Evaluation of measurement uncertainties of an analytical method for the determination of aflatoxin M<Subscript>1</Subscript> in milk and milk powder

The mycotoxin aflatoxin M₁ (AfM₁) is a serious food safety hazard for which the European Commission has already established a maximum permissible level of 0.05 μg/kg AfM₁ in milk and products thereof. For control analysis laboratories are increasingly asked to submit full uncertainties of their analytical results.The evaluation of measurement uncertainties of an analytical method for the determination of AfM₁ in milk and milk powder on the basis of ‘in-house’ validation data in compliance with the ‘Guide to the Expression of Uncertainty in Measurement (GUM)’ [1] and the ‘EURACHEM Guide’ [2] is described. A similar approach will be used to assess the performance of methods employed by laboratories participating in the certification of reference materials for AfM₁ in milk powder.     

Gender-Specific Differences in Outcome of Ascending Aortic Aneurysm Surgery

Objectives

Gender specific differences receive increasing attention and are known to affect the outcome of cardiovascular diseases. We investigated possible risk-factors for gender-specific differences in ascending aortic aneurysm surgery.

Methods

548 consecutive patients (male: n = 390, age: 58.3±14.4 years; female: n = 158, age: 65.3±12.9 years) with aneurysms of the ascending aorta eligible for cardiac surgery were retrospectively analyzed.

Results

Women were significantly older when operation was indicated (p<0.001) and presented with significantly more hypertension (p=0.04) and chronic obstructive pulmonary disease (COPD; p = 0.017), whereas men had significantly more previous cardiac operations (p = 0.016). Normalized aortic diameters (diameter / body surface area) were significantly larger in women (3.10±0.6 cm) vs. (2.75±0,5 cm, p≤0.001) in men, without differences in absolute values (5.74±1.04 cm vs. 5.86±1.34 cm). The aortic arch was significantly more involved in aneurysm formation in women (p = 0.04). Follow-up was available in 93% of the patients with a mean follow-up time of 3.9±3.9 (0-17.8) years. 30-day mortality was 3.5% in men (n=12) and 7.9% in women (n=11; p = 0.058). Univariate regression analysis shows gender specific risk factors for 30-day mortality in men to be age: p = 0.028; myocardial infarction: p = 0.0.24 and in women diameter of the ascending aorta: p=0.014; renal insufficiency: p=0.007. Long-term survival was significantly reduced in women (log-rank p = 0.0052).

Conclusions

The outcome after surgery for ascending aortic aneurysm is less favourable in women with significantly reduced long-term survival and a trend to increased 30-day mortality in this cohort. Larger normalized aortic diameters, higher incidence of involvement of the aortic arch and differences in comorbidities may contribute to gender differences. Women undergo surgery at higher age and more progressed state of aortic disease. Therefore, gender-specific guidelines for ascending replacement may be useful to improve outcome in women.     

An invasive crayfish affects egg survival and the potential recovery of an endangered population of Arctic charr

1. Many fish stocks have declined, because of overharvesting, habitat destruction and introduced species. Despite efforts to rehabilitate some of these stocks, not all are responding or are recovering only slowly. 2. In freshwater systems, introduced crayfish are often problematic, and it has been suggested that their egg predation could reduce recruitment in depleted stocks of native fish. 3. Here, we report the results of a field experiment, using experimental cages, on the extent of predation on eggs of great Arctic charr (Salvelinus umbla) in Lake Vättern, Europe’s fifth largest lake. Here, the great Arctic charr has declined dramatically and is listed as critically endangered. 4. We were able to partition the total loss rate of eggs into background mortality, predation by introduced signal crayfish (Pacifastacus leniusculus) and predation by native fish. The mortality rate of charr eggs because of crayfish was estimated at more than five times that because of native fish. Of the total loss of eggs, 80% is believed to be caused by crayfish and 14% by fish, with 6% being natural background mortality. 5. In a worst case scenario, our data infer that only 25% of the original number of eggs would survive, compared with 75% in the absence of crayfish. This could impair recovery of the stock of the endangered great Arctic charr in Lake Vättern. 6. Contrary to earlier claims that crayfish predation on eggs of great Arctic charr is insignificant, our results indicate that crayfish predation may exceed fish predation and suggest that the abundance of signal crayfish on the spawning sites of great Arctic charr should be managed.     

Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy

Objectives

Marfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1) in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR) in order to reduce elastolysis.

Methods

We performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group). Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6) were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1) or β-galactosidase (Ad.β-Gal). As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI), and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM).

Results

IHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43), but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00). Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001). As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001). However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1 (compared to untreated Marfan aorta: Ad.hTIMP-1 p = 0.902; control Ad.β-Gal. p = 0.165). The virus-untreated and not transplanted mgR/mgR aorta revealed a significant increase of albumin diffusion through the endothelial barrier (p = 0.037). TEM analysis of adenovirus-exposed mgR/mgR aortas displayed disruption of the basement membrane and basolateral space.

Conclusions

Murine Marfan aortic grafts developed severe inflammation after adenoviral contact. We demonstrated that fibrillin-1 deficiency is associated with relevant dysfunction of the endothelial barrier that enables adenovirus to induce vessel-harming inflammation. Endothelial dysfunction may play a pivotal role in the development of the vascular phenotype of Marfan syndrome.     

