High fidelity extrachromosomal recombination and gene targeting in plants

The precision of extrachromosomal homologous recombination and gene targeting in plant cells was investigated. Recombination was directed to introns of selectable marker genes where potential changes could persist without affecting the function and therefore the selectability of the genes. Approximately 9 kb of crossover regions was rescued and sequenced. Changes were detected at a frequency below one point mutation per 1000 bp, indicating that extrachromosomal recombination and gene targeting both appear to occur with high fidelity.     

Proteomic analysis reveals USP7 as a novel regulator of palmitic acid-induced hepatocellular carcinoma cell death

Nutrient surplus and consequent free fatty acid accumulation in the liver cause hepatosteatosis. The exposure of free fatty acids to cultured hepatocyte and hepatocellular carcinoma cell lines induces cellular stress, organelle adaptation, and subsequent cell death. Despite many studies, the mechanism associated with lipotoxicity and subsequent cell death still remains poorly understood. Here, we have used the proteomics approach to circumvent the mechanism for lipotoxicity using hepatocellular carcinoma cells as a model. Our quantitative proteomics data revealed that ectopic lipids accumulation in cells severely affects the ubiquitin-proteasomal system. The palmitic acid (PA) partially lowered the expression of deubiquitinating enzyme USP7 which subsequently destabilizes p53 and promotes mitotic entry of cells. Our global phosphoproteomics analysis also provides strong evidence of an altered cell cycle checkpoint proteins’ expression that abrogates early G2/M checkpoints recovery with damaged DNA and induced mitotic catastrophe leading to hepatocyte death. We observe that palmitic acid prefers apoptosis-inducing factor (AIF) mediated cell death by depolarizing mitochondria and translocating AIF to the nucleus. In summary, the present study provides evidence of PA-induced hepatocellular death mediated by deubiquitinase USP7 downregulation and subsequent mitotic catastrophe.Subject terms: Apoptosis, Protein-protein interaction networks     

Floristic diversity patterns in the White Carpathians biosphere reserve,Czech Republic

The flora of the White Carpathians, a mountain range in the south-east of the Czech Republic, is documented by about 485,000 records of vascular plant occurrences collected since the mid-19^th century. A total of 1299 species recorded in 93 grid cells of 2.8 × 3.1 km were used for an analysis of spatial patterns of floristic diversity in the White Carpathians. Multivariate statistical techniques such as ordination and classification were used to reveal the main gradients in floristic composition and species richness, and measured environmental data and Ellenberg indicator values were used to assess underlying environmental factors. There is a striking floristic contrast between the western and eastern part of the study area, which is associated with differences in climate, mean altitude, topographic heterogeneity measured as altitudinal range, and land use. The western part is characterised by thermophilous, continental and calcicolous species of open habitats. In contrast, the more forested eastern part along the state border with Slovakia and the north-eastern part of the area are characterised by acidophilous species with higher moisture requirements. This pattern is consistent with the established phytogeographical division of the Czech Republic into the phytogeographical regions of Thermophyticum and Mesophyticum. The further division of the area into four regions, based on classified grid data, is also similar to the current division into phytogeographical districts, except for the Javorníky district. There are two distinct hot spots of species richness, in the western and the extreme north-eastern part. A poorer flora was found in landscapes with intensive agriculture. Species richness is associated with different environmental factors than species composition, namely with soil types and land-use categories. Alien species are more common in areas with a higher incidence of arable land and built-up areas, and less common in areas dominated by grasslands and forests.     

