Folding of the cocaine aptamer studied by EPR and fluorescence spectroscopies using the bifunctional spectroscopic probe Ç

The cocaine aptamer is a DNA molecule that binds cocaine at the junction of three helices. The bifunctional spectroscopic probe Ç was incorporated independently into three different positions of the aptamer and changes in structure and dynamics upon addition of the cocaine ligand were studied. Nucleoside Ç contains a rigid nitroxide spin label and can be studied directly by electron paramagnetic resonance (EPR) spectroscopy and fluorescence spectroscopy after reduction of the nitroxide to yield the fluoroside Ç^f. Both the EPR and the fluorescence data for aptamer 2 indicate that helix III is formed before cocaine binding. Upon addition of cocaine, increased fluorescence of a fully base-paired Ç^f, placed at the three-way junction in helix III, was observed and is consistent with a helical tilt from a coaxial stack of helices II and III. EPR and fluorescence data clearly show that helix I is formed upon addition of cocaine, concomitant with the formation of the Y-shaped three-way helical junction. The EPR data indicate that nucleotides in helix I are more mobile than nucleotides in regular duplex regions and may reflect increased dynamics due to the short length of helix I.     

Glyphosate complexation to aluminium(III). An equilibrium and structural study in solution using potentiometry, multinuclear NMR, ATR-FTIR, ESI-MS and DFT calculations

The stoichiometries and stability constants of a series of Al³⁺-N-phosponomethyl glycine (PMG/H₃L) complexes have been determined in acidic aqueous solution using a combination of precise potentiometric titration data, quantitative ²⁷Al and ³¹P NMR spectra, ATR-FTIR spectrum and ESI-MS measurements (0.6 M NaCl, 25 °C). Besides the mononuclear AlH₂L²⁺, Al(H₂L)(HL), and Al(HL)L²⁻, dimeric Al₂(HL)L⁺ and trinuclear complexes have been postulated.¹H and ³¹P NMR data show that different isomers co-exist in solution and the isomerization reactions are slow on the ³¹P NMR time scale. The geometries of monomeric and dimeric complexes likely double hydroxo bridged and double phosphonate bridged isomers have been optimized using DFT ab initio calculations starting from rational structural proposals. Energy calculations using the PCM solvation method also support the co-existence of isomers in solutions.     

A habitat-based model for the spread of hantavirus between reservoir and spillover species

New habitat-based models for spread of hantavirus are developed which account for interspecies interaction. Existing habitat-based models do not consider interspecies pathogen transmission, a primary route for emergence of new infectious diseases and reservoirs in wildlife and man. The modeling of interspecies transmission has the potential to provide more accurate predictions of disease persistence and emergence dynamics. The new models are motivated by our recent work on hantavirus in rodent communities in Paraguay. Our Paraguayan data illustrate the spatial and temporal overlaps among rodent species, one of which is the reservoir species for Jabora virus and others which are spillover species. Disease transmission occurs when their habitats overlap. Two mathematical models, a system of ordinary differential equations (ODE) and a continuous-time Markov chain (CTMC) model, are developed for spread of hantavirus between a reservoir and a spillover species. Analysis of a special case of the ODE model provides an explicit expression for the basic reproduction number, , such that if , then the pathogen does not persist in either population but if , pathogen outbreaks or persistence may occur. Numerical simulations of the CTMC model display sporadic disease incidence, a new behavior of our habitat-based model, not present in other models, but which is a prominent feature of the seroprevalence data from Paraguay. Environmental changes that result in greater habitat overlap result in more encounters among various species that may lead to pathogen outbreaks and pathogen establishment in a new host.     

Forest history and the development of old‐growth characteristics in fragmented boreal forests

Questions: Can small and isolated high‐conservation value forests (e.g. designated woodland key habitats) maintain old‐growth forest characteristics and functionality in fragmented landscapes? To what extent have past disturbances (natural and anthropogenic) influenced the development of old‐growth characteristics of these forests? How long does it take for selectively cut stands to attain conditions resembling old‐growth forests? Location: Southern boreal zone of central Sweden. Methods: We linked multiple lines of evidence from historical records, biological archives, and analyses of current forest structure to reconstruct the forest history of a boreal landscape, with special emphasis on six remaining core localities of high‐conservation value forest stands. Results: Our reconstructions revealed that several of these stands experienced wildfires up to the 1890s; all had been selectively harvested in the late 1800s; and all underwent substantial structural and compositional reorganization over the following 100‐150 years. This time interval was sufficient to recover considerable amounts of standing and downed dead wood (mean 60.3 m³ ha^?1), a range of tree ages and sizes (mean basal area 32.6 m² ha^?1), and dominance of shade‐tolerant spruce. It was insufficient to obtain clearly uneven tree age structures and large (>45 cm diameter) living and dead trees. Thus, these forests contain some, but not all, important compositional and structural attributes of old‐growth forests, their abundance being dependent on the timing and magnitude of past natural and anthropogenic disturbances. Our landscape‐level analysis showed marked compositional and structural differences between the historical forest landscape and the present landscape, with the latter having a greater proportion of young forests, introduction of non‐native species, and lack of large trees and dead wood. Conclusions: The remnant high‐conservation value stands were not true representatives of the pre‐industrial forests, but represent the last vestige of forests that have regenerated naturally and maintained a continuous tree cover. These traits, coupled with their capacity for old‐growth recovery, make them valuable focal areas for conservation.     

