Real-time nucleic acid sequence-based amplification in nanoliter volumes

Real-time nucleic acid sequence-based amplification (NASBA) is an isothermal method specifically designed for amplification of RNA. Fluorescent molecular beacon probes enable real-time monitoring of the amplification process. Successful identification, utilizing the real-time NASBA technology, was performed on a microchip with oligonucleotides at a concentration of 1.0 and 0.1 microM, in 10- and 50-nL reaction chambers, respectively. The microchip was developed in a silicon-glass structure. An instrument providing thermal control and an optical detection system was built for amplification readout. Experimental results demonstrate distinct amplification processes. Miniaturized real-time NASBA in microchips makes high-throughput diagnostics of bacteria, viruses, and cancer markers possible, at reduced cost and without contamination.     

KlROM2 encodes an essential GEF homologue in Kluyveromyces lactis

Cellular integrity in yeasts is ensured by a rigid cell wall whose synthesis is controlled by a MAP kinase signal transduction cascade. In Saccharomyces cerevisiae upstream regulatory components of this MAP kinase pathway involve a single protein kinase C, which is regulated in part by interaction with the small GTPase Rho1p. This small G protein is in turn rendered inactive (GDP-bound) or is activated (GTP-bound) by the influence of GTPase activating proteins (GAPs) and the GDP/GTP exchange factors (GEFs), respectively. We report here on the isolation of a gene from Kluyveromyces lactis, KlROM2, which encodes a member of the latter protein family. The nucleotide sequence contains an open reading frame of 1227 amino acids, with an overall identity of 57% to the Rom2 protein of S. cerevisiae. Four conserved sequence motifs could be identified: a RhoGEF domain, a DEP sequence, a CNH domain and a less conserved pleckstrin homology (PH) sequence. Klrom2 null mutants show a lethal phenotype, which indicates that the gene may encode the only functional GEF regulating the cellular integrity pathway in K. lactis. Conditional genomic expression of KlROM2 resulted in sensitivity towards caffeine and Calcofluor white as typical phenotypes of mutants defective in this pathway. Overexpression of KIROM2 from multicopy plasmids under the control of the ScGAL1 promoter severely impaired growth in both S. cerevisiae and in K. lactis. The fact that the lethal phenotype was not prevented in mpk1 deletion mutants indicates that growth inhibition is not simply caused by hyperactivation of the Pkc1p signal transduction pathway.     

On-line coupling of size exclusion chromatography with electrospray ionization-tandem mass spectrometry for the analysis of aquatic fulvic and humic acids

A method was developed for the analysis of humic and fulvic acids by size-exclusion chromatography-electrospray ionization-tandem mass spectrometry using a completely volatile eluent. Humic and fulvic acids were separated into three peaks. These fractions occupied different mass ranges and showed differences in the fine structure of their mass spectra. The low-molecular-weight (LMW) fraction of fulvic acids is most sensitively determined by ESI-MS, and it appears that previous results obtained by infusion-ESI-MS were primarily determined by this fulvic acid fraction. The average molecular weight of this fractions turned out to be lower than that reported from infusion-ESI-MS measurements. Its scan spectra and the product ion spectra of some of its molecular anions perfectly match those previously obtained from whole fulvic acid mixtures. Obviously, a class of well-defined polycarboxylated molecules exist that occurs in all fulvic acid fractions thus far investigated. With decreasing elution time and increasing molecular weight, detection by ESI-MS loses sensitivity as compared to the parallel UV recording, and the fine structure of the scan spectra becomes increasingly uniform for both fulvic and humic acids. The average molecular weight of the HMW fraction exceeds those values calculated from infusion experiments. Scan spectra and product ion spectra of the high-molecular-weight (HMW) fraction of both the humic and the fulvic acids suggest that the HMW fraction consists of several subunits that originate from the LMW fraction.     

Interactive lectures: Effective teaching and learning in lectures using wireless networks

Among the severe instructional shortcomings of lectures is their lack of interactivity. To overcome these problems, the learning environment of the interactive lecture is introduced, in which students and lecturer interact by using mobile computers in a wireless network. In an experimental study, 44 students participated in interactive and in conventional lecture meetings. A quiz service that allowed question to be posted, students’ answers to be evaluated, and the results to be presented graphically, was implemented on mobile computers. The participants rated the interactive condition better, also reporting higher levels of attention, activity and perceived learning success for this condition. Objective measures indicated a slight but insignificant difference toward better learning results in the interactive condition.     