Insecticidal potential of natural zeolite and diatomaceous earth formulations against rice weevil (Coleoptera: Curculionidae) and red flour beetle (Coleoptera: Tenebrionidae)

Insecticidal potential of natural zeolites and diatomaceous earths originating from Serbia against Sitophilus oryzae (L.) and Tribolium castaneum (Herbst) was evaluated. Two natural zeolite formulations (NZ and NZ Modified) were applied to wheat at rates of 0.50, 0.75, and 1.0 g/kg, while two diatomaceous earth (DE) formulations (DE S-1 and DE S-2) were applied at rates of 0.25, 0.50, 0.75, and 1.0 g/kg. A bioassay was conducted under laboratory conditions: temperature of 24 +/- 1 degrees C, relative humidity in the range 50-55%, in tests with natural zeolites, and 60-65%, in tests with DEs, and in all combinations for progeny production. Mortality was assessed after 7, 14, and 21 d of insect contact with treated wheat, and the total mortality after an additional 7-d recovery on untreated broken wheat. Progeny production was also assessed after 8 wk for S. oryzae and 12 wk for T. castaneum. The highest mortality for S. oryzae and T. castaneum was found after the longest exposure period and 7 d of recovery, on wheat treated with NZ at the highest rate and DEs at rates of 0.50 -1.0 g/kg. Progeny reduction higher than 90% was achieved after 14 and 21 d of contact of both beetle pests with wheat treated with DE S-1 at 0.50-1.0 g/kg and DE S-2 at 0.75-1.0 g/kg, while the same level of reduction was achieved only for T. castaneum after its contact with the highest rate of NZ formulation. NZ Modified, applied even at the highest rate, revealed much lower insecticidal potential.     

The Fap1 fimbrial adhesin is a glycoprotein: antibodies specific for the glycan moiety block the adhesion of Streptococcus parasanguis in an in vitro tooth model

Streptococcus parasanguis is a primary colonizer of the tooth surface and plays a pivotal role in the formation of dental plaque. The fimbriae of S. parasanguis are important in mediating adhesion to saliva-coated hydroxylapatite (SHA), an in vitro tooth adhesion model. The Fap1 adhesin has been identified as the major fimbrial subunit, and recent studies suggest that Fap1 is a glycoprotein. Monosaccharide analysis of Fap1 purified from the culture supernatant of S. parasanguis indicated the presence of rhamnose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. A glycopeptide moiety was isolated from a pronase digest of Fap1 and purified by immunoaffinity chromatography. The monosaccharide composition of the purified glycopeptide was similar to that of the intact molecule. The functionality of the glycan moiety was determined using monoclonal antibodies (MAbs) specific for the intact Fap1 glycoprotein. These antibodies were grouped into two categories based on their ability to block adhesion of S. parasanguis to SHA and their corresponding specificity for either protein or glycan epitopes of the Fap1 protein. 'Non-blocking' MAb epitopes were mapped to unique protein sequences in the N-terminus of the Fap1 protein using non-glycosylated recombinant Fap1 proteins (rFap1 and drFap1) expressed in Escherichia coli. In contrast, the 'blocking' antibodies did not bind to the recombinant Fap1 proteins, and were effectively competed by the binding to the purified glycopeptide. These data suggest that the 'blocking' antibodies are specific for the glycan moiety and that the adhesion of S. parasanguis is mediated by sugar residues associated with Fap1.     

Spread and population structure of <Emphasis Type=Italic>Cryphonectria parasitica</Emphasis> in a young chestnut orchard in Slovakia

The chestnut blight pathogen Cryphonectria parasitica was studied in a chestnut collection composed of both seedlings and grafts derived from selected Castanea sativa and C. sativa × C. crenata trees located in south-east Slovakia, near village Príbelce on an area of approximately 3.5 ha. The study was conducted during eight years (2003–2010). During this period 133 trees were infected, which represents 59.82% of chestnut trees of all chestnut accessions. Based on the phenotype of the fungus culture and the type of cankers in the field, all isolates were determined to be virulent. No hypovirulent strains were found. No vegetative compatibility (vc) type diversity was observed. More than 130 isolates were analyzed for vc and all were in single vc type, which was identical with EU 12. All isolates assayed for mating type were MAT-1. No perithecia were observed. No significant differences were found between the proportion of cankered and dead cankered trees in seedlings and grafts of hybrid origin (C. sativa × C. crenata) and of C. sativa origin. However, particular seedlings and grafts of hybrid origin seemed to exhibit certain resistance to chestnut blight.