A multi-level analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

Diaminofluoresceins are widely used probes for detection and intracellular localization of NO formation in cultured/isolated cells and intact tissues. The fluorinated derivative, 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), has gained increasing popularity in recent years due to its improved NO-sensitivity, pH-stability, and resistance to photo-bleaching compared to the first-generation compound, DAF-2. Detection of NO production by either reagent relies on conversion of the parent compound into a fluorescent triazole, DAF-FM-T and DAF-2-T, respectively. While this reaction is specific for NO and/or reactive nitrosating species, it is also affected by the presence of oxidants/antioxidants. Moreover, the reaction with other molecules can lead to the formation of fluorescent products other than the expected triazole. Thus additional controls and structural confirmation of the reaction products are essential. Using human red blood cells as an exemplary cellular system we here describe robust protocols for the analysis of intracellular DAF-FM-T formation using an array of fluorescence-based methods (laser-scanning fluorescence microscopy, flow cytometry and fluorimetry) and analytical separation techniques (reversed-phase HPLC and LC-MS/MS). When used in combination, these assays afford unequivocal identification of the fluorescent signal as being derived from NO and are applicable to most other cellular systems without or with only minor modifications.

The Natural History of Slapton Ley Nature Reserve XVIII: nitrogen and phosphorus losses from the catchment - an export coefficient modelling approach

Nitrogen and phosphorus losses from the catchment of Slapton Ley, a small coastal lake in SW England, were calculated using an adaptation of a model developed by Jorgensen (1980). A detailed survey of the catchment revealed that its land use is dominated by both permanent and temporary grassland (respectively 38 and 32% of its total area), and that the remainder is made up of the cultivation of cereals and field vegetables, and market gardening. Livestock numbers in the catchment constitute ca. 6600 head of cattle, 10,000 sheep, 590 pigs, 1700 poultry and 58 horses. The permanent human population of the area is ca. 2000, served by two small gravity-fed sewage treatment works (STWs). Inputs to, and losses from, farmland in the catchment were computed using Jorgensen’s model, and coefficients derived from the data of Cooke (1976), Gostick (1982), Rast and Lee (1983) and Vollenweider (1968). Allowing for outputs from STWs, the total annual external load of N and P upon Slapton Ley is 160 t (35 kg ha-1) a-1 N, and 4.8 t (1.05 kg ha-1) a-1 P. Accordingly to Vollenweider (1968, 1975), such loadings exceed OECD permissible level by a factor of ca. 50 in the case of N, and ca. 5 in that of P. In order to reduce nutrient loads, attention would need to be paid to both STW and agricultural sources.

Forecasting extinction risk of ectotherms under climate warming: an evolutionary perspective

1. It has been postulated that climate warming may pose the greatest threat species in the tropics, where ectotherms have evolved more thermal specialist physiologies. Although species could rapidly respond to environmental change through adaptation, little is known about the potential for thermal adaptation, especially in tropical species. 2. In the light of the limited empirical evidence available and predictions from mutation-selection theory, we might expect tropical ectotherms to have limited genetic variance to enable adaptation. However, as a consequence of thermodynamic constraints, we might expect this disadvantage to be at least partially offset by a fitness advantage, that is, the ‘hotter-is-better’ hypothesis. 3. Using an established quantitative genetics model and metabolic scaling relationships, we integrate the consequences of the opposing forces of thermal specialization and thermodynamic constraints on adaptive potential by evaluating extinction risk under climate warming. We conclude that the potential advantage of a higher maximal development rate can in theory more than offset the potential disadvantage of lower genetic variance associated with a thermal specialist strategy. 4. Quantitative estimates of extinction risk are fundamentally very sensitive to estimates of generation time and genetic variance. However, our qualitative conclusion that the relative risk of extinction is likely to be lower for tropical species than for temperate species is robust to assumptions regarding the effects of effective population size, mutation rate and birth rate per capita. 5. With a view to improving ecological forecasts, we use this modelling framework to review the sensitivity of our predictions to the model’s underpinning theoretical assumptions and the empirical basis of macroecological patterns that suggest thermal specialization and fitness increase towards the tropics. We conclude by suggesting priority areas for further empirical research.

