Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species

High-density oligonucleotide (oligo) arrays are a powerful tool for transcript profiling. Arrays based on GeneChip® technology are amongst the most widely used, although GeneChip® arrays are currently available for only a small number of plant and animal species. Thus, we have developed a method to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species and tested the method by analysing the transcriptome of Brassica oleracea L., a species for which no GeneChip® array is available, using a GeneChip® array designed for Arabidopsis thaliana (L.) Heynh. Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip® array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genomic DNA on the basis of the perfect-match (PM) probe signal were then selected for subsequent B. oleracea transcriptome analysis using a .cel file parser script to generate probe mask files. The transcriptional response of B. oleracea to a mineral nutrient (phosphorus; P) stress was quantified using probe mask files generated for a wide range of gDNA hybridisation intensity thresholds. An example probe mask file generated with a gDNA hybridisation intensity threshold of 400 removed > 68 % of the available PM probes from the analysis but retained >96 % of available A. thaliana probe-sets. Ninety-nine of these genes were then identified as significantly regulated under P stress in B. oleracea, including the homologues of P stress responsive genes in A. thaliana. Increasing the gDNA hybridisation intensity thresholds up to 500 for probe-selection increased the sensitivity of the GeneChip® array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold. Our open-source software to create probe mask files is freely available http://affymetrix.arabidopsis.info/xspecies/ webcite and may be used to facilitate transcriptomic analyses of a wide range of plant and animal species in the absence of custom arrays.

Detection and molecular characterisation of Pyrenopeziza brassicae isolates resistant to methyl benzimidazole carbamates

BACKGROUND Methyl benzimidazole carbamate (MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in β-tubulin, will enable resistance diagnostics and thereby inform resistance management strategies. RESULTS P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. The MBC-target encoding gene β-tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with β-tubulin amino acid substitutions L240F and E198A. The highest level of MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. The substitutions E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. The frequencies of L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. There were no differences in the frequencies of these alleles between P. brassicae populations sampled from different locations or after fungicide treatment regimes. CONCLUSIONS The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations.

Post-normal science and the art of nature conservation

Nature conservation may be considered a post-normal science in that the loss of biodiversity and increasing environmental degradation require urgent action but are characterised by uncertainty at every level. An ‘extended peer community’ with varying skills, perceptions and values are involved in decision-making and implementation of conservation, and the uncertainty involved limits the effectiveness of practice. In this paper we briefly review the key ecological, philosophical and methodological uncertainties associated with conservation, and then highlight the uncertainties and gaps present within the structure and interactions of the conservation community, and which exist mainly between researchers and practitioners, in the context of nature conservation in the UK. We end by concluding that an openly post-normal science framework for conservation, which acknowledges this uncertainty but strives to minimise it, would be a useful progression for nature conservation, and recommend ways in which knowledge transfer between researchers and practitioners can be improved to support robust decision making and conservation enactment.

Individual differences and the role of the L1 in L2 processing: an ERP investigation

This study used ERP (event-related potentials) to examine both the role of the L1 and the role of individual differences in the processing of agreement violations. Theories of L2 acquisition differ with regard to whether or not native-like acquisition of L2 features is possible (Schwartz and Sprouse, 1994, 1996; Tsimpli and Mastropavlou, 2007), and the results of previous ERP studies are inconsistent when it comes to whether or not native-like processing is observed in response to L2 agreement violations (e.g., Sabourin, 2003; Tokowicz and MacWhinney, 2005). Furthermore, studies of learners in early stages of L2 acquisition have found variability in the emergence of native-like responses (e.g., McLaughlin et al., 2010; Tanner et al., 2009), but sources of variability have not been investigated. The current study examines responses to gender and number agreement violations in English-speaking learners of Spanish (n=24). Stimuli targeted agreement in three conditions: subject-verb agreement (el barco flota/*flotan), which is similar in Spanish and English; number agreement on adjectival predicates (la isla rocosa/*rocosas), a context in which agreement is not instantiated in English; and gender agreement on adjectival predicates (la isla rocosa/*rocoso), which is unique to Spanish. Grammaticality judgments and ERP responses were also tested for correlations with aptitude scores on the Modern Languages Aptitude Test (MLAT; Carroll and Sapon, 1959) and the Raven Advanced Progressive Matrices (Raven, 1965). Results are in line with theories that claim native-like processing is acquirable, since learners demonstrated similar ERP responses to a control group of native Spanish-speakers (n=8) with regard to all three agreement types. Additionally, the MLAT (but not the Raven) was significantly correlated with sensitivity to number violations, both in terms of grammaticality judgments and ERP amplitudes, indicating a role for verbal but not nonverbal aptitude in L2 processing.

