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.     

How Climate and Vegetation Influence the fire Regime of the Alaskan Boreal Biome: The Holocene Perspective

We synthesize recent results from lake-sediment studies of Holocene fire-climate-vegetation interactions in Alaskan boreal ecosystems. At the millennial time scale, the most robust feature of these records is an increase in fire occurrence with the establishment of boreal forests dominated by Picea mariana: estimated mean fire-return intervals decreased from ≥300 yrs to as low as ∼80 yrs. This fire-vegetation relationship occurred at all sites in interior Alaska with charcoal-based fire reconstructions, regardless of the specific time of P. mariana arrival during the Holocene. The establishment of P. mariana forests was associated with a regional climatic trend toward cooler/wetter conditions. Because such climatic change should not directly enhance fire occurrence, the increase in fire frequency most likely reflects the influence of highly flammable P. mariana forests, which are more conducive to fire ignition and spread than the preceding vegetation types (tundra, and woodlands/forests dominated by Populus or Picea glauca). Increased lightning associated with altered atmospheric circulation may have also played a role in certain areas where fire frequency increased around 4000 calibrated years before present (BP) without an apparent increase in the abundance of P. mariana. When viewed together, the paleo-fire records reveal that fire histories differed among sites in the same modern fire regime and that the fire regime and plant community similar to those of today became established at different times. Thus the spatial array of regional fire regimes was non-static through the Holocene. However, the patterns and causes of the spatial variation remain largely unknown. Advancing our understanding of climate-fire-vegetation interactions in the Alaskan boreal biome will require a network of charcoal records across various ecoregions, quantitative paleoclimate reconstructions, and improved knowledge of how sedimentary charcoal records fire events. In this paper, charcoal refers to macroscopic (≥180 μm) as opposed to microscopic (< 180 μm) particles unless indicated otherwise. Radiocarbon ages were converted to calibrated years before AD 1950 using the atmospheric calibration data set (Stuiver et al. 1998).     

Controlling Air Toxics Emissions From Remediation by Monitoring of Surrogate Parameters

Particulate matter (dust) from remediation and demolition sites may pose a potential risk to the nearby public due to the presence of various contaminants. Unfortunately, there are no good options for real‐time monitoring of metals, polychlorinated biphenyls, etc. in wind‐blown dust. A three‐tiered approach involving a combination of real‐time monitoring and off‐site analysis that has proved successful is described in this article. PM₁₀ is used as a surrogate parameter for metals and other nonvolatile pollutants, and real‐time monitoring is used to guide dust control measures. If dust emissions are controlled, air emissions of metals and other nonvolatile pollutants also are controlled. © 2014 Wiley Periodicals, Inc.     

Human K10 epithelial keratin is the most abundant protein in airborne dust of both occupied and unoccupied school rooms

Previously it was demonstrated that the levels of large particles (>2 micron) and associated bacterial cell envelope markers increase greatly on occupation in schools; it was hypothesized that the source of both was shed human skin. In the current work to test this hypothesis, room air cleaners were used to collect airborne dust (>50-100 mg) from occupied and unoccupied school rooms which was then subjected to proteomic analysis. Proteins were extracted from the dust and separated using two dimensional gel electrophoresis (2D GE). In situ digestion of protein spots with trypsin released peptides, which were subsequently analyzed by matrix assisted laser desorption/deionization, time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry (MALDI-TOF-MS-MS). In Coomassie blue stained gels, a single spot generally dominated the 2D gels; this protein was identified by tandem mass spectrometry as K10 epithelial keratin. The results experimentally confirm previous anecdotal reports that human skin is readily shed into air and suggest that increased levels of microbial markers and large particles observed in occupied rooms are also derived from skin.     

The value of early third-trimester maternal serum alpha-fetoprotein determination

The value of determination of maternal serum of alpha-fetoprotein (MSAFP) concentration in i he second trimester is well established. In addition to open neural tube defects, pregnancies associated with elevated second-trimester MSAFP have been shown to be at increased risk for a variety of problems, including low birth weight, preterm delivery, and pregnancy-induced hypertension (PIH). We evaluated the potential usefulness of MSAFP in the early third trimester. MSAFP concentration was determined in over 200 women at the time of glucose screening. Results were analysed with regard to gestational age at sampling, maternal weight, race, diabetes, and presence of twins. MSAFP was twice as high in twin gestation, but not affected by race or the presence of diabetes. In contrast to levels in early gestation, third-trimester MSAFP does not appear to be predictive of preterm delivery, low birth weight, or PIH.     

Reproducibility of the Pulmonary Function Response of Older Men and Women to a 2-Hour Ozone Exposure.

