Characterisation of benzimidazole resistance of Cercospora beticola in Serbia using PCR-based detection of resistance-associated mutations of the β-tubulin gene

A survey to detect and characterise benzimidazole resistance within populations of Cercospora beticola in Serbia was performed. From 52 field isolates collected from sugar beet and beet root, only eight were found to be benzimidazole-sensitive based on the inhibition of mycelial growth by discriminatory concentrations of carbendazim and thiophanate-methyl. Sensitivity tests revealed the presence of three resistant phenotypes among the tested isolates: high-resistance (HR), low-resistance (LR) and moderate-resistance (MR). The benzimidazole resistant isolates were characterised based on the DNA sequence of the β-tubulin gene and temperature sensitivity. The HR isolates showed no temperature sensitivity regardless of carbendazim concentration, whereas the LR and MR isolates were sensitive at lower temperatures. Analysis of the β-tubulin gene sequence revealed two amino acid replacements in the benzimidazole-resistant isolates of C. beticola. One was a glutamic acid to alanine change at position 198 (codon GAG to GCG) that was identified in HR isolates; this mutation has previously been reported to be associated with the development of benzimidazole resistance in C. beticola. The second replacement was a novel point mutation of phenylalanine (TTC) to tyrosine (TAC) at position 167, identified in low and moderate benzimidazole-resistant isolates, sharing a single LR/MR β-tubulin genotype. A diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole sensitive and LR/MR genotypes but absent in the HR genotype was developed for the routine detection of high resistance. A mutation-specific PCR assay was developed for the diagnosis of LR/MR genotype based on a mutation from T to A at codon 167, which is unique to this genotype.     

Insect pest management agents: hormonogen esters (juvenogens)

The chemical part of this investigation focused on designing structures and synthesizing a series of six new esters (juvenogens), derived from biologically active insect juvenile hormone bioanalogues (juvenoids, JHAs) and unsaturated short-chain linear and branched fatty acids for possible application as biochemically targeted insect hormonogen agents. The structures of the new compounds were assigned on the basis of a detailed NMR analysis of their (1)H and (13)C NMR spectra. The biological part of this investigation focused on introductory biological screening tests with these compounds against the red firebug (Pyrrhocoris apterus), termites (Reticulitermes santonensis and Prorhinotermes simplex), and the blowfly (Neobellieria bullata). The biological activity of the juvenogens was studied in relation to the fatty acid functionality in the structures. Notable biological activity in topical tests and medium activity in peroral tests was found for the juvenogens 3 and 7 with P. apterus. The compounds 6 and 8 showed the lowest activity in both topical and oral assays with P. apterus. Considerable effect of all tested juvenogens was observed in P. simplex; however, the juvenogens 5 and 6 (derivatives of the only branched short-chain fatty acid) showed no activity against R. santonensis. The effect of the compounds 3-8 on larval hatching of N. bullata was only moderate (larval hatching 80-90%); however, the proliferation effect caused by 5, 6, and 8 is more pronounced than the effect caused by 3, 4, and 7.     

Development of a simplified method for the determination of folates in baker's yeast by HPLC with ultraviolet and fluorescence detection

A simplified HPLC method for rapid determination of folates in yeast with ultraviolet and fluorescence detection without sample purification has been developed. By use of the column Aquasil C(18), specially designed for polar analytes, and gradient elution, it was possible to separate and determine five folate derivatives: tetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate with fluorescence detection, and 10-formylfolic acid and folic acid with ultraviolet detection. The sample preparation required only a small amount of dry yeast (25-50 mg) and included an extraction of folates by heat treatment and deconjugation of folate polyglutamates to monoglutamates with the use of rat serum conjugase. Validation involved investigation of matrix effects, determination of recovery by standard addition method, repeatability, and stability tests. The dominating folate forms in commercial dry baker's yeast were found to be tetrahydrafolate and 5-methyltetrahydrofolate with a total folate content of 2890 microg/100 g (63.4 nmol/g). The simplicity of the method makes it suitable for folate screening studies of different yeast strains.     

