New modules for PCR-based gene targeting in Candida albicans: rapid and efficient gene targeting using 100 bp of flanking homology region

The use of PCR-based techniques for directed gene alterations has become a standard tool in Saccharomyces cerevisiae. In our efforts to increase the speed of functional analysis of Candida albicans genes, we constructed a modular system of plasmid vectors and successfully applied PCR-amplified functional analysis (FA)-cassettes in the transformation of C. albicans. These cassettes facilitate: (a) gene disruptions; (b) tagging of 3'-ends of genes with green fluorescent protein (GFP); and (c) replacements of endogenous promoters to achieve regulated expression. The modules consists of a core of three selectable marker genes, CaURA3, CaHIS1 and CaARG4. Modules for C-terminal GFP-tagging were generated by adding GFP-sequences flanked at the 5'-end by a (Gly-Ala)3-linker and at the 3'-end by the S. cerevisiae URA3-terminator to these selection markers. Promoter exchange modules consist of the respective marker genes followed by the regulatable CaMAL2 or CaMET3 promoters at their 3'-ends. In order to ensure a reliably high rate of homologous gene targeting, the flanking homology regions required a size of 100 bp of gene-specific sequences, which were provided with the oligonucleotide primers. The use of shorter flanking homology regions produced unsatisfactory results with C. albicans strain BWP17. With these new modules only a minimal set of primers is required to achieve the functional analysis of C. albicans genes and, therefore, provides a basic tool to increase the number of functionally characterized C. albicans genes of this human pathogen in the near future.     

The effect of temperature and radiation on flavonol aglycones and flavonol glycosides of kale (Brassica oleracea var. sabellica)

The winter crop kale has a complex profile of different glycosylated and acylated flavonol glycosides which may be affected by global warming. To the best of our knowledge, compound–climate relationships for flavonol aglycones and flavonol glycosides were established for the first time. The investigated 10 major flavonol glycosides responded structure-dependent in the investigated temperature range between 0 and 12 °C and the photosynthetic active radiation range between 4 and 20 mol m⁻² d⁻¹, e.g. the decrease in temperature led to an increase in sinapic acid monoacylated and diacylated quercetin glycosides, while the sinapic acid monoacylated kaempferol glycosides showed a maximum at 4.5 °C. Furthermore, the hydroxycinnamic acid residues and the different number of glucose moieties in the 7-O position affected the response of kaempferol triglucosides. Consequently, global warming would result in lower concentrations of antioxidant-relevant quercetin glycosides in winter crops, suggesting a production at e.g. higher altitudes due to lower temperature.     

Non-targeted analysis of tea by hydrophilic interaction liquid chromatography and high resolution mass spectrometry

Tea is the second most consumed beverage in the world and its consumption has been associated with numerous potential health benefits. Factors such as fermentation methods, geographical origin and season can affect the primary and secondary metabolite composition of tea. In this study, a hydrophilic interaction liquid chromatography (HILIC) method coupled to high resolution mass spectrometry in both positive and negative ionisation modes was developed and optimised. The method when combined with principal component analysis to analyse three different types of tea, successfully distinguished samples into different categories, and provided evidence of the metabolites which differed between them. The accurate mass and high resolution attributes of the mass spectrometric data were utilised and relative quantification data were extracted post-data acquisition on 18 amino acids, showing significant differences in amino acid concentrations between tea types and countries. This study highlights the potential of HILIC chromatography combined with non-targeted mass spectrometric methods to provide a comprehensive understanding of polar metabolites in plant extracts.     

Species‐specific fish larvae drift in anthropogenically constructed riparian zones on the Vienna impoundment of the River Danube,Austria: Species occurrence,frequencies, and seasonal patterns based on DNA barcoding

As a result of river regulations over several centuries, followed by restoration measures in recent decades, most of the River Danube shoreline is man‐made, primarily riprap, but some reconstructed gravel banks and riparian side arms. We investigated the effects of these different structures on fish larval dispersal over a 20‐km stretch in Vienna via the use of drift nets. The habitats examined were created 18 years ago when the impoundment of the Danube hydropower station Vienna/Freudenau was constructed. About 15,000 fish larvae were trapped, and a subsample was determined to species level by DNA barcoding. In total, 26 different species were detected, including 10 species that are endangered or in danger of extinction. When species composition was considered, cyprinids become dominant at sites downstream of gravel bars, whereas in riprap sections, the majority of the larvae consist of invasive Gobiidae. Side arm habitats provide spawning and nursery grounds for additional species. Furthermore, clear species‐related seasonal patterns were observed with peak densities and multiple spawning periods of some species being recorded. The largest peak of Percidae occurred in the first half of May, followed by Cyprinidae at the end of May and Gobiidae in mid‐June.     

Ein Planungsansatz zum Umgang mit Störungen bei der Produktion: Die flexible Produktionsfeinplanung mithilfe von Planungsschritten

Disruptions like the breakdown of machines or delays in the delivery of materials cause generated production plans to be useless, at least in some parts. A new planning approach for coping with this uncertainty caused by disruptions is presented. Planning steps that are described by properties, preconditions and consequences are used for material requirements planning and scheduling. If their preconditions are satisfied in a certain state of environment, planning steps will be applicable. The consequences of planning steps change the state of environment. Each customer order establishes a goal of a new planning problem to be achieved by applying planning steps. Plan execution concerns all planning steps applicable in a certain state of environment. In choosing among applicable planning steps and among available resources the flexibility of plan execution is preserved.     

Reliability against fracture of butt welds as inferred from inspection data

Butt welds under static stress may suffer fracture due to a crack growing quickly from some defect in the weld. Nondestructive inspection for defects needs a rational criterion for acceptance of a weld. The paper demonstrates that recent results in structural reliability theory applied to defect fracture systems may constitute a basis for a criterion for highly reliable welds. It takes into account uncertainty from several sources. Essential uncertainty is due to the high degree of idealization of the widely used standard fracture mechanical criterion of crack initiation. Also the indirect measurement of the defect sizes contributes significantly. Finally the statistical uncertainty due to small sample sizes cannot be neglected. Besides the distribution of defect sizes the number of defects in the weld has importance. Only an estimate is needed of the mean number of defects. Statistical uncertainty of this estimate has very low importance for the total reliability of the weld.     

The fake of reliability measures as absolutes

It is claimed that any reliability measure defined in connection with a limit state theory of a high reliability technological system is a purely formal comparative measure of safety. It only makes sense to make comparisons within classes of “similar” technological systems which are all accessible to the same theory. Interpretation of calculated formal failure probabilities as predictions of real failure rates are generally not justified.     

Attomole peptide analysis by high-pressure matrix-assisted laser desorption/ionization Fourier transform mass spectrometry

A new high-pressure matrix-assisted laser desorption/ionization (HP-MALDI) source for FTMS has recently been described (O'Connor et al. J. Am. Soc. Mass Spectrom., in press). Improvements to the source design, including the incorporation of a new high-pressure gas channel plate, resulted in ions devoid of metastable fragmentation and also in increased sensitivity compared to the HP-MALDI prototype source design. The focus of this contribution is the evaluation of the current HP-MALDI FTMS configuration. The use of nonconductive sample surfaces, such as Parafilm and Teflon, was explored, and spectra from 30 amol of peptide applied to these surfaces were routinely obtained. In addition, the current limit of detection for this configuration is demonstrated to be 300 zmol for the phosphopeptide RRREEE(pS)EEEAA using multishot accumulation of the ions from 15 laser shots in the hexapole and 1 scan. In addition, the performance of the new HP-MALDI FTMS configuration and its potential application for high-throughput proteomics analyses are discussed.