Common structure of rare replication-deficient E1-positive particles in adenoviral vector batches

The use of the PER.C6 adenovirus packaging cell line in combination with a designated vector plasmid system, whereby the cell line and vector with E1 deleted have no sequence overlap, eliminates the generation of replication-competent adenovirus during vector production. However, we have found cytopathic effect (CPE)-inducing particles in 2 out of more than 40 large-scale manufacturing lots produced in PER.C6 cells. The CPE inducer was detected at a frequency of 1 event in 7.5 x 10(12) vector particles. Despite amplification, it was not readily purified, indicating that the agent itself is replication deficient and requires the parental recombinant adenovirus serotype 5 (rAd5) vector for replication and packaging. Therefore, we designated the agent as a helper-dependent E1-positive region containing viral particle (HDEP). Here, we report the molecular structure of the HDEP genome, revealing an Ad comprised of E1 sequences derived from PER.C6 cells flanked by inverted terminal repeat, packaging signal, and transgene sequences. These sequences form a palindromic structure devoid of E2, E3, E4, and late genes. Since only 5 bp were shared between E1 sequences in the PER.C6 genome and viral vector sequences, the data strongly suggested that insertion of genomic DNA into an adenoviral genome had occurred essentially via nonhomologous recombination. HDEPs have been found in unrelated virus batches and appear to share a common structure that may explain their mechanism of generation. This finding allowed development of an HDEP assay to screen batches of rAd5 produced on the PER.C6 cell line and resulted in detection of seven HDEP agents from four different transgene-virus vector constructs in separate batches of Ad.     

Effects of sham air and cigarette smoke on A549 lung cells: implications for iron-mediated oxidative damage

Inhalation of airborne pollution particles that contain iron can result in a variety of detrimental changes to lung cells and tissues. The lung iron burden can be substantially increased by exposure to cigarette smoke, and cigarette smoke contains iron particulates, as well as several environmental toxins, that could influence intracellular iron status. We are interested in the effects of environmental contaminants on intracellular iron metabolism. We initiated our studies using lung A549 type II epithelial cells as a model, and we evaluated the effects of iron dose and smoke treatment on several parameters of intracellular iron metabolism. We show that iron at a physiological dose stimulates ferritin synthesis without altering the transferrin receptor (TfR) mRNA levels of these cells. This is mediated primarily by a reduction of iron regulatory protein 2. Higher doses of iron reduce iron regulatory protein-1 binding activity and are accompanied by a reduction in TfR mRNA. Thus, for A549 cells, different mechanisms influencing IRP-IRE interaction allow ferritin translation in the presence of TfR mRNA to provide for iron needs and yet prevent excessive iron uptake. More importantly, we report that smoke treatment diminishes ferritin levels and increases TfR mRNA of A549 cells. Ferritin serves as a cytoprotective agent against oxidative stress. These data suggest that exposure of lung cells to low levels of smoke as are present in environmental pollutants could result in reduced cytoprotection by ferritin at a time when iron uptake is sustained, thus enhancing the possibility of lung damage by iron-mediated oxidative stress.     

Economic and greenhouse gas costs of Miscanthus supply chains in the United Kingdom

Miscanthus has been identified as one of the most promising perennial grasses for renewable energy generation in Europe and the United States [Mitigation and Adaptation Strategies for Global Change 9 (2004) 433]. However, the decision to use Miscanthus depends to a considerable degree on its economic and environmental performance [Soil Use and Management 24 (2008) 235; Renewable and Sustainable Energy Reviews 13 (2009) 1230]. This article assessed the spatial distribution of the economic and greenhouse gas (GHG) costs of producing and supplying Miscanthus in the UK. The average farm‐gate production cost of Miscanthus in the UK is estimated to be 40 £ per oven‐dried tonne (£ odt^?1), and the average GHG emissions from the production of Miscanthus are 1.72 kg carbon equivalent per oven‐dried tonnes per year (kg CE odt^?1 yr^?1). The production cost of Miscanthus varies from 35 to 55 £ odt^?1 with the lowest production costs in England, Wales and Northern Ireland, and the highest costs in Scotland. Sensitivity analysis shows that yield of Miscanthus is the most influential factor in its production cost, with precipitation the most crucial input in determining yield. GHG emissions from the production of Miscanthus range from 1.24 to 2.11 kg CE odt^?1 yr^?1. To maximize the GHG benefit, Miscanthus should be established preferentially on croplands, though other considerations obviously arise concerning suitability and value of the land for food production.     

