Continuing influences of introduced hedgehogs <Emphasis Type="Italic">Erinaceus europaeus</Emphasis> as a predator of wader (Charadrii) eggs four decades after their release on the Outer Hebrides,Scotland

Non-native predators can cause major declines or even localised extinctions in prey populations across the globe, especially on islands. The removal of non-native predators can, therefore, be a crucial conservation management tool but there can be challenges when they are viewed as charismatic in their own right. Four decades after their introduction to islands in the Outer Hebrides, Scotland, European hedgehogs Erinaceus europaeus continue to be an important nest predator for a declining population of breeding waders. Where hedgehogs were rare, clutch survival rates (assessed using nest temperature loggers) of five species of waders (dunlin Calidris alpina, lapwing Vanellus vanellus, redshank Tringa totanus, snipe Gallinago gallinago and ringed plover Charadrius hiaticula) were higher than where hedgehogs were relatively more abundant. Hedgehogs were the most frequent nest predator identified using cameras. However, factors influencing population sizes of breeding waders are complex and unlikely to be attributable to a single species of predator. The interactions between predation, land use, habitat and the changes in each deserve further attention.     

Perineural local anaesthetic catheter after major lower limb amputation trial (PLACEMENT): study protocol for a randomised controlled pilot study

Background

Pain after major lower limb amputation for peripheral arterial disease (PAD) is a significant problem. A perineural catheter (PNC) can be placed adjacent to the major nerve at the time of amputation with a continuous local anaesthetic infusion given postoperatively to try and reduce pain. Although low-quality observational data suggest that PNC usage reduces postoperative opioid requirements, there are limited data regarding its effect on pain. The aim of PLACEMENT is to explore the feasibility of running an effectiveness trial to assess the impact of a PNC with continuous local anaesthetic infusion, inserted at the time of amputation, on short and medium-term postoperative outcomes.

Methods/design

Fifty patients undergoing a major lower limb amputation (below or above the knee) for PAD will be recruited from two centres. Patients will be randomised in a 1:1 ratio to receive standard postoperative analgesia, with or without insertion of a PNC and local anaesthetic infusion for the first 5 postoperative days. Outcome data will be captured for the first 5 days, including pain scores (primary outcome, captured three times a day), opioid use, nausea or vomiting, itching, dizziness and complications. Patients will be contacted 2 and 6 months after surgery to assess quality of life, phantom limb pain, chronic stump pain and total healthcare costs. Semi-structured interviews will be conducted with at least 10 patients (dependent on saturation of analytic themes on preliminary coding) purposefully sampled to achieve variation in site and study arm. Interviews will explore patients’ perception of post-amputation pain and its treatment, and experience of study processes. Semi-structured interviews with 5–10 health professionals will explore feasibility, fidelity, and acceptability of the study. Data from this pilot will be used to assess feasibility of, and estimate parameters to calculate the sample size for an effectiveness trial. Full ethical approval has been granted (Wales Research Ethics Committee 3 reference number 16/WA/0353).

Discussion

PLACEMENT will be the first study to explore the feasibility of running an effectiveness trial on PNC usage for postoperative pain in amputees, and provide parameters to calculate the appropriate sample size for this study.

Trial registration

ISRCTN.com, ISRCTN85710690. Registered on 21 October 2016.European Clinical Trials Database (EudraCT), 2016-003544-37. Registered on 24 August 2016.

     

Inundation,Vegetation, and Sediment Effects on Litter Decomposition in Pacific Coast Tidal Marshes

The cycling and sequestration of carbon are important ecosystem functions of estuarine wetlands that may be affected by climate change. We conducted experiments across a latitudinal and climate gradient of tidal marshes in the northeast Pacific to evaluate the effects of climate- and vegetation-related factors on litter decomposition. We manipulated tidal exposure and litter type in experimental mesocosms at two sites and used variation across marsh landscapes at seven sites to test for relationships between decomposition and marsh elevation, soil temperature, vegetation composition, litter quality, and sediment organic content. A greater than tenfold increase in manipulated tidal inundation resulted in small increases in decomposition of roots and rhizomes of two species, but no significant change in decay rates of shoots of three other species. In contrast, across the latitudinal gradient, decomposition rates of Salicornia pacifica litter were greater in high marsh than in low marsh. Rates were not correlated with sediment temperature or organic content, but were associated with plant assemblage structure including above-ground cover, species composition, and species richness. Decomposition rates also varied by litter type; at two sites in the Pacific Northwest, the grasses Deschampsia cespitosa and Distichlis spicata decomposed more slowly than the forb S. pacifica. Our data suggest that elevation gradients and vegetation structure in tidal marshes both affect rates of litter decay, potentially leading to complex spatial patterns in sediment carbon dynamics. Climate change may thus have direct effects on rates of decomposition through increased inundation from sea-level rise and indirect effects through changing plant community composition.     