Computer simulation and SERR detection of cytochrome c dynamics at SAM-coated electrodes

In this paper we present a combined experimental and theoretical study of the heterogeneous electron transfer reaction of cytochrome c electrostatically adsorbed on metal electrodes coated with monolayers of 6-mercaptohexanoic acid. Molecular dynamics simulations and pathways calculations show that adsorption of the protein leads to a broad distribution of orientations and, thus, to a correspondingly broad distribution of electron transfer rate constants due to the orientation-dependence of the electronic coupling parameter. The adsorbed protein exhibits significant mobility and, therefore, the measured reaction rate is predicted to be a convolution of protein dynamics and tunnelling probabilities for each orientation. This prediction is confirmed by time-resolved surface enhanced resonance Raman which allows for the direct monitoring of protein (re-)orientation and electron transfer of the immobilised cytochrome c. The results provide a consistent explanation for the non-exponential distance-independence of electron transfer rates usually observed for proteins immobilized on electrodes.     

Microstructure and Compression Strength of Novel TRIP‐Steel/Mg‐PSZ Composites

Novel composites on basis of austenitic stainless TRIP‐steel as matrix with reinforcements of Mg‐PSZ are presented. Compact rods were produced by cold isostatic pressing and sintering, square honeycomb samples by the ceramic extrusion technique. The samples are characterized by optical and scanning electron microscopy before and after deformation, showing the microstructure and the deformation‐ induced martensite formation. The mechanical properties of samples with 5 vol% zirconia are superior compared to zirconia‐free samples and composites with higher zirconia contents in terms of bending and compression tests. The honeycomb samples exhibit extraordinary high specific energy absorption in compression.     

Determination of the overall migration from silicone baking moulds into simulants and food using 1H-NMR techniques

Different silicone baking moulds (37 samples) were characterized with respect to potential migrating substances using ¹H-NMR, RP-HPLC–UV/ELSD and GC techniques. In all cases cyclic organosiloxane oligomers with the formula [Si(CH₃)₂–O] _n were identified (n = 6 … 50). Additionally, linear, partly hydroxyl-terminated organosiloxanes HO–[Si(CH₃)₂–O] _n –H (n = 7 … 20) were found in 13 samples. No substances other than siloxanes could be detected, meaning the migrants mainly consist of organopolysiloxanes. Based on this knowledge, a ¹H-NMR quantification method for siloxanes was established for the analysis of both simulants and foodstuffs. Validation of the ¹H-NMR method gave suitable performance characteristics: limit of detection 8.7 mg kg^–1 oil, coefficient of variation 7.8% (at a level of 1.0 mg kg^–1 food). Migration studies were carried out with simulants (olive oil, isooctane, ethanol (95%), Tenax) as well as preparation of different cakes. From the 1st to 10th experiment, siloxane migration into cakes only slightly decreased, with a significant dependence on fat content. Migration never exceeded a level of 21 mg kg^–1 (3 mg dm^–2) and was, therefore, well below the overall migration limit of 60 mg kg^–1 (10 mg dm^–2). However, migration behaviour into simulants differed completely from these results.     

Ferulic acid dehydrodimers as structural elements in cereal dietary fibre

 Investigations on insoluble dietary fibre (IDF) of wheat, rye, barley, oat, maize, rice and millet led to the identification of several new dehydrodimers of ferulic acid (DFA). These compounds arise from 8–8′, 8–5′, 8–O–4′ and 5–5′ coupling. Esterified phenolics were set free by mild alkali hydrolysis, total amounts of phenolics (ester- plus etherified) were determined by alkali hydrolysis under pressure. Phenolic acids were analysed by gas chromatography – mass spectrometry (GC-MS) as their trimethylsilyl (TMS) derivatives and by high performance liquid chromatography – diode array detection (HPLC-DAD). In esterified form 8–8′aryl DFA and 5–5′ DFA dominate in most cereal IDF with, together, 45–60% of the DFA sum. More than 60% of total bound DFA are involved in ether linkages. Highest amounts of esterified as well as etherified DFA are estimated in millet, followed by maize. DFA contents of wheat, rye and barley are about two- to threefold lower than in millet but about twofold higher than in oat or rice. Received: 27 January 2000     

Short Vapour Residence Time Catalytic Pyrolysis of Spruce Sawdust in a Bubbling Fluidized-Bed Reactor with HZSM-5 Catalysts

Catalytic pyrolysis of spruce sawdust was carried out in a bubbling fluidized-bed reactor using HZSM-5 catalysts. The effects of space velocity, catalyst deactivation, catalyst acidity and catalyst regeneration were studied. The use of catalysts decreased the yield of organic liquids compared to non-catalytic yields while the yields of pyrolytic water and gases increased. Decreasing the space velocity enhanced these effects. The rate of catalyst deactivation depended on the acidity of the catalyst, with more acidic catalysts deactivating more rapidly. Using a catalyst with a Si/Al ratio of 140 resulted in the largest changes in bio-oil properties. Periodic regeneration of the catalyst in the fluidized-bed reactor was also demonstrated using varying regeneration times and temperatures. It was shown that compared to BFB reactors, CFB reactor types would offer better operating characteristics for commercial scale catalytic pyrolysis processes in regard to vapour residence times, and catalyst activity and regeneration.