Workshop report on the extraction of foetal DNA from maternal plasma

Objective Cell free foetal DNA (cff DNA) extracted from maternal plasma is now recognized as a potential source for prenatal diagnosis but the methodology is currently not well standardized. To evaluate different manual and automated DNA extraction methods with a view to developing standards, an International Workshop was performed. Methods Three plasma pools from RhD-negative pregnant women, a DNA standard, real-time-PCR protocol, primers and probes for RHD were sent to 12 laboratories and also to one company (Qiagen, Hilden, Germany). In pre-tests, pool 3 showed a low cff DNA concentration, pool 1 showed a higher concentration and pool 2 an intermediate concentration. Results The QIAamp DSP Virus Kit, the High Pure PCR Template Preparation Kit, an in-house protocol using the QIAamp DNA Blood Mini Kit, the CST genomic DNA purification kit, the Magna Pure LC, the MDx, the M48, the EZ1 and an in-house protocol using magnetic beads for manual and automated extraction were the methods that were able to reliably detect foetal RHD. The best results were obtained with the QIAamp DSP Virus Kit. The QIAamp DNA Blood Mini Kit showed very comparable results in laboratories that followed the manufacturer's protocol and started with ≥ 500 µL plasma. One participant using the QIAamp DNA Blood Midi Kit failed to detect reliably RHD in pool 3. Conclusions This workshop initiated a standardization process for extraction of cff DNA in maternal plasma. The highest yield was obtained by the QIAamp DSP Virus Kit, a result that will be evaluated in more detail in future studies. Copyright © 2007 John Wiley & Sons, Ltd.     

A farewell to Dr Thomas Knacker (* 29.04.1951 – ? 30.10.2011): scientific contributions and personal memories

Background

The bioaccumulation potential of chemical substances is commonly determined in flow-through fish tests according to the Organization for Economic Cooperation and Development (OECD) technical guideline (TG) 305, aiming at the determination of bioconcentration factors (BCF). Investigations on aquatic organisms have shown a significant positive correlation between the accumulation of a chemical and the lipid content of organisms. Residue levels can thus differ between individuals, species, and size groups depending on their lipid content. BCF values can be normalized by lipid content; however, this requires the use of appropriate extraction techniques to ensure the complete extraction of total lipids from fish/tissue samples. The OECD TG 305 is currently under revision. A literature review was carried out to select preferred methods which shall be included in the updated OECD TG 305 along with a sampling schedule for lipid measurement in fish.

Results

An overview and appraisal of a broad range of lipid extraction methods is presented. Extraction procedures applied in bioaccumulation studies described in the literature and procedures currently used for such studies by governmental, academic, and industrial labs are surveyed. Possible impacts of unsuitable extraction techniques on lipid normalized BCF results were assessed. Default values for BCF normalization commonly used in bioaccumulation studies are described. The effect of differences in residue distribution over different organs and/or lipids on the normalization of BCF values is discussed.

Conclusions

A broad range of procedures for lipid extraction are available. However, only the use of suitable extraction techniques guarantees the complete extraction of total lipids from fish samples in bioaccumulation studies which is required to ensure the correct lipid normalization of BCF values. Preferred methods for inclusion in the updated OECD 305 test guideline as well as a standard schedule for lipid measurements are suggested.     

Increased grooming after repeated brood care provides sanitary benefits in a clonal ant

Repeated pathogen exposure is a common threat in colonies of social insects, posing selection pressures on colony members to respond with improved disease-defense performance. We here tested whether experience gained by repeated tending of low-level fungus-exposed (Metarhizium robertsii) larvae may alter the performance of sanitary brood care in the clonal ant, Platythyrea punctata. We trained ants individually over nine consecutive trials to either sham-treated or fungus-exposed larvae. We then compared the larval grooming behavior of naive and trained ants and measured how effectively they removed infectious fungal conidiospores from the fungus-exposed larvae. We found that the ants changed the duration of larval grooming in response to both, larval treatment and their level of experience: (1) sham-treated larvae received longer grooming than the fungus-exposed larvae and (2) trained ants performed less self-grooming but longer larval grooming than naive ants, which was true for both, ants trained to fungus-exposed and also to sham-treated larvae. Ants that groomed the fungus-exposed larvae for longer periods removed a higher number of fungal conidiospores from the surface of the fungus-exposed larvae. As experienced ants performed longer larval grooming, they were more effective in fungal removal, thus making them better caretakers under pathogen attack of the colony. By studying this clonal ant, we can thus conclude that even in the absence of genetic variation between colony members, differences in experience levels of brood care may affect performance of sanitary brood care in social insects.     