Genetic population structure and dispersal in Atlantic Island caddisflies

SUMMARY 1. Population genetic structure of Wormaldia tagananana, a caddis with a narrow geographic range and endemic to the Canary Islands, was investigated by studying allozyme variation at 11 putative loci in five of the eight extant populations on Tenerife and La Gomera. Genetic variability, population structure and gene flow were compared with those reported previously in more widespread Trichoptera, particularly Canarian populations of the non-endemic limnephilid Mesophylax aspersus, to examine the hypothesis that the Wormaldia , with its restricted range, would exhibit relatively little genetic variability and gene flow.
2. Despite it being a narrow-range island endemic, genetic variability in populations of W. tagananana is broadly similar to values reported for more widespread caddis.
3. Significant genetic population structure was observed in W. tagananana (overall F _ST = 0.387), greater than that seen in M. aspersus and amongst the highest reported for lotic caddis to date. Several site- and island-specific alleles were reported, providing further evidence for the relative isolation of individual Wormaldia populations.
4. Significant deviations from Hardy–Weinberg equilibrium were found in four of five populations (overall F _IS = 0.675). This could result from within-locality population substructuring, or offspring within a reach being the product of a limited number of matings.
5. This genetic evidence supports the hypothesis that the restricted range of W. tagananana is, at least in part, because of limited dispersal ability.     

Pathways mediating the nuclear import of histones H3 and H4 in yeast.

The correct assembly of chromatin is necessary for the maintenance of genomic stability in eukaryotic cells. A critical step in the assembly of new chromatin is the cell cycle-regulated synthesis and nuclear import of core histones. Here we demonstrate that the nuclear import pathway of histones H3 and H4 is mediated by at least two karyopherins/importins, Kap123p and Kap121p. Cytosolic H4 is found associated with Kap123p and H3. Kap121p is also present in the H4-PrA-associated fractions, albeit in lesser amounts than Kap123p, suggesting that this Kap serves as an additional import receptor. We further demonstrate that cytosolic Kap123p is associated with acetylated H3 and H4. H3 and H4 each contain a nuclear localization signal (NLS) in their amino-terminal domains. These amino-terminal domains were found to be essential for the nuclear accumulation of H3 and H4-green fluorescent protein reporters. Each NLS mediated direct binding to Kap123p and Kap121p, and decreased nuclear accumulation of H3 and H4 NLS-green fluorescent protein reporters was observed in specific kap mutant strains. H3 and H4 are the first histones to be assembled onto DNA, and these results show that their import is mediated by at least two import pathways.     

Geometry and water accessibility of the inhibitor binding site of Na+-pump: Pulse- and CW-EPR study

Spin labels based on cinobufagin, a specific inhibitor of the Na,K-ATPase, have proved valuable tools to characterize the binding site of cardiotonic steroids (CTSs), which also constitutes the extracellular cation pathway. Because existing literature suggests variations in the physiological responses caused by binding of different CTSs, we extended the original set of spin-labeled inhibitors to the more potent bufalin derivatives. Positioning of the spin labels within the Na,K-ATPase site was defined and visualized by molecular docking. Although the original cinobufagin labels exhibited lower affinity, continuous-wave electron paramagnetic resonance spectra of spin-labeled bufalins and cinobufagins revealed a high degree of pairwise similarity, implying that these two types of CTS bind in the same way. Further analysis of the spectral lineshapes of bound spin labels was performed with emphasis on their structure (PROXYL vs. TEMPO), as well as length and rigidity of the linkers. For comparable structures, the dynamic flexibility increased in parallel with linker length, with the longest linker placing the spin label at the entrance to the binding site. Temperature-related changes in spectral lineshapes indicate that six-membered nitroxide rings undergo boat-chair transitions, showing that the binding-site cross section can accommodate the accompanying changes in methyl-group orientation. D₂O-electron spin echo envelope modulation in pulse-electron paramagnetic resonance measurements revealed high water accessibilities and similar polarity profiles for all bound spin labels, implying that the vestibule leading to steroid-binding site and cation-binding sites is relatively wide and water-filled.     

