Interlaboratory evaluation of endotoxin analyses in agricultural dusts--comparison of LAL assay and mass spectrometry

Endotoxin exposure is associated with wheeze and asthma morbidity, while early life exposure may reduce risk of allergy and asthma. Unfortunately, it is difficult to compare endotoxin results from different laboratories and environments. We undertook this study to determine if lipopolysaccharide (LPS) extraction efficiency could account for differences among laboratories. We generated and collected aerosols from chicken and swine barns, and corn processing. We randomly allocated side-by-side filter samples to five laboratories for Limulus assay of endotoxin. Lyophilized aliquots of filter extracts were analyzed for 3-hydroxy fatty acids (3-OHFAs) as a marker of LPS using gas chromatography-mass spectrometry. There were significant differences in endotoxin assay and GC-MS (LPS) results between laboratories for all dust types (p < 0.01). Patterns of differences between labs varied by dust type. Relationships between assay and GC/MS results also depended on dust type. The percentages of individual 3-OHFA chain lengths varied across labs (p < 0.0001) suggesting that each lab recovered a different fraction of the LPS available. The presence of large amounts of particle associated LPS and absence of a freezing thawing cycle were associated with lower correlations between LPS and bioactivity, consistent with an absence of Limulus response to cell-bound endotoxin. These data suggest that extraction methods affect endotoxin measurements. The LAL methods may be most suitable when comparing exposures within similar environments; GC-MS offers additional information helpful in optimizing sample treatment and extraction. GC-MS may be of use when comparing across heterogeneous environments and should be considered for inclusion in future studies of human health outcomes.     

Major ionic compositions of fine particulate matter in an animal feeding operation facility and its vicinity

Animal feeding operations (AFOs) produce particulate matter (PM) and gaseous pollutants. Investigation of the chemical composition of PM_2.5 inside and in the local vicinity of AFOs can help to understand the impact of the AFO emissions on ambient secondary PM formation. This study was conducted on a commercial egg production farm in North Carolina. Samples of PM_2.5 were collected from five stations, with one located in an egg production house and the other four located in the vicinity of the farm along four wind directions. The major ions of NH₄⁺, Na⁺, K⁺, SO₄^2?, Cl^?, and NO₃^? were analyzed using ion chromatography (IC). In the house, the mostly abundant ions were SO₄^2?, Cl^?, and K⁺. At ambient stations, SO₄^2?, and NH₄⁺ were the two most abundant ions. In the house, NH₄⁺, SO₄^2?, and NO₃^? accounted for only 10% of the PM_2.5 mass; at ambient locations, NH₄⁺, SO₄^2?, and NO₃^? accounted for 36–41% of the PM_2.5 mass. In the house, NH₄⁺ had small seasonal variations indicating that gas-phase NH₃ was not the only major force driving its gas–particle partitioning. At the ambient stations, NH₄⁺ had the highest concentrations in summer. In the house, K⁺, Na⁺, and Cl^? were highly correlated with each other. In ambient locations, SO₄^2? and NH₄⁺ had a strong correlation, whereas in the house, SO₄^2? and NH₄⁺ had a very weak correlation. Ambient temperature and solar radiation were positively correlated with NH₄⁺ and SO₄^2?. This study suggests that secondary PM formation inside the animal house was not an important source of PM_2.5. In the vicinity, NH₃ emissions had greater impact on PM_2.5 formation.

ImplicationsThe chemical composition of PM_2.5 inside and in the local vicinity of AFOs showed the impact of the AFO emissions on ambient secondary PM_2.5 formation, and the fate and transport of air pollutants associated with AFOs. The results may help to manage in-house animal facility air quality, and to develop regional air quality control strategies and policies, especially in animal agriculture-concentrated areas.     

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.     

Prenatal diagnosis of congenital nephrosis by in utero kidney biopsy

The diagnosis of congenital nephrosis is difficult during the antepartum period. The combination of an elevated amniotic fluid alpha-fetoprotein, a negative acetylcholinesterase, and a negative ultrasound examination is highly indicative of congenital nephrosis; however, these findings can also be associated with a normal gestation. This is the first report of pathologic confirmation of congenital nephrosis from an in utero fetal kidney biopsy. Copyright © 2001 John Wiley & Sons, Ltd.     

The evolutionary position of turtles revised

Consensus on the evolutionary position of turtles within the amniote phylogeny has eluded evolutionary biologists for more than a century. This phylogenetic problem has remained unsolved partly because turtles have such a unique morphology that only few characters can be used to link them with any other group of amniotes. Among the many alternative hypotheses that have been postulated to explain the origin and phylogenetic relationships of turtles, a general agreement among paleontologists emerged in favoring the placement of turtles as the only living survivors of the anapsid reptiles (those that lack temporal fenestrae in the skull). However, recent morphological and molecular studies have radically changed our view of amniote phylogenetic relationships, and evidence is accumulating that supports the diapsid affinities of turtles. Molecular studies favor archosaurs (crocodiles and birds) as the living sister group of turtles, whereas morphological studies support lepidosaurs (tuatara, lizards, and snakes) as the closest living relatives of turtles. Accepting these hypotheses implies that turtles cannot be viewed any longer as primitive reptiles, and that they might have lost the temporal holes in the skull secondarily rather than never having had them.     

Synthetic attractants for the bark beetle parasitoid Coeloides bostrichorum Giraud (Hymenoptera: Braconidae)

Coeloides bostrichorum Giraud parasitoids (Hymenoptera: Braconidae) attack late larval stages of various bark beetle species breeding in spruce. Volatile compounds collected from Norway spruce (Picea abies) infested by Ips typographus L. (Coleoptera: Scolytidae) were analysed by coupled gas chromatography-mass spectrometry (GC-MS) and GC-electroantennographic detection (GC-EAD). Monoterpene hydrocarbons are the predominant volatile compounds of fresh Norway spruce, while the presence of oxygenated monoterpenes indicates damaged trees. Between one and eight EAD-active oxygenated monoterpenes were used, in amounts reflecting their natural abundance in spruce trees containing bark beetle larvae, to prepare five synthetic baits which were tested in wind tunnel bioassays. Odour samples collected from spruce logs containing the preferred host stage were attractive, while similar samples from uninfested logs failed to elicit any flight activity. However, when a four- or an eight-component synthetic bait was added to volatiles collected from uninfested spruce logs, this combination was as attractive as volatiles collected from infested spruce logs.     

Sex or Food? Appetetive Learning of Sex Odors in a Male Moth

Moths learn to associate a flower odor with a food reward after a few learning trials. Can a hungry, male moth learn to associate a sex attractant with food instead of with sex? We provided a hungry male with odors of single female sex pheromone components, of the full sex pheromone blend or of a flower odor component as cues in an appetitive learning assay. The male learned the single pheromone components just as well as the flower odor. Learning was, however, severely impaired when the full sex pheromone blend was used as conditioning stimulus. The "hard-wiring" between pheromone odor and sex thus seems to be restricted to those circumstances when the male moth experiences the full blend.     

Framing an independent,integrated and evidence-based evaluation of the state of Australia's biophysical and human environments

A new approach was developed for Australia's 2011 national State of the Environment (SoE) report to integrate the assessment of biophysical and human elements of the environment. A Common Assessment and Reporting Framework (CARF) guided design and implementation, responding to jurisdictional complexity, outstanding natural diversity and ecosystem values, high levels of cultural and heritage diversity, and a paucity of national-scale data. The CARF provided a transparent response to the need for an independent, robust and evidence-based national SoE report. We conclude that this framework will be effective for subsequent national SoE assessments and other integrated national-scale assessments in data-poor regions.