In-House Validation and Comparison of Two Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) Taxon-Specific Real-Time PCR Methods for GMO Quantification Supported by Droplet Digital PCR

Common wheat is one of the most important staple food crops worldwide. However, unlike other important staple crops such as maize or soybean, genetically modified (GM) wheat is not yet present in the global food market. Nonetheless, in the recent past, the adventitious presence of GM glyphosate-tolerant volunteers was reported in open wheat fields in the USA. The European Union Reference Laboratory for GM Food and Feed (EURL-GMFF) was therefore called to develop a strategy to detect such unauthorised GM wheat in wheat samples by using both taxon-specific and screening tests. Two candidate common wheat taxon-specific real-time PCR methods were suggested, one targeting ssII-D gene coding for starch synthase and the other targeting waxy-D1 gene, coding for granule-bound starch synthase. In the present study, the two above-mentioned real-time PCR taxon-specific methods were in-house verified and compared, proposing droplet digital PCR (ddPCR) as a new tool for supporting the application of the European Network of GMO Laboratories (ENGL) established method performance criteria. Preliminary performance data of waxy-D1 and ssII-D methods in ddPCR format are shown too to give a contribution to the bridging process from the consolidated to the emerging quantitative PCR methodology.     

Comparison between conjugated linoleic acid and essential fatty acids in preventing oxidative stress in bovine mammary epithelial cells

Some in vitro and in vivo studies have demonstrated protective effects of conjugated linoleic acid (CLA) isomers against oxidative stress and lipid peroxidation. However, only a few and conflicting studies have been conducted showing the antioxidant potential of essential fatty acids. The objectives of the study were to compare the effects of CLA to other essential fatty acids on the thiol redox status of bovine mammary epithelia cells (BME-UV1) and their protective role against oxidative damage on the mammary gland by an in vitro study. The BME-UV1 cells were treated with complete medium containing 50 μM of cis-9,trans-11 CLA, trans-10,cis-12 CLA, α-linolenic acid, γ-linolenic acid, and linoleic acid. To assess the cellular antioxidant response, glutathione, NADPH, and γ-glutamyl-cysteine ligase activity were measured 48 h after addition of fatty acids (FA). Intracellular reactive oxygen species and malondialdehyde production were also assessed in cells supplemented with FA. Reactive oxygen species production after 3 h of H₂O₂ exposure was assessed to evaluate and to compare the potential protection of different FA against H₂O₂-induced oxidative stress. All FA treatments induced an intracellular GSH increase, matched by high concentrations of NADPH and an increase of γ-glutamyl-cysteine ligase activity. Cells supplemented with FA showed a reduction in intracellular malondialdehyde levels. In particular, CLA isomers and linoleic acid supplementation showed a better antioxidant cellular response against oxidative damage induced by H₂O₂ compared with other FA.     

Alloying Behaviour of Zinc with Rare Earth Metals: The Er-Pr-Zn System

Zinc based alloys are among the most commonly used light alloys. To improve the knowledge of the ternary systems constituted by zinc with two lanthanides the isothermal section at 400 °C of the Er-Pr-Zn system was studied. Some selected samples at the Er:Pr ratio?=?1:1 were investigated by differential thermal analysis to draw the vertical section of the system. The experimental techniques used were x-ray powder diffraction, scanning electron microscopy, coupled with electron probe microanalysis and differential thermal analysis. No ternary compounds were found in the system. As regard the intermetallic phases, the (Er, Pr)Zn (Er, Pr)Zn₂, (Er, Pr)Zn_3, (Er, Pr)₁₃Zn₅₈ and (Er, Pr)₂Zn₁₇ solid solutions form in the full field of composition, while the ErZn₁₂, Pr₃Zn₁₁ and PrZn₁₁ compounds partially dissolve the third element. The metastable intermetallic binary phases ErZn₅, PrZn₅ and Er₆Zn₂₃ were not found either as ternary solid solutions or as binary compounds in the ternary Er-Pr-Zn system at 400 °C.     