Cloning and Characterization of Three Fatty Alcohol Oxidase Genes from Candida tropicalis Strain ATCC 20336

Candida tropicalis (ATCC 20336) converts fatty acids to long-chain dicarboxylic acids via a pathway that includes among other reactions the oxidation of ω-hydroxy fatty acids to ω-aldehydes by a fatty alcohol oxidase (FAO). Three FAO genes (one gene designated FAO1 and two putative allelic genes designated FAO2a and FAO2b), have been cloned and sequenced from this strain. A comparison of the DNA sequence homology and derived amino acid sequence homology between these three genes and previously published Candida FAO genes indicates that FAO1 and FAO2 are distinct genes. Both genes were individually cloned and expressed in Escherichia coli. The substrate specificity and K_m values for the recombinant FAO1 and FAO2 were significantly different. Particularly striking is the fact that FAO1 oxidizes ω-hydroxy fatty acids but not 2-alkanols, whereas FAO2 oxidizes 2-alkanols but not ω-hydroxy fatty acids. Analysis of extracts of strain H5343 during growth on fatty acids indicated that only FAO1 was highly induced under these conditions. FAO2 contains one CTG codon, which codes for serine (amino acid 177) in C. tropicalis but codes for leucine in E. coli. An FAO2a construct, with a TCG codon (codes for serine in E. coli) substituted for the CTG codon, was prepared and expressed in E. coli. Neither the substrate specificity nor the K_m values for the FAO2a variant with a serine at position 177 were radically different from those of the variant with a leucine at that position.     

Denitrification in the periphyton associated with plant shoots and in the sediment of a wetland system supplied with sewage treatment plant effluent

Seasonal variation in denitrification and major factors controlling this process were determined in sediment, microbial communities attached to plant shoots (periphyton) and in the water of a Phragmites and an Elodea-dominated stand of a constructed wetland system between May 1997 and February 1998. The wetland was supplied with effluent from a sewage treatment plant. The denitrification rate in periphyton on plants shoots (expressed per shoot area) was always considerably higher than in the sediment and varied with the chlorophyll-a content of the periphyton in the course of the year. The algae in the periphyton provided attachment surfaces and probably also organic compounds to the denitrifying bacteria. Decreases in periphyton biomass and denitrification rate in the Phragmites and Elodea-dominated stands during the growing season were associated with enhanced shading by Phragmites shoots or a floating layer of macro-algae and Lemna spp., respectively. Light availability and the denitrification rate of periphyton increased again after the Phragmites shoots were cut in October. Nitrate appeared to limit the denitrification rate in the sediment. Periphyton denitrification rates were mostly lower on Elodea shoots than on Phragmites shoots, in spite of the higher living algal biomass on Elodea shoots. This difference was associated with lower nitrate concentrations in the Elodea-dominated stand. In the two stands, the daily denitrification rates in periphyton on shoots of Phragmites australis (44.4–121 mg N m^–2 stand area d^–1) and Elodea nuttallii (14.8–33.1 mg N m^–2 d^–1) were clearly more important than rates in the sediment (0.5–25.5 mg N m^–2 d^–1) or the water (0.4–3.9 mg N m^–2 d^–1). The presence of few bacteria attachment sites or low organic carbon availability possibly resulted in low denitrification rates in the water. Denitrification appeared to be a major process in nitrate removal from the through-flowing water in this wetland system.     

Transformation of Fatty Acids Catalyzed by Cytochrome P450 Monooxygenase Enzymes of Candida tropicalis

Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in β-oxidation convert these substrates to long-chain α,ω-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is ω-hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C_18:1), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to ω-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C_14:0) much more effectively. Both enzymes, in particular CYP52A17, also oxidized ω-hydroxy fatty acids, ultimately generating the α,ω-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for β-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.