Disrupting the methionine biosynthetic pathway in Candida guilliermondii: characterization of the MET2 gene as counter‐selectable marker

Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the fungal CTG clade. This yeast remains actively studied as a result of its moderate clinical importance and most of all for its potential uses in biotechnology. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both a methionine auxotroph recipient strain and a functional MET gene as selection marker. We first disrupted the MET2 and MET15 genes encoding homoserine‐O‐acetyltransferase and O‐acetylserine O‐acetylhomoserine sulphydrylase, respectively. The met2 mutant was shown to be a methionine auxotroph in contrast to met15 which was not. Interestingly, met2 and met15 mutants formed brown colonies when cultured on lead‐containing medium, contrary to the wild‐type strain, which develop as white colonies on this medium. The MET2 wild‐type allele was successfully used to transfer a yellow fluorescent protein (YFP) gene‐expressing vector into the met2 recipient strain. In addition, we showed that the loss of the MET2‐containing YFP‐expressing plasmid can be easily observed on lead‐containing medium. The MET2 wild‐type allele, flanked by two short repeated sequences, was then used to disrupt the LYS2 gene (encoding the α‐aminoadipate reductase) in the C. guilliermondii met2 recipient strain. The resulting lys2 mutants displayed, as expected, auxotrophy for lysine. Unfortunately, all our attempts to pop‐out the MET2 marker (following the recombination of the bordering repeat sequences) from a target lys2 locus were unsuccessful using white/brown colony colour screening. Nevertheless, this MET2 transformation/disruption system represents a new versatile genetic tool for C. guilliermondii. Copyright © 2014 John Wiley & Sons, Ltd.     

High levels of dietary arachidonic acid triglyceride exhibit no subchronic toxicity in rats

Arachidonic acid (AA), an n?6 long-chain polyunsaturated fatty acid (LC-PUFA), serves an important role in the body as a structural fatty acid of many tissues including neurological tissues. It is also a precursor of the n?6 class of eicosanoids and is the most abundant n?6 LC-PUFA found in human breast milk. We have optimized the production of a microfungal source of a triglyceride oil (ARASCO^®) which is enriched in AA to about 40% by weight. To establish the safety of this oil as a food, we evaluated the effect of ARASCO^® in Sprague-Dawley rats (20/sex/group) gavaged at dose levels of 1.0 and 2.5 g/kg/d for a period of 90 d, paying special attention to any potential neurotoxicity of the oil. Two groups of control animals received either untreated standard laboratory diet (untreated control) or the same diet and vehicle oil at the same dose volume administered to the treated animals (vehicle control). Physical observations, ophthalmoscopic examinations, body weight, food consumption, clinical chemistry, hematology parameters, neurobehavioral assessments, and macroscopic as well as microscopic postmortem evaluations were performed. Tissue fatty acid analyses indicated that the AA levels in the brain, heart, and liver of the high-dose ARASCO^®-fed animals increased by 8, 59, and 76%, respectively, indicating that the AA in the oil was readily incorporated into tissue lipids. In spite of this high elevation in tissue AA levels, no developmental, histopathological, or neuropathological differences were seen in the animals administered ARASCO^® compared with the vehicle control animals. Being highly enriched in AA, ARASCO^® offers the means to study the effect of this fatty acid in experimental settings and in human metabolic studies.     

Finite element analysis and contact modelling considerations of interference fits for fretting fatigue strength calculations

This paper presents a 3D finite element study of an interference fit assembly subjected to bending. The results accuracy and the solution convergence are governed by mesh size and contact algorithms options. Their influences were investigated to evaluate displacements and stresses near the contact edge where fretting fatigue failure occurs. Four contact algorithms were tested: Penalty function, Augmented Lagrangian, Normal Lagrange and Pure Lagrange. Performance criteria such as precision and time were highlighted and specific convergence control parameters were found. Finally, best practice rules for interference fit FEA are specified.     

Fretting fatigue strength reduction factor for interference fits

This work is based on a comparison between experimental data and finite element analysis for many configurations of interference fits in rotating bending and alternated torsion. Refined mesh and sub modeling techniques were used to obtain converged results. Contact convergence was achieved using augmented Lagrangian algorithm, with penetration control equal to 0.1% on radial interference and allowable elastic slip of 0.1 μm. Fatigue results are presented for 4 different fatigue criteria: Von Mises, Sine, Crossland and Dang Van. Dang Van criterion gives the most consistent results and shows no evidence of a correlation between sliding amplitude and fatigue life. Relative slip seems to be a consequence rather than the cause, excluding cases with important wear. A fretting fatigue strength reduction factor was calculated for the fretting zone. Its value is between 0.35 and 0.69 depending on the chosen criterion, but varies for different materials and/or loading types. Results give a good fatigue life approximation and can easily be used to optimize the shape of the interference fits.     

Calculation of Winding Losses in Shell-Form Transformers for Improved Accuracy and Reliability Part-2: Results and Program Verification

This part of the paper presents calculated results of the program for eddy current losses and circulating currents and losses of several large power transformers. In order to demonstrate the improved accuracy and the new and valuable information provided by the program, the 3D results are compared to 2D results, results of approximate classical formulas of losses, and finally test results on several commercial transformers. The results of a study on the effect of braze splitting and transposition on circulating currents using the 3D loss program are also presented.     

Parasitic diffuse reflection in a Fourier transform spectrometer yielding subharmonic ghosts and line-shape distortion

When using a high-resolution Fourier transform spectrometer (FTS) in a cube-corner configuration, subharmonic ghosts are observed in the spectrum. These ghosts are attributable to parasitic diffuse reflections on the mirrors of the FTS arm. The reflected beams skip a part of the interferometer and travel a different path from the main beam thus experiencing a smaller optomechanical gain. These reflections are present in the reference laser channel as well as on the measurement channel, and each affect the estimated spectrum differently. The sampling grid generated by the reference laser has periodic errors that are synchronized with the fringe signal. The measured spectrum can therefore exhibit sampling jitter ghosts at submultiples of the reference laser wavenumber in addition to its own additive subharmonics. The diffuse reflection experiencing the nominal optomechanical gain, such as in a plane-mirror configuration, will impact directly on the instrument line shape and on the radiometric accuracy of the spectrometer since some radiation is not propagating at the expected angles in the instrument.     

Effects of reference laser intensity noise in a Fourier transform spectrometer

Many noise sources deteriorate the performance of a Fourier-transform spectrometer. When such an instrument utilizes a reference source such as an HeNe laser to sample the measurement data, it is desirable to determine how the intensity fluctuations of the reference source will affect the final spectrum. This text shows how the intensity noise of the reference laser is transformed at the output of the interferometer, how it translates into a sampling jitter by a simple linear relation under a small bandwidth approximation, and how it is manifested in the final spectrum as a noise floor. Analytical and simulation results show that for an instrument designed for optical communication applications, the intensity noise of the reference laser may be one of the dominant noise source limiting the overall performance of the instrument.