Recombineering reagents for improved inducible expression and selection marker re-use in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is an excellent model organism for cell biology. However, its genetic toolbox is less developed than that of Saccharomyces cerevisiae. In the first part of this study we describe an improved inducible expression vector based on tetracycline regulation of the CaMV35S promoter, which is also capable of chromosomal integration and therefore works in minimal and in rich media. We found that anhydrotetracycline is a superior ligand for induction. Maximum expression levels were observed after 12 h in minimal media (EMM) and after 9 h in rich media (YES), which is faster than the nmt1 promoter system. The system was combined with a convenient recombineering-based subcloning strategy for ease of cloning. In the second part we present four template plasmids, pSVEM-bsd, pSVEM-nat, pSVEM-kan and pSVEM-hph, which harbour four recyclable disruption cassettes based on the Cre recombinase lox71/66 strategy for use in PCR targeting methods. Cre-mediated excision leaves a non-functional mutant lox site in the genome, allowing the reiterative usage of these cassettes for multiple targetings. These cassettes are also configured with dual eukaryotic/prokaryotic promoters so that they can be used for recombineering in E. coli. Amongst other purposes, this permits the rapid and convenient creation of targeting constructs with much longer homology arms for difficult and complex targetings in the Sz. pombe genome.     

Disposable indicators for monitoring lighting conditions in museums

Photoinduced alterations of light-sensitive artifacts represent one of the main problems that conservators and curators have to face for environmental control in museums and galleries. Therefore, increasing attention has been recently devoted to developing strategies of indoor light monitoring, especially aimed at minimizing the cumulated light exposure for the objects on exhibit. In this work a prototype of a light dosimeter, constituted by a photosensitive dyes/polymer mixture applied on a paper substrate, is presented. This indicator, specially designed for a preventive assessment of the risk of damage for highly light-sensitive objects, undergoes a progressive color variation as its exposure to the light increases. Different, easily distinguishable color steps are exhibited depending on the light dose received, so that the dosimeter can be used straightforwardly to have a first, instrumentation-free estimation of the total light exposure. A reflectance spectroscopy study in the 350-860 nm range was carried out on prototype dosimeters exposed to light emitted from a tungsten-halogen lamp to investigate the response of the dosimeter to the light and to study the fading mechanism. Two different approaches were evaluated for the calibration of the prototype: colorimetry and principal component analysis of the reflectance spectra. The usefulness of the two methods in providing a quantitative indication of the light dose received was evaluated.     

Structure and degree of polymerisation of fructooligosaccharides present in roots and leaves of Stevia rebaudiana (Bert.) Bertoni

Fructooligosaccharides have been isolated from roots and leaves of Stevia rebaudiana, by hot aqueous extraction, followed by precipitation with ethanol. Their structure has been determined using methylation and NMR analysis, MALDI-TOF, and ESI-MS. Fructooligosaccharides contained almost exclusively (2→1)-linked β-fructofuranosyl, with terminal α-glucopyranosyl and β-fructofuranosyl units. MALDI-TOF and ESI-MS analyses showed the wide range of degree of polymerisation (DP) present in various extracts. From roots and leaves, three different fractions gave profiles of homologous series, with DPs ranging up to 17 with MALDI-TOF and 19 using ESI-MS. These inulin-type fructooligosaccharides were the major component of extracts from S. rebaudiana roots and significant components from the leaves.     

Melatonin in Mediterranean diet,a new perspective

The health‐promoting properties of the Mediterranean diet have been attributed, at least in part, to the chemical diversity of plant foods. Among phytochemicals, polyphenols represent the paradigm of the relationship between healthy foods and reduced risk of chronic‐degenerative diseases, although, in the past few years, a new element has enriched this scenario. Melatonin, and possibly other indoleamines recently discovered in some relevant Mediterranean foods, may represent a new factor contributing to the elucidation of the protective effects of diets rich in plant products. Therefore, in synergy with polyphenols and other bioactive phytochemicals (e.g. carotenoids and glucosinolates), melatonin may contribute to maximizing the benefits of healthy dietary styles. This brief survey deals with the occurrence of melatonin in the Mediterranean diet, with an emphasis on grape products, and focuses on the biological significance of dietary melatonin, an emerging and exciting topic in the field of nutritional sciences. © 2014 Society of Chemical Industry     

Diet supplementation with donkey milk upregulates liver mitochondrial uncoupling,reduces energy efficiency and improves antioxidant and antiinflammatory defences in rats

Dietary PUFA, mainly those of the n‐3 family, are known to play essential roles in the maintenance of energy balance and in the reduction of body fat deposition through the upregulation of mitochondrial uncoupling that is the main source of reactive oxygen species. We hypothesized that rat supplementation with raw donkey's milk (DM), characterized by low‐fat content and higher n3:n6 ratio, may affect energy balance, lipid metabolism, and prooxidant status as compared to animals treated with cow's milk. In the present study, the effects of drinking raw DM (for 4 weeks) on energy balance, lipid metabolism, antiinflammatory, and antioxidant/detoxifying defences was compared to that produced by rat intake of an iso‐energetic amount of raw cow's milk. The hypolipidemic effect produced by DM paralleled with the enhanced mitochondrial activity/proton leakage and with the increased activity or expression of mitochondrial markers namely, carnitine palmitoyl transferase and uncoupling protein 2. The association of decreased energy efficiency with reduced proinflammatory signs (TNF‐α and LPS levels) with the significant increase antioxidant (total thiols) and detoxifying enzyme activities (glutathione‐S‐transferase NADH quinone oxidoreductase) in DM‐treated animals, indicated that beneficial effects were attributable, at least in part, to the activation of nuclear factor 2 erythroid‐related factor 2 pathway.     

Assessing the Potential Content of Ethyl Carbamate in White,Red, and Rosé Wines as a Key Factor for Pursuing Urea Degradation by Purified Acid Urease

The ethyl carbamate (EC) content of a wine after a given temperature‐time storage was theoretically predicted from the potential concentration of ethyl carbamate (PEC), as determined via an accelerated EC formation test. Such information was used to decide whether an enzymatic treatment was needed to reduce the wine urea level before bottling/aging. To this end, 6 white, red, and rosé wines, manufactured in Italy as such or enriched with urea, were tested for their PEC content either before or after enzymatic treatment using a purified acid urease preparation derived from Lactobacillus fermentum. The treatment was severely affected by the total phenolic content (TP) of the wine, the estimated pseudo‐first‐order kinetic rate constant for NH₃ formation reducing by a factor of approximately 2000 as the TP increased from 0 to 1.64 g L^‐1. Such a sensitivity to TP was by far greater than that pertaining to a killed cell‐based enzyme preparation used previously. Urea hydrolysis was successful at reducing EC concentration in wines with low levels of TP and other EC precursors.