequences (including parking). Major employers should have an important role to play in the consideration of more sensible and sustainable uses of the car including the promotion of alternatives to car use. Using a case study at a large institution in Oxford this paper examines findings from a survey into staff travel behaviour in order to identify how staff currently behave. It explores the potential for transport and non-transport solutions to reduce car-based journeys to work and concludes that there is real potential to encourage a reduction in car-based travel. The paper seeks to widen the debate about who should take responsibility for achieving such a reduction. It argues that whilst travel awareness campaigns primarily push responsibility onto individuals an 'integrated package' is required with employers also assuming responsibility and thereby enabling individuals to adapt.     

NABat: A top-down,bottom-up solution to collaborative continental-scale monitoring

Collaborative monitoring over broad scales and levels of ecological organization can inform conservation efforts necessary to address the contemporary biodiversity crisis. An important challenge to collaborative monitoring is motivating local engagement with enough buy-in from stakeholders while providing adequate top-down direction for scientific rigor, quality control, and coordination. Collaborative monitoring must reconcile this inherent tension between top-down control and bottom-up engagement. Highly mobile and cryptic taxa, such as bats, present a particularly acute challenge. Given their scale of movement, complex life histories, and rapidly expanding threats, understanding population trends of bats requires coordinated broad-scale collaborative monitoring. The North American Bat Monitoring Program (NABat) reconciles top-down, bottom-up tension with a hierarchical master sample survey design, integrated data analysis, dynamic data curation, regional monitoring hubs, and knowledge delivery through web-based infrastructure. NABat supports collaborative monitoring across spatial and organizational scales and the full annual lifecycle of bats.     

Kosetice, Czech Republic--ten years of air pollution monitoring and four years of evaluating the origin of persistent organic pollutants

The regional observatory Kosetice is a central European background station. Unique continuous monitoring from 1988 on is held here. POP (persistent organic pollutant) concentration values of air samples from Kosetice taken between 1996 and 2005 were statistically processed. Values of Czech ambient air quality standards were not exceeded. Concentrations of polycyclic aromatic hydrocarbons reached two maxima, in 1996 and 2001-2002. Polychlorinated biphenyls concentrations reached the highest values in 1997 and 1998 and hexachlorocyclohexanes concentrations in 1998. DDTs, hexachlorobenzene and pentachlorobenzene were analysed as well. Long-range transport of pollutants between 2002 and 2005 was evaluated using the Potential Source Contribution Function hybrid receptor model. Indicated potential source areas of PCBs coincide with many well-known urban and industrialised areas, while the indicated potential source areas of HCHs and DDTs coincide with many agricultural and/or forested regions and the potential source areas of HCB comprise all land use types.     

Modulation of steroidogenesis by coastal waters and sewage effluents of Hong Kong, China, using the H295R assay

