A universal primer for isolation of fragments of a gene encoding phytoene desaturase for use in virus-induced gene silencing (VIGS) studies

We have been using Virus-Induced Gene Silencing (VIGS) to test the function of genes that are candidates for involvement in floral senescence. Although VIGS is a powerful tool for assaying the effects of gene silencing in plants, relatively few taxa have been studied using this approach, and most that have are in the Solanaceae. We typically use silencing of phytoene desaturase (PDS) in preliminary tests of the feasibility of using VIGS. Silencing this gene, whose product is involved in carotene biosynthesis, results in a characteristic photobleaching phenotype in the leaves. We have found that efficient silencing requires the use of fragments that are more than 90% homologous to the target gene. To simplify testing the effectiveness of VIGS in a range of species, we designed a set of universal primers to a region of the PDS gene that is highly conserved among species, and that therefore allows an investigator to isolate a fragment of the homologous PDS gene from the species of interest. We report the sequences of these primers and the results of VIGS experiments in horticultural species from the Asteraceae, Leguminosae, Balsaminaceae and Solanaceae.

Hydrogen-bond assisted stabilization of the less favored conformation of a tridentate Schiff base ligand in dinuclear nickel(II) complex: an experimental and theoretical study

The Schiff base ligand, HL (2-[1-(3-methylamino-propylimino)-ethyl]-phenol), the 1:1 condensation product of 2-hydroxy acetophenone and N-methyl-1,3-diaminopropane, has been synthesized and characterized by X-ray crystallography as the perchlorate salt [H2L]ClO4 (1). The structure consists of discrete [H2L](+) cations and perchlorate anions. Two dinuclear Ni-II complexes, [Ni2L2(NO2)(2)] (2), [Ni2L2(NO3)(2)] (3) have been synthesized using this ligand and characterized by single crystal X-ray analyses. Complexes 2 and 3 are centrosymmetric dimers in which the Ni-II ions are in distorted fac- and mer-octahedral environments, respectively, bridged by two mu(2)-phenolate ions of deprotonated ligand, L. The plane of the phenyl rings and the Ni2O2 basal plane are nearly coplanar in 2 but almost perpendicular in 3. We have studied and explained this different behavior using high level DFT calculations (RI-BP86/def2-TZVP level of theory). The conformation observed in 3, which is energetically less favorable, is stabilized via intermolecular non-covalent interactions. Under the excitation of ultraviolet light, characteristic fluorescence of compound 1 was observed; by comparison fluorescence intensity decreases in case of compound 3 and completely quenched in compound 2.

Isolation of Actinomyces hyovaginalis from sheep and comparison with isolates obtained from pigs

Actinomyces hyovaginalis, an organism initially described from pigs, was recovered from nine sheep and a moufflon. Further strains of A. hyovaginalis were recovered from five samples from pigs over the same period. 16S rRNA sequencing and extensive phenotyping demonstrated high similarity between the ovine and porcine isolates; however differences with respect to erythritol, adonitol and l-arabitol fermentation were detected. Ovine isolates were made from various sample sites including abscesses and highlight the importance of the accurate identification of the various coryneform isolates which affect sheep. A. hyovaginalis can be added to the growing list of coryneforms which can cause disease in sheep including Corynebacterium pseudotuberculosis, Trueperella pyogenes and Arcanobacterium pluranimalium.

Isolation of recombinant antibodies against EspA and intimin of Escherichia coli O157:H7

Intimin, Tir, and EspA proteins are expressed by attaching-effacing Escherichia coli, which include enteropathogenic and enterohemorrhagic E. coli pathotypes. EspA proteins are part of the type three secretion system needle complex that delivers Tir to the host epithelial cell, while surface arrayed intimin docks the bacterium to the translocated Tir. This intimate attachment leads to attaching and effacing lesions. Recombinant forms of these effector proteins from enterohemorrhagic E. coli O157:H7 were produced by using E. coli expression vectors. Binding of intimin and Tir fragments in enzyme-linked immunosorbent assay (ELISAs) demonstrated the interaction of intimin fragments containing the C-terminal 282 or 188 amino acids to a Tir fragment containing amino acid residues 258 to 361. Recombinant intimin and EspA proteins were used to elicit immune responses in rabbits and immune phage-display antibody libraries were produced. Screening of these immune libraries by conventional phage-antibody panning and colony filter screening produced a panel of antibodies with specificity for EspA or intimin. Antibodies recognizing different C-terminal epitopes on intimin bound specifically to the gamma intimin of O157:H7 and not to other classes of intimin. Antibodies recognizing EspA from E. coli O157 also recognized the protein from the eae-deficient O157 mutant DM3 and from E. coli O111. Anti-intimin antibodies were also produced as fusion proteins coupled to the reporter molecule alkaline phosphatase, allowing the one-step detection of gamma intimin. The isolated recombinant monoclonal antibodies were functional in a range of assay formats, including ELISA, Western blotting, and dot blots, thus demonstrating their diagnostic potential.

