Trapping DNA–protein binding reactions with neutral osmolytes for the analysis by gel mobility shift and self-cleavage assays

We take advantage of our previous observation that neutral osmolytes can strongly slow down the rate of DNA–protein complex dissociation to develop a method that uses osmotic stress to ‘freeze’ mixtures of DNA–protein complexes and prevent further reaction enabling analysis of the products. We apply this approach to the gel mobility shift assay and use it to modify a self-cleavage assay that uses the nuclease activity of the restriction endonucleases to measure sensitively their specific binding to DNA. At sufficiently high concentrations of neutral osmolytes the cleavage reaction can be triggered at only those DNA fragments with initially bound enzyme. The self-cleavage assay allows measurement of binding equilibrium and kinetics directly in solution avoiding the intrinsic problems of gel mobility shift and filter binding assays while providing the same sensitivity level. Here we compare the self-cleavage and gel mobility shift assays applied to the DNA binding of EcoRI and BamHI restriction endonucleases. Initial results indicate that BamHI dissociation from its specific DNA sequence is strongly linked to water activity with the half-life time of the specific complex increasing ∼20-fold from 0 to 1 osmolal betaine

Geochemical features and formation conditions of the cupriferous sandstones of the Orenburg Pre-Urals

Object. The article describes a new geological and geochemical data for Permian host rocks and copper sandstones (CS) in Orenburg’s Pre-Urals. Methods . X-ray fluorescence, atomic absorption for Au and Ag, and ICP-MS analysis were used during investigations. Results. The cupriferous sandstones are enriched in a quite broad range of microelements: Cu, Ag, Au, Cd, Сr, Ni, Mn, Co, V, U, Sc, and Pb, compared to the average values of the upper crust. The Co/Ni ratio in the CS is very low, which is typical of low-thermal fluids of meteoric origin. The CS are characterized by a REE spectrum without Eu-minima and maxima, which is close to the chondrite spectrum. Like in the host rocks, the “cerium” group of REE dominates in the CS over the “yttrium” and “scandium” groups. The spectra of REE of the CS and the host rocks have definitely similar morphology. The δCe and δEu combination indicates that CS was formed under oxidizing conditions. On geological and genetic characteristics and geochemical indicators installed the similarity of the CS in the southern Pre-Urals deposits and the Manto-type Cu-Ag deposits of Chile and Iran, and also with the CS of Iran and Cu-shales of Kupferschiefer. Conclusion. Proposed geological-genetic model of CS, points to possible high rise development of the Cu-Ag mineralization at depth as in the fields of the Manto-type copper deposits in Chile and Iran. Geochemical data show that accommodating the Permian strata could serve as a source of REE and other trace elements for ore-forming fluids. Perm CS of Pre Urals can be considered as a very promising new (“old”) source of raw materials for the development of the copper industry of the region. The obtained results can be used to predict new ore fields in the Eastern Russia region

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children