Isolation and computer analysis of the 5'-regulatory region of the seed storage protein gene from buckwheat (Fagopyrum esculentum Moench)

Using the modified rapid amplification of cDNA ends (5'-RACE) approach, a fragment containing the 955 bp long 5'-regulatory region of the buckwheat storage globulin gene (FeLEG1) has been amplified from the genomic DNA of buckwheat. The entire fragment was sequenced, and the sequence was analyzed by computer prediction of cis-regulatory elements possibly involved in tissue-specific and developmentally controlled seed storage protein gene expression. The promoter obtained might be interesting not only for fundamental research but also as a useful tool for biotechnological application.     

Characterisation, Availability, and Risk Assessment of the Metals in Sediment after Aging

The behavior of metals in sediments after their disposal to land has important implications for the environmental management. The sediment from the Carska Bara (Serbia) was polluted with adequate metal salts in order to reach severe contamination based on the pseudo-total metal content of Pb, Cd, Ni, Zn, Cu, and Cr according to the corresponding Dutch standards and Canadian guidelines. The toxicity and fate of the metal in sediment depend on its chemical form, and therefore, quantification of the different forms of a metal is more meaningful than the estimation of its total concentration. In this study, fractionation of metals in sediment has been investigated to determine its speciation and ecotoxic potential, as well as evaluation of metal potential toxicity based on the simultaneously extracted metals (SEMs) and acid volatile sulfides (AVSs) analysis at the beginning of the experiment and after 5?weeks of sediment aging. The investigations suggest that Cd, Pb, and Zn have a tendency to associate with labile fraction, the most mobile and most dangerous fraction for the environment. Risk assessment code revealed their high risk. Copper and chromium showed low to medium risk to the aquatic environment. Nickel showed no risk to the aquatic environment. This was the case at the beginning and after 5?weeks of aging. Aging yielded an increased mobility of all metals based on the increased proportion in mobile fractions. The ??[SEM _i ]/[AVS] ratio was found to be >1 both at the beginning and after 5?weeks of aging, with the ratio showing an increase with time. This ratio indicates the potential availability/toxicity and, according to the US EPA criteria, the samples belong to the group with probable negative effect. If particular metals are considered, only the ??[SEM _i ]/[AVS] ratio for zinc was >1 at the beginning. After 5?weeks, the ratio was >1 for zinc, lead, and copper. Comparison of the results of sequential extraction and the results of SEM and AVS analysis showed good agreement for lead and zinc.     

Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth

Selective autophagy can be mediated via receptor molecules that link specific cargoes to the autophagosomal membranes decorated by ubiquitin-like microtubule-associated protein light chain 3 (LC3) modifiers. Although several autophagy receptors have been identified, little is known about mechanisms controlling their functions in vivo. In this work, we found that phosphorylation of an autophagy receptor, optineurin, promoted selective autophagy of ubiquitin-coated cytosolic Salmonella enterica. The protein kinase TANK binding kinase 1 (TBK1) phosphorylated optineurin on serine-177, enhancing LC3 binding affinity and autophagic clearance of cytosolic Salmonella. Conversely, ubiquitin- or LC3-binding optineurin mutants and silencing of optineurin or TBK1 impaired Salmonella autophagy, resulting in increased intracellular bacterial proliferation. We propose that phosphorylation of autophagy receptors might be a general mechanism for regulation of cargo-selective autophagy.     

Synthesis and structure of sila-adamantane

An organosilicon cage compound containing the sila-adamantane structural unit, which is the building block of crystalline silicon, was synthesized by the Lewis acid-catalyzed rearrangement reaction of a structural isomer.     

Controlled phase shifts with a single quantum dot

Optical nonlinearities enable photon-photon interaction and lie at the heart of several proposals for quantum information processing, quantum nondemolition measurements of photons, and optical signal processing. To date, the largest nonlinearities have been realized with single atoms and atomic ensembles. We show that a single quantum dot coupled to a photonic crystal nanocavity can facilitate controlled phase and amplitude modulation between two modes of light at the single-photon level. At larger control powers, we observed phase shifts up to pi/4 and amplitude modulation up to 50%. This was accomplished by varying the photon number in the control beam at a wavelength that was the same as that of the signal, or at a wavelength that was detuned by several quantum dot linewidths from the signal. Our results present a step toward quantum logic devices and quantum nondemolition measurements on a chip.