Emotional reasoning and anxiety sensitivity: associations with social anxiety disorder in childhood

Background Two specific cognitive constructs that have been implicated in the development and maintenance of anxiety symptoms are anxiety sensitivity and emotional reasoning, both of which relate to the experience and meaning of physical symptoms of arousal or anxiety. The interpretation of physical symptoms has been particularly implicated in theories of social anxiety disorder, where internal physical symptoms are hypothesized to influence the individual's appraisals of the self as a social object. Method The current study compared 75 children on measures of anxiety sensitivity and emotional reasoning: 25 with social anxiety disorder, 25 with other anxiety disorders, and 25 nonanxious children (aged 7–12 years). Results Children with social anxiety disorder reported higher levels of anxiety sensitivity and were more likely than both other groups to view ambiguous situations as anxiety provoking, whether physical information was present or not. There were no group differences in the extent to which physical information altered children's interpretation of hypothetical scenarios. Limitations This study is the first to investigate emotional reasoning in clinically anxious children and therefore replication is needed. In addition, those in both anxious groups commonly had comorbid conditions and, consequently, specific conclusions about social anxiety disorder need to be treated with caution. Conclusion The findings highlight cognitive characteristics that may be particularly pertinent in the context of social anxiety disorder in childhood and which may be potential targets for treatment. Furthermore, the findings suggest that strategies to modify these particular cognitive constructs may not be necessary in treatments of some other childhood anxiety disorders.

Sensitivity of direct radiative forcing by mineral dust to particle characteristics

Airborne dust affects the Earth's energy balance — an impact that is measured in terms of the implied change in net radiation (or radiative forcing, in W m-2) at the top of the atmosphere. There remains considerable uncertainty in the magnitude and sign of direct forcing by airborne dust under current climate. Much of this uncertainty stems from simplified assumptions about mineral dust-particle size, composition and shape, which are applied in remote sensing retrievals of dust characteristics and dust-cycle models. Improved estimates of direct radiative forcing by dust will require improved characterization of the spatial variability in particle characteristics to provide reliable information dust optical properties. This includes constraints on: (1) particle-size distribution, including discrimination of particle subpopulations and quantification of the amount of dust in the sub-10 µm to <0.1 µm mass fraction; (2) particle composition, specifically the abundance of iron oxides, and whether particles consist of single or multi-mineral grains; (3) particle shape, including degree of sphericity and surface roughness, as a function of size and mineralogy; and (4) the degree to which dust particles are aggregated together. The use of techniques that measure the size, composition and shape of individual particles will provide a better basis for optical modelling.

What Tenants Want: UK occupiers’ requirements when renting commercial property and strategic implications for landlords

Businesses need property in order to generate turnover and profits. If real estate owners are to be able to provide properties and related services that are desirable, it is crucial that they understand tenants’ requirements and preferences. Changes in the way businesses operate might well lead to an overall reduction in space requirements in all sectors. Faced with reductions in demand, landlords will find themselves in an increasingly competitive marketplace for tenants. Of the array of strategies available to landlords, what strategies should they employ for maximum effect? This paper examines what United Kingdom tenants want from commercial property (retail, industrial and office). The first part provides an analysis of data from several hundred interviews with occupiers of commercial properties owned by some of the largest UK real estate investment companies. Results are presented for each of the asset classes separately. The second part compares the findings with previous research and discusses the strategic implications for landlords.

Intra-crystalline protein diagenesis (IcPD) in Patella vulgata. Part II: Breakdown and temperature sensitivity

Artificial diagenesis of the intra-crystalline proteins isolated from Patella vulgata was induced by isothermal heating at 140 °C, 110 °C and 80 °C. Protein breakdown was quantified for multiple amino acids, measuring the extent of peptide bond hydrolysis, amino acid racemisation and decomposition. The patterns of diagenesis are complex; therefore the kinetic parameters of the main reactions were estimated by two different methods: 1) a well-established approach based on fitting mathematical expressions to the experimental data, e.g. first-order rate equations for hydrolysis and power-transformed first-order rate equations for racemisation; and 2) an alternative model-free approach, which was developed by estimating a “scaling” factor for the independent variable (time) which produces the best alignment of the experimental data. This method allows the calculation of the relative reaction rates for the different temperatures of isothermal heating. High-temperature data were compared with the extent of degradation detected in sub-fossil Patella specimens of known age, and we evaluated the ability of kinetic experiments to mimic diagenesis at burial temperature. The results highlighted a difference between patterns of degradation at low and high temperature and therefore we recommend caution for the extrapolation of protein breakdown rates to low burial temperatures for geochronological purposes when relying solely on kinetic data.