Quantifying global greenhouse gas emissions from land‐use change for crop production

Many assessments of product carbon footprint (PCF) for agricultural products omit emissions arising from land‐use change (LUC). In this study, we developed a framework based on IPCC national greenhouse gas inventory methodologies to assess the impacts of LUC from crop production using oil palm, soybean and oilseed rape as examples. Using ecological zone, climate and soil types from the top 20 producing countries, calculated emissions for transitions from natural vegetation to cropland on mineral soils under typical management ranged from ?4.5 to 29.4 t CO₂‐eq ha^?1 yr^?1 over 20 years for oil palm and 1.2–47.5 t CO₂‐eq ha^?1 yr^?1 over 20 years for soybeans. Oilseed rape showed similar results to soybeans, but with lower maximum values because it is mainly grown in areas with lower C stocks. GHG emissions from other land‐use transitions were between 62% and 95% lower than those from natural vegetation for the arable crops, while conversions to oil palm were a sink for C. LUC emissions were considered on a national basis and also expressed per‐tonne‐of‐oil‐produced. Weighted global averages indicate that, depending on the land‐use transition, oil crop production on newly converted land contributes between ?3.1 and 7.0 t CO₂‐eq t oil production^?1 yr^?1 for palm oil, 11.9–50.6 t CO₂‐eq t oil production^?1 yr^?1 for soybean oil, and 7.7–31.4 t CO₂‐eq t oil production^?1 yr^?1 for rapeseed oil. Assumptions made about crop and LUC distribution within countries contributed up to 66% error around the global averages for natural vegetation conversions. Uncertainty around biomass and soil C stocks were also examined. Finer resolution data and information (particularly on land management and yield) could improve reliability of the estimates but the framework can be used in all global regions and represents an important step forward for including LUC emissions in PCFs.     

Which cropland greenhouse gas mitigation options give the greatest benefits in different world regions? Climate and soil‐specific predictions from integrated empirical models

Major sources of greenhouse gas (GHG) emissions from agricultural crop production are nitrous oxide (N₂O) emissions resulting from the application of mineral and organic fertilizer, and carbon dioxide (CO₂) emissions from soil carbon losses. Consequently, choice of fertilizer type, optimizing fertilizer application rates and timing, reducing microbial denitrification and improving soil carbon management are focus areas for mitigation. We have integrated separate models derived from global data on fertilizer‐induced soil N₂O emissions, soil nitrification inhibitors, and the effects of tillage and soil inputs of soil C stocks into a single model to determine optimal mitigation options as a function of soil type, climate, and fertilization rates. After Monte Carlo sampling of input variables, we aggregated the outputs according to climate, soil and fertilizer factors to consider the benefits of several possible emissions mitigation strategies, and identified the most beneficial option for each factor class on a per‐hectare basis. The optimal mitigation for each soil‐climate‐region was then mapped to propose geographically specific optimal GHG mitigation strategies for crops with varying N requirements. The use of empirical models reduces the requirements for validation (as they are calibrated on globally or continentally observed phenomena). However, as they are relatively simple in structure, they may not be applicable for accurate site‐specific prediction of GHG emissions. The value of this modelling approach is for initial screening and ranking of potential agricultural mitigation options and to explore the potential impact of regional agricultural GHG abatement policies. Given the clear association between management practice and crop productivity, it is essential to incorporate characterization of the yield effect on a given crop before recommending any mitigation practice.     