Hetero-metallic trinuclear nickel(II)–cadmium(II) complexes of a salicylaldimine ligand with thiocyanate, cyanate and azide ions: isolation of a pair of polymorphs with thiocyanate ion

Four new trinuclear hetero-metallic nickel(II)-cadmium(II) complexes [(NiL)(2)Cd(NCS)(2)] (1A and 1B), [(NiL)(2)Cd(NCO)(2)] (2) and [(NiL)(2)Cd(N-3)(2)] (3) have been synthesized using [NiL] as a so-called "ligand complex" (where H2L = N,N'-bis(salicylidene)-1,3-propanediamine) and structurally characterized. Crystal structure analyses reveal that all four complexes contain a trinuclear moiety in which two square planar [NiL] units are bonded to a central cadmium(II) ion through double phenoxido bridges. The Cd(II) is in a six-coordinate distorted octahedral environment being bonded additionally to two mutually cis nitrogen atoms of terminal thiocyanate (in 1A and 1B), cyanate (in 2) and azide (in 3). Complexes 1A and 1B have the same molecular formula but crystallize in very different monoclinic unit cells and can be considered as polymorphs. On the other hand, the two isoelectronic complexes 2 and 3 are indeed isomorphous and crystallize only in one form. Their conformation is similar to that observed in 1A.

Facilities management carbon footprints: an audit of critical elements of management and reporting

Concern for the environmental impact of organizations’ activities has led to the recognition and demand for organizations to manage and report on their carbon footprint. However, there is no limit as to the areas of carbon footprints required in such annual environmental reports. To deliver improvements in the quality of carbon footprint management and reporting, there is a need to identify the main elements of carbon footprint strategy that can be endorsed, supported and encouraged by facility managers. The study investigates carbon footprint elements managed and reported upon by facility manager in the UK. Drawing on a questionnaire survey of 256 facility managers in the UK, the key elements of carbon footprints identified in carbon footprint reports are examined. The findings indicate that the main elements are building energy consumption, waste disposal and water consumption. Business travel in terms of using public transport, air travel and company cars are also recognized as important targets and objectives for the carbon footprint strategy of several FM (facilities management) organizations.

Patterns of discourse semantics: A corpus-assisted study of financial crisis in British newspaper discourse in 2009

Corpus-assisted analyses of public discourse often focus on the lexical level. This article argues in favour of corpus-assisted analyses of discourse, but also in favour of conceptualising salient lexical items in public discourse in a more determined way. It draws partly on non-Anglophone academic traditions in order to promote a conceptualisation of discourse keywords, thereby highlighting how their meaning is determined by their use in discourse contexts. It also argues in favour of emphasising the cognitive and epistemic dimensions of discourse-determined semantic structures. These points will be exemplified by means of a corpus-assisted, as well as a frame-based analysis of the discourse keyword financial crisis in British newspaper articles from 2009. Collocations of financial crisis are assigned to a generic matrix frame for ‘event’ which contains slots that specify possible statements about events. By looking at which slots are more, respectively less filled with collocates of financial crisis, we will trace semantic presence as well as absence, and thereby highlight the pragmatic dimensions of lexical semantics in public discourse. The article also advocates the suitability of discourse keyword analyses for systematic contrastive analyses of public/political discourse and for lexicographical projects that could serve to extend the insights drawn from corpus-guided approaches to discourse analysis.

Cross-country effects of regulatory capital arbitrage

One reason for the recent asset price bubbles in many developed countries could be regulatory capital arbitrage. Regulatory and legal changes can help traditional banks to move their assets off their balance sheets into the lightly regulated shadows and thus enable regulatory arbitrage through the securitized sector. This paper adopts a global vector autoregression (GVAR) methodology to assess the effects of regulatory capital arbitrage on equity prices, house prices and economic activity across 11 OECD countries/ regions. A counterfactual experiment disentangles the effects of regulatory arbitrage following a change in the net capital rule for investment banks in April 2004 and the adoption of the Basel II Accord in June 2004. The results provide evidence for the existence of an international finance multiplier, with about half of the countries overshooting U.S. impulse responses. The counterfactual shows that regulatory arbitrage via the U.S. securitized sector may enhance the cross-country reallocation of capital from housing markets towards equity markets.

Distribution and availability of trace elements in municipal solid waste composts

Trace element contamination is one of the main problems linked to the quality of compost, especially when it is produced from urban wastes, which can lead to high levels of some potentially toxic elements such as Cu, Pb or Zn. In this work, the distribution and bioavailability of five elements (Cu, Zn, Pb, Cr and Ni) were studied in five Spanish composts obtained from different feedstocks (municipal solid waste, garden trimmings, sewage sludge and mixed manure). The five composts showed high total concentrations of these elements, which in some cases limited their commercialization due to legal imperatives. First, a physical fractionation of the composts was performed, and the five elements were determined in each size fraction. Their availability was assessed by several methods of extraction (water, CaCl2–DTPA, the PBET extract, the TCLP extract, and sodium pyrophosphate), and their chemical distribution was assessed using the BCR sequential extraction procedure. The results showed that the finer fractions were enriched with the elements studied, and that Cu, Pb and Zn were the most potentially problematic ones, due to both their high total concentrations and availability. The partition into the BCR fractions was different for each element, but the differences between composts were scarce. Pb was evenly distributed among the four fractions defined in the BCR (soluble, oxidizable, reducible and residual); Cu was mainly found in the oxidizable fraction, linked to organic matter, and Zn was mainly associated to the reducible fraction (iron oxides), while Ni and Cr were mainly present almost exclusively in the residual fraction. It was not possible to establish a univocal relation between trace elements availability and their BCR fractionation. Given the differences existing for the availability and distribution of these elements, which not always were related to their total concentrations, we think that legal limits should consider availability, in order to achieve a more realistic assessment of the risks linked to compost use.