Influence of UV radiation on chlorophyll, and antioxidant enzymes of wetland plants in different types of constructed wetland

A surface- and vertical subsurface-flow-constructed wetland were designed to study the response of chlorophyll and antioxidant enzymes to elevated UV radiation in three types of wetland plants (Canna indica, Phragmites austrail, and Typha augustifolia). Results showed that (1) chlorophyll content of C. indica, P. austrail, and T. augustifolia in the constructed wetland was significantly lower where UV radiation was increased by 10 and 20 % above ambient solar level than in treatment with ambient solar UV radiation (p < 0.05). (2) The malondialdehyde (MDA) content, guaiacol peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities of wetland plants increased with elevated UV radiation intensity. (3) The increased rate of MDA, SOD, POD, and CAT activities of C. indica, P. australis, and T. angustifolia by elevated UV radiation of 10 % was higher in vertical subsurface-flow-constructed wetland than in surface-flow-constructed wetland. The sensitivity of MDA, SOD, POD, and CAT activities of C. indica, P. austrail, and T. augustifolia to the elevated UV radiation was lower in surface-flow-constructed wetland than in the vertical subsurface-flow-constructed wetland, which was related to a reduction in UV radiation intensity through the dissolved organic carbon and suspended matter in the water. C. indica had the highest SOD and POD activities, which implied it is more sensitive to enhanced UV radiation. Therefore, different wetland plants had different antioxidant enzymes by elevated UV radiation, which were more sensitive in vertical subsurface-flow-constructed wetland than in surface-flow-constructed wetland.

Assessing and monitoring impacts of genetically modified plants on agro-ecosystems: the approach of AMIGA project

The environmental impacts of genetically modified crops is still a controversial issue in Europe. The overall risk assessment framework has recently been reinforced by the European Food Safety Authority(EFSA) and its implementation requires harmonized and efficient methodologies. The EU-funded research project AMIGA − Assessing and monitoring Impacts of Genetically modified plants on Agro-ecosystems − aims to address this issue, by providing a framework that establishes protection goals and baselines for European agro-ecosystems, improves knowledge on the potential long term environmental effects of genetically modified (GM) plants, tests the efficacy of the EFSA Guidance Document for the Environmental Risk Assessment, explores new strategies for post market monitoring, and provides a systematic analysis of economic aspects of Genetically Modified crops cultivation in the EU. Research focuses on ecological studies in different EU regions, the sustainability of GM crops is estimated by analysing the functional components of the agro-ecosystems and specific experimental protocols are being developed for this scope.

Temporal sensitivity of rice seed development from spikelet fertility to viable mature seed to extreme-temperature

Extreme temperature during reproductive development affects rice (Oryza sativa L.) yield and seed quality. A controlled-environment reciprocal-transfer experiment was designed where plants from two japonica cultivars were grown at 28/24 ⁰C and moved to 18/14 ⁰C and vice versa, or from 28/24 to 38/34 ⁰C and vice versa, for 7-d periods to determine the respective temporal pattern of sensitivity of spikelet fertility, yield, and seed viability to each temperature extreme. Spikelet fertility and seed yield per panicle were severely reduced by extreme temperature in the 14 d period prior to anthesis; and both cultivars were affected at 38/34 ⁰C while only cv. Gleva was affected at 18/14 ºC. The damage was greater the earlier the panicles were stressed within this period. Later-exserted panicles compensated only partly for yield loss. Seed viability was significantly reduced by 7-d exposure to 38/34 ⁰C or 18/14 ⁰C at 1 to 7 and 1 to 14 d after anthesis, respectively, in cv. Gleva. Cultivar Taipei 309 was not affected by 7 d exposure at 18/14 ⁰C; and no consistent temporal pattern of sensitivity was evident at 38/34 ⁰C. Hence, brief exposure to low or high temperature was most damaging to spikelet fertility and yield 14 to 7 d before anthesis, coinciding with microsporogenesis; and it was almost as damaging around anthesis. Seed viability was most vulnerable to low or high temperature in the 7 or 14 d after anthesis, when histodifferentiation occurs.

Insiders, outsiders, and the role of local enforcement in forest management: an example from Tanzania

In low-income countries, both nearby local villagers, “insiders”, and non-locals, “outsiders”, extract products from protected forests even though their actions are illegal. Forest managers typically combine enforcement and livelihood projects offered to nearby communities to reduce this illegal activity, but with limited budgets cannot deter all extraction. We develop a game theoretic model of a forest manager's decision interacting with the extraction decisions of insiders and outsiders. Our analysis suggests that, depending on the relative ecological damage caused by each group, budget-constrained forest managers may reduce total forest degradation by legalizing “insider” extraction in return for local villagers' involvement in enforcement activities against outsiders.

