Adsorption kinetics and mechanical properties of thiol-modified DNA-oligos on gold investigated by microcantilever sensors

Immobilised DNA-oligo layers are scientifically and technologically appealing for a wide range of sensor applications such as DNA chips. Using microcantilever-based sensors with integrated readout, we demonstrate in situ quantitative studies of surface-stress formation during self-assembly of a 25-mer thiol-modified DNA-oligo layer. The self-assembly induces a surface-stress change, which closely follows Langmuir adsorption model. The adsorption results in compressive surface-stress formation, which might be due to intermolecular repulsive forces in the oligo layer. The rate constant of the adsorption depends on the concentration of the oligo solution. Based on the calculated rate constants a surface free energy of the thiol-modified DNA-oligo adsorption on gold is found to be -32.4 kJ mol(-1). The adsorption experiments also indicate that first a single layer of DNA-oligos is assembled on the gold surface after which a significant unspecific adsorption takes place on top of the first DNA-oligo layer. The cantilever-based sensor principle has a wide range of applications in real-time local monitoring of chemical and biological interactions as well as in the detection of specific DNA sequences, proteins and particles.     

Reversible acetonitrile-induced inactivation/activation of thermolysin

Thermolysin is catalytically inactive in mixtures of 10-15 % acetonitrile in aqueous buffer. Unexpectedly, dilution of the inactive enzyme with acetonitrile leads to complete recovery of the catalytic activity in a similar way to dilution with aqueous buffer. Circular dichroism and fluorescence studies of thermolysin in the same solvent mixtures reveal discontinuous changes in the overall secondary and tertiary protein structure that correlate well with the reversible differences in catalytic activity. The spectra on either side of the minimum activity point are different from each other, a fact indicating that the enzyme may be able to access two active conformations which are thermodynamically stable in different solvent environments.     

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.     

Snow-Covered Area Estimation Using Satellite Radar Wide-Swath Images

Satellite radar-based remote sensing of snow cover during the snow-melt season has been widely studied for different geographical regions, such as mountainous, open, and forested areas. However, a single method has not been found to function well on all regions. The investigations on boreal forest zone have allowed the Helsinki University of Technology (TKK) to develop a snow-covered area (SCA) method that is feasible using spatially limited European Remote Sensing-1/2 Satellite data. This paper investigates the use of wide-swath radar data for boreal forest SCA estimation for the first time. The TKK SCA method is adapted here for HH-polarization Radarsat data. The predominant aspect originated by the use of wide-swath synthetic aperture radar (SAR) data is the large variation in the radar incidence angle. The effect of incidence angle variation on SCA estimation is characterized in this paper. The foundation for operational implementation of the TKK SCA method is also established by an error propagation analysis presented in this paper. The error propagation analysis is compared with accuracy characteristics acquired between SAR and optical SCA evaluation. The performance of forest compensation, which is a key element of the TKK method, was analyzed for the wide-swath radar data. Furthermore, the correlation between the topography and the SCA estimation accuracy was examined in this paper. This paper lays the foundation for operational SCA estimation on boreal forest zone using wide-swath SAR data     

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.     

Fabrication and Characterization of Large Free-Standing Polarizer Grids for Millimeter Waves

Construction of large aperture free-standing metal wire grids is demonstrated for the lower end of the millimeter wave spectral region. For the constructed two grids the co-polarized and cross-polarized components of transmitted power were measured at 45° oblique incidence. The measurements were performed as a function of wire orientation angle and in more detail at selected angles. The results are in good agreement with the theoretical results presented in the literature. In order to save time and costs the construction apparatus was simplified from those reported previously by other authors. It was shown that for this frequency range the grid characteristics are not degraded when such an apparatus is applied. One of the constructed grids will be used in a calibration system of the Helsinki University of Technology (HUT) polarimetric radiometer.     

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.