YHp as a highly stable,hyper-copy,hyper-expression plasmid constructed using a full 2-μm circle sequence in cir0 strains of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the yeast episomal plasmid (YEp), containing a partial sequence from a natural 2-μm plasmid, has been frequently used to induce high levels of gene expression. In this study, we used Japanese sake yeast natural cir⁰ strain as a host for constructing an entire 2-μm plasmid with an expression construct using the three-fragment gap-repair method without Escherichia coli manipulation. The 2-μm plasmid contains two long inverted repeats, which is problematic for the amplification by polymerase chain reaction. Therefore, we amplified it by dividing into two fragments, each containing a single repeat together with an overlapping sequence for homologous recombination. TDH3 promoter-driven yEmRFP (TDH3p-yEmRFP) and the URA3 were used as a reporter gene and a selection marker, respectively, and inserted at the 3′ end of the RAF1 gene on the 2-μm plasmid. The three fragments were combined and used for the transformation of sake yeast cir⁰ ura3^- strain. The resulting transformant colonies showed a red or purple coloration, which was significantly stronger than that of the cells transformed with YEp-TDH3p-yEmRFP. The 2-μm transformants were cultured in YPD medium and observed by fluorescence microscopy. Almost all cells showed strong fluorescence, suggesting that the plasmid was preserved during nonselective culture conditions. The constructed plasmid maintained a high copy state similar to that of the natural 2-μm plasmid, and the red fluorescent protein expression was 54 fold compared with the chromosomal integrant. This vector is named YHp, the Yeast Hyper expression plasmid.     

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non‐homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR‐amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour‐intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ‐mediated integrative transformation with PCR‐amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.     

Fabrication and characterization of nanoporous copper through chemical dealloying of cold-rolled and annealed Mn–Cu alloy

Journal of Porous Materials - This study investigated the effects of the manufacturing process on the microstructure of nanoporous copper (NPC) produced using cold-rolled Mn–30 at.% Cu and...     

Nickel Sensitivity Is Associated with GH-IGF1 Axis Impairment and Pituitary Abnormalities on MRI in Overweight and Obese Subjects

Nickel (Ni) is a ubiquitous metal, the exposure of which is implied in the development of contact dermatitis (nickel allergic contact dermatitis (Ni-ACD)) and Systemic Ni Allergy Syndrome (SNAS), very common among overweight/obese patients. Preclinical studies have linked Ni exposure to abnormal production/release of Growth Hormone (GH), and we previously found an association between Ni-ACD/SNAS and GH-Insulin-like growth factor 1 (IGF1) axis dysregulation in obese individuals, altogether suggesting a role for this metal as a pituitary disruptor. We herein aimed to directly evaluate the pituitary gland in overweight/obese patients with signs/symptoms suggestive of Ni allergy, exploring the link with GH secretion; 859 subjects with overweight/obesity and suspected of Ni allergy underwent Ni patch tests. Among these, 106 were also suspected of GH deficiency (GHD) and underwent dynamic testing as well as magnetic resonance imaging for routine follow up of benign diseases or following GHD diagnosis. We report that subjects with Ni allergies show a greater GH-IGF1 axis impairment, a higher prevalence of Empty Sella (ES), a reduced pituitary volume and a higher normalized T2 pituitary intensity compared to nonallergic ones. We hypothesize that Ni may be detrimental to the pituitary gland, through increased inflammation, thus contributing to GH-IGF1 axis dysregulation.     

Fabrication of metallic nanopatterns using the vacuum type UV‐NIL equipment

Nanoscale dot patterns of cobalt alloy were formed on a silicon substrate using the ultra‐violet nanoimprint lithography (UV‐NIL) technology in combination with an electrodeposition process. We developed an improved UV‐NIL equipment that can evacuate the chamber during imprinting. Using this equipment, we successfully imprinted 240‐nm dot patterns with 500 nm pitch on a photocurable resin with high dimensional accuracy. Thickness control of the resin and imprinting under vacuum are important issues to obtain fine nanopatterns. Using these resin patterns as a mask layer, 300‐nm cobalt alloy patterns are successfully formed by the electrodeposition process. Copyright © 2007 Institute of Electrical Engineers of Japan© 2007 Institute of Electrical Engineers of Japan. Published by John Wiley & Sons, Inc.     