Migratory timing, marine survival and growth of anadromous brown trout Salmo trutta in the River Imsa, Norway

The aim of the paper was to study sea migration, growth and survival of brown trout Salmo trutta of the River Imsa, 1976–2005. The migratory S. trutta were individually tagged and fish leaving or entering the river were monitored daily in traps located near the river mouth. The mean annual duration of the sea sojourn was 6–9 months for first-time migrants moving to sea between January and June. It was 8–18 months for those migrating to sea between July and December. Veteran migrants stayed 12 months or less at sea and most returned to the river in August. Early ascending fish stayed the longest in fresh water because most returned to sea in April to May. The day number of 50% cumulative smolt descent correlated negatively with mean water temperature in February to March and the February North Atlantic Oscillation index (NAOI). Mean annual sea growth during the first 2 years after smolting was higher for S. trutta spending the winter at sea than those wintering in the River Imsa. First year's sea growth was lower for S. trutta descending in spring than autumn. For first-time migrants, it correlated negatively with the February NAOI of the smolt year. Sea survival was higher for spring than autumn descending first-time migratory S. trutta with a maximum in May (14·9%). Number of anadromous S. trutta returning to the river increased linearly with the size of the cohort moving to sea, with no evidence of density-dependent sea mortality. Sea survival of S. trutta smolts moving to sea between January and June correlated positively both with the annual number of Atlantic Salmo salar smolts, the specific growth rate at sea, and time of seaward migration in spring. This is the first study indicating how environmental factors at the time of seaward migration influence the sea survival of S. trutta .     

Insect Colonisation of Fruiting Bodies of the Wood-decaying Fungus Fomitopsis pinicola at Different Distances from an Old-growth Forest

We studied the colonisation rate of insects inhabiting fruiting bodies of the wood-decaying fungus Fomitopsis pinicola both within and at different distances (up to 1610 m) from an old-growth forest reserve. The colonisation rate of most species was not affected by the distance from the reserve, and none of the species were affected by the size of local sources of fruiting bodies in the managed forest. We suggest that many insect species inhabiting fruiting bodies of wood-decaying fungi can colonise fruiting bodies at a high enough rate to persist in managed forests of Fennoscandia. However, the colonisation rates of the fungivorous beetle Cis quadridens and the predatory fly Medetera apicalis were negatively affected by distance from the reserve. Cis quadridens is rare in many managed forests, but often quite common at sites with high substrate densities. The rarity of this species may therefore be due to weak ability to colonize distant patches. The same may also be true for M. apicalis, but less is known about the biology of this species. Medetera apicalis was the most common insect predator in the old-growth forest, but it was rare at the largest distances from it in the managed forest. Therefore, it seems likely that the overall pressure from natural enemies significantly declined with distance from the reserve.     

Phi-analysis at the experimental limits: mechanism of beta-hairpin formation

The 37-residue Formin-binding protein, FBP28, is a canonical three-stranded beta-sheet WW domain. Because of its small size, it is so insensitive to chemical denaturation that it is barely possible to determine accurately a denaturation curve, as the transition spans 0-7 M guanidinium hydrochloride (GdmCl). It is also only marginally stable, with a free energy of denaturation of just 2.3 kcal/mol at 10 degrees Celsius so only small changes in energy upon mutation can be tolerated. But these properties and relaxation times for folding of 25 micros-400 micros conspire to allow the rapid acquisition of accurate and reproducible kinetic data for Phi-analysis using classical temperature-jump methods. The transition state for folding is highly polarized with some regions having Phi-values of 0 and others 1, as readily seen in chevron plots, with Phi-values of 0 having the refolding arms overlaying and those of 1 the unfolding arms superimposable. Good agreement is seen with transition state structures identified from independent molecular dynamics (MD) simulations at 60, 75, and 100 degrees Celsius, which allows us to explore further the details of the folding and unfolding pathway of FBP28. The first beta-turn is near native-like in the transition state for folding (experimental) and unfolding (MD and experiment). The simulations show that there are transient contacts between the aromatic side-chains of the beta-strands in the denatured state and that these interactions provide the driving force for folding of the first beta-hairpin of this three-stranded sheet. Only after the backbone hydrogen bonds are formed between beta1 and beta2 does a hydrogen bond form to stabilize the intervening turn, or the first beta-turn.     

Inactivation of the Ecs ABC transporter of Staphylococcus aureus attenuates virulence by altering composition and function of bacterial wall

Background

Ecs is an ATP-binding cassette (ABC) transporter present in aerobic and facultative anaerobic Gram-positive Firmicutes. Inactivation of Bacillus subtilis Ecs causes pleiotropic changes in the bacterial phenotype including inhibition of intramembrane proteolysis. The molecule(s) transported by Ecs is (are) still unknown.

Methodology/Principal Findings

In this study we mutated the ecsAB operon in two Staphylococcus aureus strains, Newman and LS-1. Phenotypic and functional characterization of these Ecs deficient mutants revealed a defect in growth, increased autolysis and lysostaphin sensitivity, altered composition of cell wall proteins including the precursor form of staphylokinase and an altered bacterial surface texture. DNA microarray analysis indicated that the Ecs deficiency changed expression of the virulence factor regulator protein Rot accompanied by differential expression of membrane transport proteins, particularly ABC transporters and phosphate-specific transport systems, protein A, adhesins and capsular polysaccharide biosynthesis proteins. Virulence of the ecs mutants was studied in a mouse model of hematogenous S. aureus infection. Mice inoculated with the ecs mutant strains developed markedly milder infections than those inoculated with the wild-type strains and had consequently lower mortality, less weight loss, milder arthritis and decreased persistence of staphylococci in the kidneys. The ecs mutants had higher susceptibility to ribosomal antibiotics and plant alkaloids chelerythrine and sanguinarine.

Conclusions/Significance

Our results show that Ecs is essential for staphylococcal virulence and antimicrobial resistance probably since the transport function of Ecs is essential for the normal structure and function of the cell wall. Thus targeting Ecs may be a new approach in combating staphylococcal infection.