Propidium monoazide–quantitative polymerase chain reaction for viable Escherichia coli and Pseudomonas aeruginosa detection from abundant background microflora

Nucleic acid-based techniques represent a promising alternative to cultivation-based microbial water quality assessment methods. However, their application is hampered by their innate inability to differentiate between living and dead organisms. Propidium monoazide (PMA) treatment was proposed as an efficient approach for alleviating this limitation. In this study, we demonstrate the performance of PMA–quantitative polymerase chain reaction (qPCR) for the detection of indicator organisms (Escherichia coli and Pseudomonas aeruginosa) in a background of a highly abundant and complex microflora. Treatment with 10 μM PMA resulted in the complete or significant reduction of the false positive signal arising from the amplification of DNA from dead cells.     

An allelopathic investigation of the domination of the introduced invasive Conyza canadensis L.

The introduced, invasive species Conyza canadensis L. covers large areas of the sandy levees next to the River Tamiš (Serbia), forming dense microcomplexes and dominating the other herbaceous species in the ruderal phytocoenosis with its aboveground mass and abundance. In addition to this species, a further 28 plant species were found, but the abundance and cover of these was significantly lower. The allelopathic influence of the species C. canadensis was investigated through analyzing the total phenolics and phenolic acids, as the main allelochemicals, in dead and vegetative parts and the soil beneath them. Seed germination and seedling growth of the target plants (Dactylis glomerata L. and Trifolium repens L.), which grow in this community, served as a measure of the inhibitory capacity of this species. It was established that the content of total phenolics and phenolic acids (p-coumaric, ferulic, p-hydroxybenzoic, vanillic and syringic) varies, following the order: vegetative plant parts > dead plant parts > sandy soil under C. canadensis. Water leachate and soils inhibited seed germination and seedling growth of the test plants to varying degrees, following the order: vegetative parts > dead parts > sandy soil, which is directly related to the content of total phenolics and phenolic acids in them. It was concluded that the pioneer species C. canadensis plays a decisive role in the first phases of vegetation succession and the process of soil formation on the barren sandy levees, owing to the synthesis of secondary phenolic metabolites.     

Symmetry-based self-assembled nanotubes constructed using native protein structures: the key role of flexible linkers

We construct nanotubes using native protein structures and their native associations from structural databases. The construction is based on a shape-guided symmetric self-assembly concept. Our strategy involves fusing judiciously-selected oligomerization domains via peptide linkers. Linkers are inherently flexible, hence their choice is critical: they should position the domains in three-dimensional space in the desired orientation while retaining their own natural conformational tendencies; however, at the same time, retain the construct stability. Here we outline a design scheme which accounts for linker flexibility considerations, and present two examples. The first is HIV-1 capsid protein, which in vitro self-assembles into nanotubes and conical capsids, and its linker exists as a short flexible loop. The second involves novel nanotubes construction based on antimicrobial homodimer Magainin 2, employing linkers of distinct lengths and flexibility levels. Our strategy utilizes the abundance of unique shapes and sizes of proteins and their building blocks which can assemble into a vast number of combinations, and consequently, nanotubes of distinct morphologies and diameters. Computational design and assessment methodologies can help reduce the number of candidates for experimental validation. This is an invited paper for a special issue on protein dynamics, here focusing on flexibility in nanotube design based on protein building blocks.     

Verocytotoxin-producing Escherichia coli in chamois (Rupicapra rupicapra) and cattle in Austria

We assessed the prevalence of verotoxigenic Escherichia coli (VTEC) in chamois (Rupicapra rupicapra) and livestock grazing on a mountain pasture in Austria during June-August 2009. We detected VTEC throughout the sampling period in high numbers in cattle as well as in chamois, leading to the assumption that the degree of contamination of the environment is high. This is the first report of pathogenic E. coli identified in chamois, implicating chamois as a new potential reservoir of these zoonotic pathogens. Because the study area also serves recreational purposes, there is a risk of humans acquiring infection via direct or indirect contact.     

Two new phenylpiperazines with atypical antipsychotic potential

Two new series of substituted arylpiperazines with heterocyclic 3-propoxy-benzimidazole or 3-propoxy-benzimidazole-2-thione groups were synthesized and their in vitro binding affinities for the D(2), 5-HT(1A), 5-HT(2A), and alpha(1)-adrenergic receptors determined. Among them, only two compounds with phenyl aryl-constituent (8a and 9a) showed 5-HT(2A)/D(2) pK(i) binding ratios proposed for atypical neuroleptics. As to their behavioral screening on rodents, both compounds exhibited a non-cataleptic action in rats and antagonized D-amphetamine-induced hyperlocomotion in mice, suggesting their possible atypical antipsychotic potency.     

Identification of the Substrate Recognition and Transport Pathway in a Eukaryotic Member of the Nucleobase-Ascorbate Transporter (NAT) Family

Using the crystal structure of the uracil transporter UraA of Escherichia coli, we constructed a 3D model of the Aspergillus nidulans uric acid-xanthine/H(+) symporter UapA, which is a prototype member of the Nucleobase-Ascorbate Transporter (NAT) family. The model consists of 14 transmembrane segments (TMSs) divided into a core and a gate domain, the later being distinctly different from that of UraA. By implementing Molecular Mechanics (MM) simulations and quantitative structure-activity relationship (SAR) approaches, we propose a model for the xanthine-UapA complex where the substrate binding site is formed by the polar side chains of residues E356 (TMS8) and Q408 (TMS10) and the backbones of A407 (TMS10) and F155 (TMS3). In addition, our model shows several polar interactions between TMS1-TMS10, TMS1-TMS3, TMS8-TMS10, which seem critical for UapA transport activity. Using extensive docking calculations we identify a cytoplasm-facing substrate trajectory (D360, A363, G411, T416, R417, V463 and A469) connecting the proposed substrate binding site with the cytoplasm, as well as, a possible outward-facing gate leading towards the substrate major binding site. Most importantly, re-evaluation of the plethora of available and analysis of a number of herein constructed UapA mutations strongly supports the UapA structural model. Furthermore, modeling and docking approaches with mammalian NAT homologues provided a molecular rationale on how specificity in this family of carriers might be determined, and further support the importance of selectivity gates acting independently from the major central substrate binding site.