BACKGROUND, AIM, AND SCOPE: The presence of a variety of pollutants in the aquatic environment that can potentially interfere with the production of sex steroid hormones in wildlife and humans has been of increasing concern. The aim of the present study was to investigate the effects of extracts from Hong Kong marine waters, and influents and effluents from wastewater treatment plants on steroidogenesis using the H295R cell bioassay. After exposing H295R cells to extracts of water, the expression of four steroidogenic genes and the production of three steroid hormones were measured. MATERIALS AND METHODS: Water samples were collected during the summer of 2005 from 24 coastal marine areas and from the influents and effluents of two major waste water treatment plants (WWTPs) in Hong Kong, China. Samples were extracted by solid phase extraction (SPE). H295R cells were exposed for 48 h to dilutions of these extracts. Modulations of the expression of the steroidogenic genes CYP19, CYP17, 3betaHSD2, and CYP11beta2 were determined by measuring mRNA concentrations by real-time polymerase chain reaction (Q-RT-PCR). Production of the hormones progesterone (P), estradiol (E2), and testosterone (T) was quantified using enzyme linked immunosorbent assays (ELISA). RESULTS: Extracts from samples collected in two fish culture areas inhibited growth and proliferation of H295R cells at concentrations greater or equal to 10(5) L equivalents. The cells were exposed to the equivalent concentration of active substances in 10,000 L of water. Thus, to observe the same level of effect as observed in vitro on aquatic organisms would require a bioaccumulation factor of this same magnitude. None of the other 22 marine samples affected growth of the cells at any dilution tested. Twelve of the marine water samples completely inhibited the expression of CYP19 without affecting E2 production; inhibition of CYP17 expression was observed only in one of the samples while expression of CYP11beta2 was induced as much as five- and ninefold after exposure of cells to extracts from two locations. The expression of the progesterone gene 3betaHSD2 was not affected by any of the samples; only one sample induced approximately fourfold the production of E2. Although more than twofold inductions were observed for P and T production, none of these values were statistically significant to conclude effects on the production of these two hormones. While influents from WWTPs did not affect gene expression, an approximately 30% inhibition in the production of E2 and a 40% increase in P occurred for the exposure with influents from the Sha Tin and Stonecutters WWTPs, respectively. Effluents from WWTPs did not affect the production of any of the studied hormones, but a decrement in the expression of the aldosterone gene CYP11beta2 was observed for the Sha Tin WWTP exposure. No direct correlation could be established between gene expression and hormone production. DISCUSSION: Observed cytotoxicity in the two samples from fish culture areas suggest the presence of toxic compounds; chemical analysis is required for their full identification. Although effluents from WWTPs did not affect hormone production, other types of endocrine activity such as receptor-mediated effects cannot be ruled out. Interactions due to the complexity of the samples and alternative steroidogenic pathways might explain the lack of correlation between gene expression and hormone production results. CONCLUSIONS: Changes observed in gene expression and hormone production suggest the presence in Hong Kong coastal waters of pollutants with endocrine disruption potential and others of significant toxic effects. The aromatase and aldosterone genes seem to be the most affected by the exposures, while E2 and P are the hormones with more significant changes observed. Results also suggest effectiveness in the removing of compounds with endocrine activity by the WWTPs studied, as effluent samples did not significantly affect hormone production. The H295R cell showed to be a valuable toll in the battery required for the analysis of endocrine disrupting activities of complex environmental samples. RECOMMENDATIONS AND PERSPECTIVES: Due to the intrinsic complexity of environmental samples, a combination of analytical tools is required to realistically assess environmental conditions, especially in aquatic systems. In the evaluation of endocrine disrupting activities, the H295R cell bioassay should be used in combination with other genomic, biological, chemical, and hydrological tests to establish viable modes for endocrine disruption and identify compounds responsible for the observed effects.     

Nitrogen and phosphorus status of soils and trophic state of lakes associated with forage-based beef cattle operations in Florida

Forage-based livestock systems have been implicated as major contributors to deteriorating water quality, particularly for phosphorus (P) from commercial fertilizers and manures affecting surface and ground water quality. Little information exists regarding possible magnitudes of nutrient losses from pastures that are managed for both grazing and hay production and how these might impact adjacent bodies of water. We examined the changes that have occurred in soil fertility levels of rhizoma peanut (Arachis glabrata Benth.)-based beef cattle pastures (n = 4) in Florida from 1988 to 2002. These pastures were managed for grazing in spring followed by haying in late summer and were fertilized annually with P (39 kg P2O5 ha(-1)) and K (68 kg K2O ha(-1)). Additionally, we investigated trends in water quality parameters and trophic state index (TSI) of lakes (n = 3) associated with beef cattle operations from 1993 to 2002. Overall, there was no spatial or temporal buildup of soil P and other crop nutrients despite the annual application of fertilizers and daily in-field loading of animal waste. In fact, soil fertility levels showed a declining trend for crop nutrient levels, especially soil P (y = 146.57 - 8.14 x year; r2= 0.75), even though the fields had a history of P fertilization and the cattle were rotated into the legume fields. Our results indicate that when nutrients are not applied in excess, cow-calf systems are slight exporters of P, K, Ca, and Mg through removal of cut hay. Water quality in lakes associated with cattle production was "good" (30-46 TSI) based on the Florida Water Quality Standard. These findings indicate that properly managed livestock operations may not be major contributors to excess loads of nutrients (especially P) in surface water.