Milk from cows fed a diet with a high forage:concentrate ratio improves inflammatory state,oxidative stress,and mitochondrial function in rats

Excessive energy intake may evoke complex biochemical processes characterized by inflammation, oxidative stress, and impairment of mitochondrial function that represent the main factors underlying noncommunicable diseases. Because cow milk is widely used for human nutrition and in food industry processing, the nutritional quality of milk is of special interest with respect to human health. In our study, we analyzed milk produced by dairy cows fed a diet characterized by a high forage:concentrate ratio (high forage milk, HFM). In view of the low n-6:n-3 ratio and high content of conjugated linoleic acid of HFM, we studied the effects of this milk on lipid metabolism, inflammation, mitochondrial function, and oxidative stress in a rat model. To this end, we supplemented for 4 wk the diet of male Wistar rats with HFM and with an isocaloric amount (82 kJ, 22 mL/d) of milk obtained from cows fed a diet with low forage:concentrate ratio, and analyzed the metabolic parameters of the animals. Our results indicate that HFM may positively affect lipid metabolism, leptin:adiponectin ratio, inflammation, mitochondrial function, and oxidative stress, providing the first evidence of the beneficial effects of HFM on rat metabolism.     

Characterization of major and trace minerals,fatty acid composition,and cholesterol content of Protected Designation of Origin cheeses

Cheese provides essential nutrients for human nutrition and health, such as minerals and fatty acids (FA). Its composition varies according to milk origin (e.g., species and breed), rearing conditions (e.g., feeding and management), and cheese-making technology (e.g., coagulation process, addition of salt, ripening period). In recent years, cheese production has increased worldwide. Italy is one of the main producers and exporters of cheese. This study aimed to describe mineral, FA, and cholesterol content of 133 samples from 18 commercial cheeses from 4 dairy species (buffalo, cow, goat, and sheep) and from 3 classes of moisture content (hard, <35% moisture; semi-hard, 35–45%; and soft, >45%). Mineral concentrations of cheese samples were determined by inductively coupled plasma optical emission spectrometry, and FA and cholesterol contents were determined by gas chromatography. Moisture and species had a significant effect on almost all traits: the highest levels of Na, Ca, and Fe were found in cheeses made from sheep milk; the greatest level of Cu was found in cow milk cheese, the lowest amount of K was found in buffalo milk cheese, and the lowest amount of Zn was found in goat cheeses. In all samples, Cr and Pb were not detected (below the level of detection). In general, total fat, protein, and minerals significantly increased when the moisture decreased. Buffalo and goat cheeses had the highest saturated FA content, and sheep cheeses showed the highest content of unsaturated and polyunsaturated FA, conjugated linoleic acid, and n-3 FA. Goat and sheep cheeses achieved higher proportions of minor FA than did cow and buffalo cheeses. Buffalo cheese exhibited the lowest cholesterol level. Our results confirm that cheese mineral content is mainly affected by the cheese-making process, whereas FA profile mainly reflects the FA composition of the source milk. This study allowed the characterization of mineral and FA composition and cholesterol content and revealed large variability among different commercial cheeses.     

Extraction of pure lycopene from industrial tomato by‐products in water using a new high‐pressure process

BACKGROUND: Lycopene, a precursor of β‐carotene with a well‐known antioxidant activity, contained in many natural products such as tomato (Lycopersicon esculentum Mill.), watermelon, red pepper and papaya, is usually recovered from natural vegetal sources using organic solvents and a purification step. In this paper an innovative process for the extraction of pure lycopene from tomato waste in water that uses the Naviglio^® extractor and water as extracting phase is presented. RESULTS: Lycopene was obtained in the all‐trans form at a very high grade of purity, not less than 98% (w/w), with an average recovery of 14% (w/w). The availability of high‐purity trans‐lycopene allowed measurement of the molar absorption coefficient. An alternative procedure for high‐performance liquid chromatographic analysis using a phenyl‐hexyl silicone phase as inverse phase and a linear gradient in water and acetonitrile is also described. CONCLUSIONS: The use of water as extracting phase considerably reduces the cost of the entire process when compared with the commonly used solvent‐based procedure or with the newer supercritical extraction process of lycopene from tomato waste. Lycopene, not soluble in water, was recovered in a quasi‐crystalline solid form and purified by solid‐phase extraction using a small amount of organic solvent. Copyright © 2008 Society of Chemical Industry This article was published online on September 15, 2008. Errors in Figures 2 ‐ 4 were subsequently identified. The publishers wish to apologise for these errors. This notice is included in the online and print versions to indicate that both have been corrected [September 19, 2008]     