Influence of fatty acids on the tocopherol stability in vegetable oils during microwave heating

Effects of 0, 0.05, 0.25, 0.50 and 1.0% levels of fatty acids (caproic, caprylic, capric and lauric) or hydrocarbons (decane and dodecane) on tocopherol stability in vegetable oils during microwave heating were determined by measuring tocopherol losses and carbonyl and anisidine values. The fatty acids showed similar prooxidant activities toward tocopherols in purified vegetable, oils when heated in a microwave oven. However, decane or dodecane, which had the same number of carbons as capric or lauric acid but no carboxylic group, did not show prooxidant activity. The shorter the chainlength and the higher the level of fatty acids, the greater was the reduction of tocopherols in the oils. The addition of low-molecular weight fatty acids resulted in greater acceleration in the oxidation of to pay attention to these free fatty acids produced in the oils when heated in a microwave oven.     

High docosahexaenoic acid levels in both neutral and polar lipids of a highly migratory fish: <Emphasis Type="Italic">Thunnus tonggol</Emphasis> (Bleeker)

The lipid and FA compositions of various organs (light muscle, dark muscle, liver, pyloric cecum, and the orbital region) and of the stomach contents of a highly migratory fish species Thunnus tonggol (Bleeker) were analyzed. TAG and phospholipids (PE and PC) were the major lipid classes in the total lipids of T. tonggol. DHA was characteristically the major FA of all the major classes of all its organs except for only one case of liver TAG. The mean DHA contents of the various organs accounted for more than 20% of the total FA (TFA), even though it is a neutral depot lipid. However, DHA in the stomach contents, originating from their prey, fluctuated and was generally low. DHA levels were generally higher in a year (2000) when water temperatures were colder than in one when it was warmer (1998). Furthermore, DHA levels in muscle TAG were consistently high in spite of the fluctuation of those in the visceral TAG, which might be directly influenced by the prey lipids. This phenomenon suggests the physiological selective accumulation of DHA in the muscle, after the migration of the digested FA in the vascular system and absorption of the prey lipids in the intestine. In contrast, the FA composition of other species is generally variable and their DHA contents of TAG are usually less than 20% of TFA.     

Hyphal surface architecture and cell morphology of Trichoderma reesei

The filamentous fungus Trichoderma reesei which is known to secrete high amounts of cellulolytic enzymes was found to produce a massive amount of fibrous material at the outer surface of the cell wall as observed by ultrahigh-resolution low-voltage scanning electron microscopy. Using transmission electron microscopy, the cell wall ornamentation of the hyper-cellulosic mutant PC-3-7 was found to be less massive and much thinner than for QM9414. A significant amount of fibrous material was produced in Avicel-grown cultures that were less abundant in glucose-grown cultures and Avicel was occasionally found entangled within the cell wall-associated fibrous layer.     

Parallelization of genetic operations that takes building-block linkage into account

We propose a performance enhancement using parallelization of genetic operations that takes highly fit schemata (building-block) linkages into account. Previously, we used the problem of solving Sudoku puzzles to demonstrate the possibility of shortening processing times through the use of many-core processors for genetic computations. To increase accuracy, we proposed a genetic operation that takes building-block linkages into account. Here, in an evaluation using very difficult problems, we show that the proposed genetic operations are suited to fine-grained parallelization; processing performance increased by approximately 30?% (four times) with fine-grained parallel processing of the proposed mutation and crossover methods on Intel Core i5 (NVIDIA GTX5800) compared with non-parallel processing on a CPU. Increasing GPU resources will diminish the conflicts with thread usage in coarse-grained parallelization of individuals and will enable faster processing.     

Synthesis of the Right-Side Structure of Type B Physalins

We present a full account of our synthetic studies on the racemic DEFGH-ring moiety of physalins, featuring domino ring transformation of a tricyclic key intermediate. We also report the results of a detailed mechanistic examination of the domino ring transformation, as well as a reoptimization of the 2,3-Wittig rearrangement and methylation steps. Furthermore, we have newly established a method for the preparation of an optically active synthetic intermediate by enzymatic kinetic resolution. Our work provides access to both natural and nonnatural right-side physalin structures.