To test the reproducibility of the pulmonary function response to ozone exposure in older individuals, eight men and eight women, average age 62.8 years, participated in three 2-hour exposures to 0.45 ppm ozone at 23.3 C and 62.5 percent relative humidity. The first and second exposures were separated by an average time of 17.2 days, and 27.3 days separated the second and third exposures. Subjects alternated riding a bicycle ergometer for 20 minutes at an average minute ventilation of 26 liters BTPS (body temperature pressure, saturated), with 20-minute rest periods. Forced vital capacity (FVC), forced expiratory volume in 1 sec (FEV-1), and forced expiratory flow between 25 and 75% of FVC (FEF 25- 75%) were measured pre and post exposure. The reproducibility of individual pre-post changes were assessed by calculation of the best linear fit and correlation coefficients between exposures. For FVC and FEV-1, the slopes were significantly different from 1, and correlation coefficients not significantly different from zero, implying that older individuals may not respond consistently to similar ozone exposures.     

The good soldier: who is s(he)?

This paper examines how perceptions of Organizational Citizenship Behaviors (OCBs) are affected by socially constructed gender roles. We argue that gender roles are important for the perception, categorization, and consequences of OCBs. We suggest that the dimensions of OCBs (altruism, courtesy, sportsmanship, and civic virtue) are related to gender stereotypes. Combining social identity theory with gender role theory suggests that the ‘gender’ of these behaviors, the job, the job incumbent, and the gender identity of the observer interact, potentially broaden the breadth of requisite job behaviors defined as either in‐ or extra‐role. Implications are discussed. Copyright © 2001 John Wiley & Sons, Ltd.     

Herbivores on a dominant understory shrub increase local plant diversity in rain forest communities

Indirect effects of trophic interactions on biodiversity can be large and common, even in complex communities. Previous experiments with dominant understory Piper shrubs in a Costa Rican rain forest revealed that increases in herbivore densities on these shrubs caused widespread seedling mortality as a result of herbivores moving from Piper to seedlings of many different plant genera. We tested components of the Janzen-Connell hypothesis by conducting focused studies on the effects of specialist and generalist Piper herbivores on local seedling diversity. Whereas specialist herbivores are predicted to increase mortality to neighboring seedlings that are closely related to the source plant, true generalists moving from source plants may cause density-dependent mortality of many species, and possibly increase richness if new species replace abundant species that have been thinned by herbivores. Therefore, we hypothesized that seedling richness would be greater in understory control plots created in patches of Piper that had normal densities of generalist herbivores compared to plots from which we removed generalist herbivores manually from all Piper shrubs. After 15 months, generalist-herbivore-removal plots had > 40% fewer seedlings, > 40% fewer species, and 40% greater seedling evenness, on average, than control plots with generalist herbivores intact. Using a complementary approach in unmanipulated plots in four forests, we used path analysis to test for a positive association between seedling diversity and herbivore damage on Piper species. In unmanipulated plots, for both generalist and specialist herbivores, our data were significant fits to the causal model that Piper herbivores decrease evenness and increase plant species richness, corroborating the experimental results. Because herbivores changed how individuals were apportioned among the species and families present (lower evenness), one interpretation of these associations between herbivores on Piper shrubs and local seedling richness is that high seedling mortality in dominant families allowed the colonization or survival of less common species. If interspecific or apparent competition allowed for a relative increase in species richness, then the Janzen-Connell hypothesis may extend its predictions to generalist seedling predators. We speculate that apparent competition may explain some of the deviations from neutral model predictions, especially at small scales.     

Effects of vegetation cover, presence of a native ant species, and human disturbance on colonization by Argentine ants

The spread of non-native invasive species is affected by human activity, vegetation cover, weather, and interaction with native species. We analyzed data from a 17-year study of the distribution of the non-native Argentine ant (Linepithema humile) and the native winter ant (Prenolepis imparis) in a preserve in northern California (U.S.A.). We conducted logistic regressions and used model selection to determine whether the following variables were associated with changes in the distribution of each species: presence of conspecifics at neighboring sites, distance to development (e.g., roads, buildings, and landscaped areas), proportion of vegetation cover taller than 0.75 m, elevation, distance to water, presence of both species at a site, temperature, and rainfall. Argentine ants colonized unoccupied sites from neighboring sites, but the probability of appearance and persistence decreased as distance to development, vegetation cover, and elevation increased. Winter ants appeared and persisted in sites with relatively high vegetation cover (i.e., highly shaded sites). Presence of the 2 species was negatively associated in sites with high vegetation cover (more winter ants) and sites near development (more Argentine ants). Probability of colonization of Argentine ants decreased where winter ants were most persistent. At sites near development within the preserve, abundant Argentine ant populations may be excluding winter ants. The high abundance of Argentine ants at these sites may be due to immigration from suburban areas outside the preserve, which are high-quality habitat for Argentine ants. In the interior of the preserve, distance from development, low-quality habitat, and interaction with winter ants may in combination exclude Argentine ants. Interactions among the variables we examined were associated with low probabilities of Argentine ant colonization in the preserve.