Sensitivity of a hydraulic model to changes in channel erosion during extreme flooding

Recent research into flood modelling has primarily concentrated on the simulation of inundation flow without considering the influences of channel morphology. River channels are often represented by a simplified geometry that is implicitly assumed to remain unchanged during flood simulations. However, field evidence demonstrates that significant morphological changes can occur during floods to mobilise the boundary sediments. Despite this, the effect of channel morphology on model results has been largely unexplored. To address this issue, the impact of channel cross-section geometry and channel long-profile variability on flood dynamics is examined using an ensemble of a 1D-2D hydraulic model (LISFLOOD-FP) of the 1:2102 year recurrence interval floods in Cockermouth, UK, within an uncertainty framework. A series of hypothetical scenarios of channel morphology were constructed based on a simple velocity based model of critical entrainment. A Monte-Carlo simulation framework was used to quantify the effects of channel morphology together with variations in the channel and floodplain roughness coefficients, grain size characteristics, and critical shear stress on measures of flood inundation. The results showed that the bed elevation modifications generated by the simplistic equations reflected a good approximation of the observed patterns of spatial erosion despite its overestimation of erosion depths. The effect of uncertainty on channel long-profile variability only affected the local flood dynamics and did not significantly affect the friction sensitivity and flood inundation mapping. The results imply that hydraulic models generally do not need to account for within event morphodynamic changes of the type and magnitude modelled, as these have a negligible impact that is smaller than other uncertainties, e.g. boundary conditions. Instead morphodynamic change needs to happen over a series of events to become large enough to change the hydrodynamics of floods in supply limited gravel-bed rivers like the one used in this research.

Sensitivity of simulated regional Arctic climate to the choice of coupled model domain

The climate over the Arctic has undergone changes in recent decades. In order to evaluate the coupled response of the Arctic system to external and internal forcing, our study focuses on the estimation of regional climate variability and its dependence on large-scale atmospheric and regional ocean circulations. A global ocean–sea ice model with regionally high horizontal resolution is coupled to an atmospheric regional model and global terrestrial hydrology model. This way of coupling divides the global ocean model setup into two different domains: one coupled, where the ocean and the atmosphere are interacting, and one uncoupled, where the ocean model is driven by prescribed atmospheric forcing and runs in a so-called stand-alone mode. Therefore, selecting a specific area for the regional atmosphere implies that the ocean–atmosphere system can develop ‘freely’ in that area, whereas for the rest of the global ocean, the circulation is driven by prescribed atmospheric forcing without any feedbacks. Five different coupled setups are chosen for ensemble simulations. The choice of the coupled domains was done to estimate the influences of the Subtropical Atlantic, Eurasian and North Pacific regions on northern North Atlantic and Arctic climate. Our simulations show that the regional coupled ocean–atmosphere model is sensitive to the choice of the modelled area. The different model configurations reproduce differently both the mean climate and its variability. Only two out of five model setups were able to reproduce the Arctic climate as observed under recent climate conditions (ERA-40 Reanalysis). Evidence is found that the main source of uncertainty for Arctic climate variability and its predictability is the North Pacific. The prescription of North Pacific conditions in the regional model leads to significant correlation with observations, even if the whole North Atlantic is within the coupled model domain. However, the inclusion of the North Pacific area into the coupled system drastically changes the Arctic climate variability to a point where the Arctic Oscillation becomes an ‘internal mode’ of variability and correlations of year-to-year variability with observational data vanish. In line with previous studies, our simulations provide evidence that Arctic sea ice export is mainly due to ‘internal variability’ within the Arctic region. We conclude that the choice of model domains should be based on physical knowledge of the atmospheric and oceanic processes and not on ‘geographic’ reasons. This is particularly the case for areas like the Arctic, which has very complex feedbacks between components of the regional climate system.

Sensitivity of a coupled climate model to canopy interception capacity

The canopy interception capacity is a small but key part of the surface hydrology, which affects the amount of water intercepted by vegetation and therefore the partitioning of evaporation and transpiration. However, little research with climate models has been done to understand the effects of a range of possible canopy interception capacity parameter values. This is in part due to the assumption that it does not significantly affect climate. Near global evapotranspiration products now make evaluation of canopy interception capacity parameterisations possible. We use a range of canopy water interception capacity values from the literature to investigate the effect on climate within the climate model HadCM3. We find that the global mean temperature is affected by up to -0.64 K globally and -1.9 K regionally. These temperature impacts are predominantly due to changes in the evaporative fraction and top of atmosphere albedo. In the tropics, the variations in evapotranspiration affect precipitation, significantly enhancing rainfall. Comparing the model output to measurements, we find that the default canopy interception capacity parameterisation overestimates canopy interception loss (i.e. canopy evaporation) and underestimates transpiration. Overall, decreasing canopy interception capacity improves the evapotranspiration partitioning in HadCM3, though the measurement literature more strongly supports an increase. The high sensitivity of climate to the parameterisation of canopy interception capacity is partially due to the high number of light rain-days in the climate model that means that interception is overestimated. This work highlights the hitherto underestimated importance of canopy interception capacity in climate model hydroclimatology and the need to acknowledge the role of precipitation representation limitations in determining parameterisations.