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.     

Novel oxidative in vitro metabolites of the mycotoxins alternariol and alternariol methyl ether

The Alternaria toxins alternariol (AOH; 3,7,9-trihydroxy-1-methyl-6H-benzo[c]chromen-6-one) and alternariol methyl ether (AME, 3,7-dihydroxy-9-methoxy-1-methyl-6H-benzo[c]chromen-6-one) are common contaminants of food and feed, but their oxidative metabolism in mammals is as yet unknown. We have therefore incubated AME and AOH with microsomes from rat, human, and porcine liver and analyzed the microsomal metabolites with HPLC and GC-MS/MS. Seven oxidative metabolites of AME and five of AOH were detected. Their chemical structures were derived from their mass spectra using deuterated trimethylsilyl (TMS) derivatives, and from the information obtained from enzymatic methylation. Several of the metabolites were identified by comparison with synthetic reference compounds. AME as well as AOH were monohydroxylated at each of the four possible aromatic carbon atoms and also at the methyl group. In addition, AME was demethylated to AOH and dihydroxylated to a small extent. As the four metabolites arising through aromatic hydroxylation of AME and AOH are either catechols or hydroquinones, the oxidative metabolism of these mycotoxins may be of toxicological significance.     

Monitoring biofilm development in a microfluidic device using modified confocal reflection microscopy

The feasibility of a method to monitor biofilm development non-destructively in a microfluidic device was addressed. Here, we report that biofilm growth could be non-destructively monitored by an image analysis technique based on modification of confocal reflection microscopy.     

Residence time distributions in a modular micro reaction system

Residence time distributions (RTDs) were investigated in a retention reactor (RR) and a coaxial heat exchanger (CHE) of a commercial modular micro reaction system, to improve processing time control and implement this system as a lab scale tool for thermal process development.     

Evaluation of Adh1 alleles and transgenic soybean seeds using Scorpion PCR and HRM analysis

The identification of both approved and non-approved genetically modified organisms (GMOs) is an integral part of GMO biosafety legislation in many countries. One aspect that may affect PCR-based detection of a GMO lies within the analysis of its genetic stability, as sequence alterations or DNA instabilities may impede quantification by PCR. Genetic stability can be analyzed using various methods, yet many of these methods have distinct disadvantages, including low sensitivity. In this study, high resolution melting (HRM) analysis and real-time PCR with Scorpion primers were used as a method to analyze the 3′ end of RR soybeans (RR 40-3-2) in a large number of samples (n = 1,034). No evidence for the occurrence of mutation events was found, implying that the nucleotide sequence of this region is unlikely to be unstable and is well suited as a target for the quantification of RR soybeans. Additionally, and as a preparative work for an optimization of the method, a 174 bp region of the first intron of the Adh1 gene was analyzed in several varieties of maize with different GMO events using the same approach. The results show that 2 alleles are present. In further experiments, the different alleles were cloned into plasmids to generate homozygous plasmids from heterozygous templates in order to generate for a more precise analysis. The overall methodological aim of these studies was to compare HRM analysis with Scorpion primer PCR. Both methods were capable of differentiating between the 2 homozygous and heterozygous alleles. For a better discrimination, however, we conclude that it is most reliable to consider the results of both methods. This dual approach is assumed to be an effective tool as an accurate, high-throughput means of the screening of GMOs for potential genetic instabilities that may interfere with the detection and identification of specific GM events.     

Hydrogen refueling station compression and storage optimization with tube-trailer deliveries

Hydrogen refueling stations require high capital investment, with compression and storage comprising more than half of the installed cost of refueling equipment. Refueling station configurations and operation strategies can reduce capital investment while improving equipment utilization. Argonne National Laboratory developed a refueling model to evaluate the impact of various refueling compression and storage configurations and tube trailer operating strategies on the cost of hydrogen refueling. The modeling results revealed that a number of strategies can be employed to reduce fueling costs. Proper sizing of the high-pressure buffer storage reduces the compression requirement considerably, thus reducing refueling costs. Employing a tube trailer to initially fill the vehicle's tank also reduces the compression and storage requirements, further reducing refueling costs. Reducing the cut-off pressure of the tube trailer for initial vehicle fills can also significantly reduce the refueling costs. Finally, increasing the trailer's return pressure can cut refueling costs, especially for delivery distances less than 100 km, and in early markets, when refueling stations will be grossly underutilized.     

Learners' questions and comprehension in a tutoring setting.

Three experiments, presented within the framework of Activity Theory, deal with the relationship between adult learners' questions and subsequent comprehension in a tutorial learning setting. Students were first given verbal instructions (acquisition) to a novel card game and then asked to play one hand with the teacher (implementation). In Experiment 1, there was no correlation between number of questions asked during acquisition and comprehension, but questions during implementation were negatively correlated with comprehension. In Experiment 2, learners whose questions were answered during acquisition scored higher than those whose questions were not answered. In Experiment 3, learners whose questions were answered during implementation showed greater gains in comprehension than those whose questions were answered during acquisition. Individual differences in question-asking during implementation but not acquisition were significantly related to comprehension. The results confirm the view that questions answered during knowledge implementation more effectively aid comprehension than those answered during acquisition. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Structural optimization of hollow-section steel trusses by differential evolution algorithm

This paper deals with the weight minimization of planar steel trusses by adopting a differential evolution-based algorithm. Square hollow sections are considered. The design optimization refers to size, shape and topology. The design variables are represented by the geometrical dimensions of the cross sections of the different components of the truss, directly involving the size of the structure, and by some geometrical parameters affecting the outer shape of the truss. The topology is included in the optimization search in a particular way, since the designer at different runs of the algorithm can change the number of bays keeping constant the total length of the truss, to successively choose the best optimal solution. The minimum weight optimum design is posed as a single-objective optimization problem subject to constraints formulated in accordance with the current Eurocode 3. The optimal solution is obtained by a Differential Evolutionary (DE) algorithm. In the DE algorithm, a particular combination of mutation and crossover operators is adopted in order to achieve the best solutions and a specific way for dealing with constraints is introduced. The effectiveness of the proposed approach is shown with reference to two case-studies. The analysis results prove the versatility of the optimizer algorithm with regard to the three optimization categories of sizing, shape, topology as well as its high computational performances and its efficacy for practical applications. In particular useful practical indications concerning the geometrical dimensions of the various involved structural elements can be deduced by the optimal solutions: in a truss girder the cross section of the top chord should be bigger than the one of the bottom chord as well as diagonals should be characterized by smaller cross sections with respect to the top and bottom chords in order to simultaneously optimize the weight and ensure an optimal structural behaviour.     

Estimating weak ratiometric signals in imaging data. II. Meta-analysis with multiple, dual-channel datasets

Ratiometric fluorescent indicators are used for making quantitative measurements of a variety of physiological variables. Their utility is often limited by noise. This is the second in a series of papers describing statistical methods for denoising ratiometric data with the aim of obtaining improved quantitative estimates of variables of interest. Here, we outline a statistical optimization method that is designed for the analysis of ratiometric imaging data in which multiple measurements have been taken of systems responding to the same stimulation protocol. This method takes advantage of correlated information across multiple datasets for objectively detecting and estimating ratiometric signals. We demonstrate our method by showing results of its application on multiple, ratiometric calcium imaging experiments.