Exploring barriers to climate change adaptation in the Swiss tourism sector

Increasingly, various sectors are affected by climate change and need to find ways to adapt with much public guidance and support. This paper examines the adaptation process of a sector that started it some time ago – Swiss Alpine tourism. It identifies barriers that may be relevant for all sectors, all along the successive phases of the adaptation process. It additionally identifies the barriers which are most important and how these can be overcome. In order to do this we use an online survey directed to Swiss tourism stakeholders. Our results indicate that both economic and social feasibility barriers are important impediments to the adjustment process, whereas the acceptability among inhabitants and the willingness to act of stakeholders appear less critical. These barriers can be overcome and adaptation facilitated with more and especially better information about the regional consequences of climate change and feasible adaptation measures, by some top-down leadership and coordination, and by providing financial support.     

Imperfect chemical female mimicry in males of the ant Cardiocondyla obscurior

Winged and wingless males coexist in the ant Cardiocondyla obscurior. Wingless (“ergatoid”) males never leave their maternal colony and fight remorselessly among each other for the access to emerging females. The peaceful winged males disperse after about 10 days, but beforehand also mate in the nest. In the first 5 days of their life, winged males perform a chemical female mimicry that protects them against attack and even makes them sexually attractive to ergatoid males. When older, the chemical profile of winged males no longer matches that of virgin females; nevertheless, they are still tolerated, which so far has been puzzling. Contrasting this general pattern, we have identified a single aberrant colony in which all winged males were attacked and killed by the ergatoid males. A comparative analysis of the morphology and chemical profile of these untypical attacked winged males and the tolerated males from several normal colonies revealed that normal old males are still performing some chemical mimicry to the virgin queens, though less perfect than in their young ages. The anomalous attacked winged males, on the other hand, had a very different odour to the females. Our study thus exemplifies that the analysis of rare malfunctioning can add valuable insight on functioning under normal conditions and allows the conclusion that older winged males from normal colonies of the ant C. obscurior are guarded through an imperfect chemical female mimicry, still close enough to protect against attacks by the wingless fighters yet dissimilar enough not to elicit their sexual interest.     

Reduction of pharmaceutically active compounds by a lagoon wetland wastewater treatment system in Southeast Louisiana

A number of pharmaceutically active compounds (PhACs) have been detected in the aquatic environment as a result of discharges of municipal wastewater. In the state of Louisiana, USA, many municipalities treat wastewater using natural systems, such as lagoons and wetlands, rather than conventional wastewater treatment technologies. Nearly all research to date has focused on the fate of PhACs in conventional treatment plants, not constructed and natural wetlands. In the wastewater treatment plant (WWTP) for Mandeville, Louisiana, USA, wastewater flows of 7600 m³ d⁻¹ are treated in a series of aeration lagoons (basins), followed by a constructed wetland and UV disinfection, before being discharged into a natural forested wetland (i.e. Bayou Chinchuba) and eventually, Lake Pontchartrain. Thirteen out of the 15 PhACs investigated were detected in the wastewater inflow to the treatment plant. Only 9 of the 13 compounds were above the detection limits at the treatment plant effluent. The concentrations of most compounds were reduced by greater than 90% within the plant, while carbamazepine and sotalol were only reduced by 51% and 82%, respectively. The percent reductions observed in the Mandeville system were greater than reduction rates reported for conventional WWTPs; perhaps due to the longer treatment time (30 days). Most target PhACs were not completely removed before discharge into Lake Pontchartrain, although their collective annual loading was reduced to less than 1 kg and down to ppb with significant potential for dilution in the large lake.