Newly resolved relationships in an early land plant lineage: Bryophyta class Sphagnopsida (peat mosses)

? Premise of the study: The Sphagnopsida, an early-diverging lineage of mosses (phylum Bryophyta), are morphologically and ecologically unique and have profound impacts on global climate. The Sphagnopsida are currently classified in two genera, Sphagnum (peat mosses) with some 350-500 species and Ambuchanania with one species. An analysis of phylogenetic relationships among species and genera in the Sphagnopsida were conducted to resolve major lineages and relationships among species within the Sphagnopsida. ? Methods: Phylogenetic analyses of nucleotide sequences from the nuclear, plastid, and mitochondrial genomes (11 704 nucleotides total) were conducted and analyzed using maximum likelihood and Bayesian inference employing seven different substitution models of varying complexity. ? Key results: Phylogenetic analyses resolved three lineages within the Sphagnopsida: (1) Sphagnum sericeum, (2) S. inretortum plus Ambuchanania leucobryoides, and (3) all remaining species of Sphagnum. Sister group relationships among these three clades could not be resolved, but the phylogenetic results indicate that the highly divergent morphology of A. leucobryoides is derived within the Sphagnopsida rather than plesiomorphic. A new classification is proposed for class Sphagnopsida, with one order (Sphagnales), three families, and four genera. ? Conclusions: The Sphagnopsida are an old lineage within the phylum Bryophyta, but the extant species of Sphagnum represent a relatively recent radiation. It is likely that additional species critical to understanding the evolution of peat mosses await discovery, especially in the southern hemisphere.     

Plants and landscape in the Vestfold Hills,Antarctica

Maps are presented showing the recorded distribution and species density of terrestrial plants in the Vestfold Hills. The distribution, biomass and species diversity of terrestrial lithic algae, mosses and lichens is influenced positively by availability of meltwater from drift snow and by additional nutrient supply (probably N and P) near bird nest sites. The terrestrial plants are affected negatively by exposure (including sand blast) and salinity. These four factors are probably the most important environmental characteristics exercising local control over plant distribution and abundance within the limits set by temperature.Large changes in salinity, degree of exposure, water supply and nutrient supply occur across the Vestfold Hills, with the most favourable conditions generally occurring in the eastern half fairly close to the ice sheet.Plant distribution and abundance are also discussed in relation to the length of time that particular areas have been exposed as a result of ice retreat. With increasing time of exposure, plant diversity and abundance rise but subsequently fall sharply as conditions become more arid, saline, or both. This temporal sequence can be explained by considering changes in the important factors that control plant growth.     

Nuclear DNA cytophotometry in prostate carcinoma

Eighty patients with prostate carcinoma underwent fine-needle aspiration biopsy for cytologic grading and DNA-single-cell fluorescence photometry before and at 6-month intervals after endocrine treatment. The histograms of DNA values showed single peaks and bimodal and scattered distributions which correlated to the different tumor grades before therapy. The DNA values were significantly different from the controls with benign prostatic hypertrophy. After start of therapy, regressive changes of the DNA-histograms were increases of diploid and hypodiploid DNA values and disappearance of secondary peaks. Progressive changes were increased scattering of DNA values and appearance of secondary peaks. Progressive changes in the histograms were closely related to clinical remission and stable disease, but related poorly to clinical progression. The survival correlated with the pretherapeutic DNA-histograms and with the DNA-median, third-quartile, and maximum parameters.     

Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization

Microfilaments were isolated from cultured mammalian cells, utilizing procedures similar to those for isolation of "native" thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK- 21), and Swiss mouse 3T3 cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin, and a high molecular weight actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS-polyacrylamide gels with both muscle and nonmuscle types of tropomyosin. In order to identify more specifically these subunits, we isolated and partially characterized tropomyosin from three cell types. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. However, tropomyosin isolated from rate embryo and 3T3 cells contained subunits that comigrated with both skeletal muscle and nonmuscle types of myosin, whereas the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition.     

Assessment of the Microbiological Potential for the Natural Attenuation of Petroleum Hydrocarbons in a Shallow Aquifer System

Abstract A multidisciplinary field study investigating the fate and transport of petroleum hydrocarbons commonly associated with jet-fuel contamination is currently underway at Columbus Air Force Base (AFB), Mississippi. Sixty sediment cores from 12 boreholes were recovered from the study aquifer. The goal of this initial sampling was to characterize the potential microbial activity using 14C-labeled substrates, as well as the presence, abundance, and distribution of specific hydrocarbon degrading genotypes using DNA:DNA hybridization. Enumeration of total microbial abundance using a 16S rDNA universal oligonucleotide probe was compared to traditional enumeration methods. Total culturable populations determined by spread plate analysis ranged from a low of 10(4) to more than 10(6) organisms per gram sediment. Microbial abundance estimated by DNA hybridization studies with 16S rDNA genes ranged from 10(7) to 10(8) organisms per gram sediment. Molecular analysis of aquifer samples using DNA probes targeting genes encoding the degradative enzymes alkane hydroxylase (alkB), catechol 2,3-dioxygenase (nahH), naphthalene dioxygenase (nahA), toluene dioxygenase (todC1C2), toluene monooxygenase (tomA), and xylene monooxygenase (xylA), as well as two probes measuring methanogenic microorganisms, codh (carbon monoxide dehydrogenase) and mcr (methyl coenzyme reductase), revealed that each target gene sequence was present in nearly all 60 samples. The presence of organisms demonstrating the phenotype to degrade BTEX and naphthalene was further supported using mineralization assays with 14C-labeled benzene, toluene, naphthalene, and phenanthrene. Minimal activity occurred during the first 24 hours. After a period of 5-7 days, greater than 40% of the target compounds were mineralized in aquifer sediments.