Electrochemical study of the XNA on Gold microarray

A novel electrode array was developed based on the XNA on Gold trade mark microarray platform. The platform combines self-assembling monolayers, thick film patterning and streptavidin based immobilization to provide a robust, versatile platform capable of analysing virtually any biomolecule including nucleic acids, proteins, carbohydrates and lipids. Electrochemical analysis of the self-assembling monolayer/streptavidin (SAMS) XNA on Gold coating revealed that the ferrocene redox current for the SAMS modified electrode was greater than that with a bare Gold electrode. The electrochemical reaction of K4Fe(CN)6 was inhibited by the SAMS coating, but was reactivated upon addition of ferrocene. These results indicate that ferrocene is involved as a mediator in the electron transfer of K4Fe(CN)6 to the SAMS modified electrode. Addition of DNA to the SAMS resulted in only a minor change in the electrochemical signal, indicating that XNA on Gold can be used for electrochemical based bioanalysis. After cycling a SAMS electrode 50 times, no signs of deterioration were detected showing that coating has excellent stability. In addition to the biosensing applications, the scheme provides a non-invasive method for accessing the quality of the SAMS coatings which is of industrial interest. These studies show that the XNA on Gold microarray platform can be used for electrochemical studies, thus providing an additional alternative for developing multianalyte biosensors as well as expanding the range of detection methods available for microarray analysis.     

Structural studies of the polysaccharide antigen of Eubacterium saburreum, Strain L 32

The structure of the polysaccharide antigen produced by Eubacterium saburreum, strain L 32, has been investigated. The principal methods used were methylation analysis, graded hydrolysis with acid, and n.m.r. spectroscopy. The polysaccharide, which contains the unusual sugar 3,6-dideoxy-D-arabino-hexose (tyvelose, Tyv), is composed of trisaccharide repeating-units having the following structure:

     

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.     

Isoprostanes, prostaglandins and tocopherols in pre-eclampsia, normal pregnancy and non-pregnancy

This study is designed to evaluate whether oxidative stress and inflammation are involved in severe pre-eclampsia compared to normal pregnancy and non-pregnancy. We have measured plasma and urinary levels of 8-iso-PGF2alpha, a major isoprostane as an indicator of oxidative stress; plasma and urinary 15-keto-dihydro-PGF2alpha, a major metabolite of cyclooxygenase-catalysed PGF2alpha as an indicator of inflammatory response, and plasma -alpha-and -gamma-tocopherol in 18 pre-eclamptic, 19 normal pregnancy and 20 non-pregnant women. Pregnant women had significantly higher levels of 8-iso-PGF2alpha and PGF2alpha metabolite as compared to the non-pregnancy. Levels of 8-iso-PGF2alpha in the pre-eclamptic women did not differ from the normal pregnancy but PGF2alpha metabolite levels were significantly higher in normal pregnancy. On the other hand, gamma-tocopherol levels were significantly lower in pre-eclampsia than normal pregnancy. In contrast, the concentration of alpha-tocopherol was very similar between the groups. alpha-and gamma-tocopherol levels were significantly lower in pregnancy compared to non-pregnancy. Although no direct evidence of oxidative stress and inflammatory response was observed in severe pre-eclampsia, a reduction of gamma-tocopherol suggests the possible precedence of oxidative stress in this condition. Higher levels of isoprostanes and prostaglandin metabolite in late pregnancy suggest the importance of both free radicals and cyclooxygenase-catalysed oxidation products in normal biological processes of pregnancy.     

Phylogenetic Properties of 50 Nuclear Loci in Medicago (Leguminosae) Generated Using Multiplexed Sequence Capture and Next-Generation Sequencing

Next-generation sequencing technology has increased the capacity to generate molecular data for plant biological research, including phylogenetics, and can potentially contribute to resolving complex phylogenetic problems. The evolutionary history of Medicago L. (Leguminosae: Trifoliae) remains unresolved due to incongruence between published phylogenies. Identification of the processes causing this genealogical incongruence is essential for the inference of a correct species phylogeny of the genus and requires that more molecular data, preferably from low-copy nuclear genes, are obtained across different species. Here we report the development of 50 novel LCN markers in Medicago and assess the phylogenetic properties of each marker. We used the genomic resources available for Medicago truncatula Gaertn., hybridisation-based gene enrichment (sequence capture) techniques and Next-Generation Sequencing to generate sequences. This alternative proves to be a cost-effective approach to amplicon sequencing in phylogenetic studies at the genus or tribe level and allows for an increase in number and size of targeted loci. Substitution rate estimates for each of the 50 loci are provided, and an overview of the variation in substitution rates among a large number of low-copy nuclear genes in plants is presented for the first time. Aligned sequences of major species lineages of Medicago and its sister genus are made available and can be used in further probe development for sequence-capture of the same markers.     

Measurement of the diffusion coefficient of ethanol in agarose gel beads: A reproducibility study

Summary The diffusion coefficient of ethanol in a 4 w/w% agarose gel at 25°C was measured, using the methods of unsteady-state diffusion into, and out of, gel beads dispersed in a solution of finite volume. The results (8.0 – 9.5×10^–6 cm²/s) agreed well with available theory. The results of method out were more reproducible than method in, but the standard deviation was in no case lower than 3.3%.     

Analysis of single Alzheimer solid plaque cores by laser capture microscopy and nanoelectrospray/tandem mass spectrometry

Aggregation of the 40-42 residue amyloid beta-peptide (Abeta) into amyloid plaques is a central event in Alzheimer's disease (AD) pathogenesis. Many proteins have by immunohistochemical techniques been shown to codeposit with Abeta in AD plaques. It is possible that some of these could seed Abeta aggregation and therefore be found in the actual core of the plaque. Here, we present a highly sensitive method for unbiased biochemical analysis of plaque cores. A mild purification protocol based on centrifugation and filtration was used to purify intact plaque cores from human AD brain. The purified plaques were dispensed on a glass slide and viewed in a laser capture microscope, and plaque cores were catapulted into a tube cap by a laser beam. After dissolution in formic acid, plaques were digested and analyzed by liquid chromatography coupled online to electrospray/tandem mass spectrometry. One single plaque was found to be sufficient for positive identification of the main amyloid component. Remarkably, Abeta was the only protein identified when 200 plaques were isolated and analyzed with the present method. Thus, it is possible that no proteins copolymerize with Abeta in the plaque cores and that Abeta alone is sufficient for formation of plaque cores. In support of this notion, core-like structures were observed after incubation of synthetic Abeta for 2 weeks. We suggest that the method described here could be used for the general analysis of amyloid aggregates and inclusion bodies found in other neurodegenerative disorders and that plaque cores in AD brain are molecularly homogeneous structures.