The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Asclepios

The impact of demographic, lifestyle, and seminal factors on the sperm chromatin structure assay (SCSA) parameters was evaluated in a population of 277 healthy Danish men. This cohort was established within the framework of a European Concerted Action on occupational hazards to male reproductive capability in order to examine the possible reproductive effects of exposure to styrene or pesticides. The SCSA measures the susceptibility of sperm DNA to in-situ acid-induced denaturation, by multiparameter flow cytometric analysis after staining with the DNA-specific fluorescent dye acridine orange. The green versus red bivariate cytogram patterns were quite variable among donors, showing a wide heterogeneity of sperm DNA denaturability. Nevertheless, in those cases where we had the possibility to measure two semen samples from the same donor, the cytogram pattern remained stable over time (0.64 < r < 0.78). Analysis of variance demonstrated that the SCSA results can be influenced by the age of the donor (P < 0.0001), smoking habits (P < 0.05), the presence of leukocytes and immature germ forms in the ejaculate (P < 0.0001), and the duration of sexual abstinence (P < 0.0001). Furthermore, the relationship between the SCSA data and sperm concentration, morphology, and vitality was weak (-0.22 < r < -0.46). Therefore, the SCSA provides independent and complementary measurements of semen quality and is thus a useful tool for epidemiological studies, but the effects of some confounders should be accounted for in the survey design and analysis.     

A novel aminopeptidase associated with the 60 kDa chaperonin in the thermophilic archaeon Sulfolobus solfataricus

The chaperonins are high-molecular-weight protein complexes having a characteristic double-ring toroidal shape; they are thought to aid the folding of denatured or newly synthesized polypeptides. These proteins exist as two functionally similar but distantly related families, one including the bacterial and organellar chaperonins and the other (termed the CCT-TRiC family) including the chaperonins of the Archaea and the eukaryotes. The CCT-TRiC chaperonins, particularly their archeal members, are less well known than their bacterial counterparts, and their main cellular function is still doubtful. In this work, we report that the chaperonin of the thermophilic archaeon Sulfolobus solfataricus interacts with several polypeptides other than the two subunits that constitute the 18-mer double-ring structure. We have cloned and sequenced the gene encoding one 90 kDa chaperonin-associated protein and have shown, using biochemical assays, that the product is an enzyme belonging to the family of zinc-dependent aminopeptidases. The Sulfolobus protein shows maximal homology to eukaryotic (yeast and mouse) aminopeptidases. It contains a leucine zipper motif and can be phosphorylated by an unidentified kinase present in the cell extracts. The possible significance of an association between an aminopeptidase and a chaperonin is discussed.     

Intracranial and spinal dissemination of an ACTH secreting pituitary neoplasia. Case report and review of the literature

Smoking is associated with insulin resistance, dyslipidaemia and markers of the insulin resistance syndrome. This study investigated adipose tissue lipolysis in situ by subcutaneous microdialysis twice in 10 healthy, male smokers after smoking four cigarettes over 2 h and after the administration of an equal amount of nicotine given as nasal spray (NNS). Glucose and insulin levels, in situ lipolysis and adipose tissue blood flow were studied in the post-absorptive state and after a 75-g oral glucose tolerance test (OGTT). Post-absorptively, acute smoking and NNS increased neither subcutaneous adipose tissue glycerol production nor plasma free fatty acid (FFA) or glycerol levels. After the OGTT, plasma insulin and lactate levels were significantly higher after smoking, whereas FFA levels were higher after NNS. Normal smoking or the administration of a normal dose of NNS caused only minor metabolic changes. Thus, it does not seem likely that increased lipolysis is an important contributor to the dyslipidaemia seen in smokers.