Structural Requirements for the Activity of the MirB Ferrisiderophore Transporter of Aspergillus fumigatus

Siderophores have been identified as virulence factors in the opportunistic fungal pathogen Aspergillus fumigatus. The 14-pass transmembrane protein MirB is postulated to function as a siderophore transporter, responsible for uptake of the hydroxamate siderophore N,N′,N″-triacetylfusarinine C (TAFC). Our aim was to identify amino acids of A. fumigatus MirB that are crucial for uptake of TAFC. Site-directed mutagenesis was used to create MirB mutants. Expression of wild-type and mutant proteins in the Saccharomyces cerevisiae strain PHY14, which lacks endogenous siderophore transporters, was confirmed by Western blotting. TAFC transport assays using ⁵⁵Fe-labeled TAFC and growth assays with Fe-TAFC as the sole iron source identified alanine 125, tyrosine 577, loop 3, and the second half of loop 7 (Loop7Del2) as crucial for function, since their substitution or deletion abrogated uptake completely. Wild-type MirB transported ferricrocin and coprogen as well as TAFC but not ferrichrysin. MirB was localized by fluorescence microscopy using antisera raised against a MirB extracellular loop peptide. Immunofluorescence microscopy showed that in yeast, wild-type MirB had a punctate distribution under the plasma membrane, as did the A125D and Y577A strains, indicating that the defect in transport of these mutants was unlikely to be due to mislocalization or degradation. MirB immunolocalization in A. fumigatus showed that the transporter was found in vesicles which cycled between the cytoplasm and the plasma membrane and was concentrated at the hyphal tips. The location of MirB was not influenced by the presence of the siderophore TAFC but was sensitive to internal iron stores.     

Causes of Ring-Related Leg Injuries in Birds – Evidence and Recommendations from Four Field Studies

One of the main techniques for recognizing individuals in avian field research is marking birds with plastic and metal leg rings. However, in some species individuals may react negatively to rings, causing leg injuries and, in extreme cases, the loss of a foot or limb. Here, we report problems that arise from ringing and illustrate solutions based on field data from Brown Thornbills (Acanthiza pusilla) (2 populations), Siberian Jays (Perisoreus infaustus) and Purple-crowned Fairy-wrens (Malurus coronatus). We encountered three problems caused by plastic rings: inflammations triggered by material accumulating under the ring (Purple-crowned Fairy-wrens), contact inflammations as a consequence of plastic rings touching the foot or tibio-tarsal joint (Brown Thornbills), and toes or the foot getting trapped in partly unwrapped flat-band colour rings (Siberian Jays). Metal rings caused two problems: the edges of aluminium rings bent inwards if mounted on top of each other (Brown Thornbills), and too small a ring size led to inflammation (Purple-crowned Fairy-wrens). We overcame these problems by changing the ringing technique (using different ring types or larger rings), or using different adhesive. Additionally, we developed and tested a novel, simple technique of gluing plastic rings onto metal rings in Brown Thornbills. A review of studies reporting ring injuries (N = 23) showed that small birds (<55 g body weight) are more prone to leg infections while larger birds (>35 g) tend to get rings stuck over their feet. We give methodological advice on how these problems can be avoided, and suggest a ringing hazard index to compare the impact of ringing in terms of injury on different bird species. Finally, to facilitate improvements in ringing techniques, we encourage online deposition of information regarding ringing injuries of birds at a website hosted by the European Union for Bird Ringing (EURING).     

Is optimism optimal? Functional causes of apparent behavioural biases

We review the use of the terms ‘optimism’ and ‘pessimism’ to characterize particular types of behaviour in non-human animals. Animals can certainly behave as though they are optimistic or pessimistic with respect to specific motivations, as documented by an extensive range of examples in the literature. However, in surveying such examples we find that these terms are often poorly defined and are liable to lead to confusion. Furthermore, when considering behaviour within the framework of optimal decision theory using appropriate currencies, it is often misleading to describe animals as optimistic or pessimistic. There are two common misunderstandings. First, some apparent cases of biased behaviour result from misidentifying the currencies and pay-offs the animals should be maximising. Second, actions that do not maximise short-term pay-offs have sometimes been described as optimistic or pessimistic when in fact they are optimal in the long term; we show how such situations can be understood from the perspective of bandit models. Rather than describing suboptimal, unrealistic behaviour, the terms optimism and pessimism are better restricted to informal usage. Our review highlights the importance of choosing the relevant currency when attempting to predict the action of natural selection.