Detection of mutations in Salmonella enterica gyrA, gyrB, parC and parE genes by denaturing high performance liquid chromatography (DHPLC) using standard HPLC instrumentation

Aims: Quinolone antibiotics are the agents of choice for treating systemic Salmonella infections. Resistance to quinolones is usually mediated by mutations in the DNA gyrase gene gyrA. Here we report the evaluation of standard HPLC equipment for the detection of mutations (single nucleotide polymorphisms; SNPs) in gyrA, gyrB, parC and parE by denaturing high performance liquid chromatography (DHPLC). Methods: A panel of Salmonella strains was assembled which comprised those with known different mutations in gyrA (n = 8) and fluoroquinolone-susceptible and -resistant strains (n = 50) that had not been tested for mutations in gyrA. Additionally, antibiotic-susceptible strains of serotypes other than Salmonella enterica serovar Typhimurium strains were examined for serotype-specific mutations in gyrB (n = 4), parC (n = 6) and parE (n = 1). Wild-type (WT) control DNA was prepared from Salmonella Typhimurium NCTC 74. The DNA of respective strains was amplified by PCR using Optimase (R) proofreading DNA polymerase. Duplex DNA samples were analysed using an Agilent A1100 HPLC system with a Varian Helix (TM) DNA column. Sequencing was used to validate mutations detected by DHPLC in the strains with unknown mutations. Results: Using this HPLC system, mutations in gyrA, gyrB, parC and parE were readily detected by comparison with control chromatograms. Sequencing confirmed the gyrA predicted mutations as detected by DHPLC in the unknown strains and also confirmed serotype-associated sequence changes in non-Typhimurium serotypes. Conclusions: The results demonstrated that a non-specialist standard HPLC machine fitted with a generally available column can be used to detect SNPs in gyrA, gyrB, parC and parE genes by DHPLC. Wider applications should be possible.

Simulated variations in atmospheric CO2 over a Wisconsin forest using a coupled ecosystem-atmosphere model

Ecosystem fluxes of energy, water, and CO2 result in spatial and temporal variations in atmospheric properties. In principle, these variations can be used to quantify the fluxes through inverse modelling of atmospheric transport, and can improve the understanding of processes and falsifiability of models. We investigated the influence of ecosystem fluxes on atmospheric CO2 in the vicinity of the WLEF-TV tower in Wisconsin using an ecophysiological model (Simple Biosphere, SiB2) coupled to an atmospheric model (Regional Atmospheric Modelling System). Model parameters were specified from satellite imagery and soil texture data. In a companion paper, simulated fluxes in the immediate tower vicinity have been compared to eddy covariance fluxes measured at the tower, with meteorology specified from tower sensors. Results were encouraging with respect to the ability of the model to capture observed diurnal cycles of fluxes. Here, the effects of fluxes in the tower footprint were also investigated by coupling SiB2 to a high-resolution atmospheric simulation, so that the model physiology could affect the meteorological environment. These experiments were successful in reproducing observed fluxes and concentration gradients during the day and at night, but revealed problems during transitions at sunrise and sunset that appear to be related to the canopy radiation parameterization in SiB2.

Private climate change reporting: an emerging discourse of risk and opportunity?

Purpose – This paper aims to explore the nature of the emerging discourse of private climate change reporting, which takes place in one-on-one meetings between institutional investors and their investee companies. Design/methodology/approach – Semi-structured interviews were conducted with representatives from 20 UK investment institutions to derive data which was then coded and analysed, in order to derive a picture of the emerging discourse of private climate change reporting, using an interpretive methodological approach, in addition to explorative analysis using NVivo software. Findings – The authors find that private climate change reporting is dominated by a discourse of risk and risk management. This emerging risk discourse derives from institutional investors' belief that climate change represents a material risk, that it is the most salient sustainability issue, and that their clients require them to manage climate change-related risk within their portfolio investment. It is found that institutional investors are using the private reporting process to compensate for the acknowledged inadequacies of public climate change reporting. Contrary to evidence indicating corporate capture of public sustainability reporting, these findings suggest that the emerging private climate change reporting discourse is being captured by the institutional investment community. There is also evidence of an emerging discourse of opportunity in private climate change reporting as the institutional investors are increasingly aware of a range of ways in which climate change presents material opportunities for their investee companies to exploit. Lastly, the authors find an absence of any ethical discourse, such that private climate change reporting reinforces rather than challenges the “business case” status quo. Originality/value – Although there is a wealth of sustainability reporting research, there is no academic research on private climate change reporting. This paper attempts to fill this gap by providing rich interview evidence regarding the nature of the emerging private climate change reporting discourse.

Estimation of optimal gravity wave parameters for climate models using data assimilation

There is a current need to constrain the parameters of gravity wave drag (GWD) schemes in climate models using observational information instead of tuning them subjectively. In this work, an inverse technique is developed using data assimilation principles to estimate gravity wave parameters. Because mostGWDschemes assume instantaneous vertical propagation of gravity waves within a column, observations in a single column can be used to formulate a one-dimensional assimilation problem to estimate the unknown parameters. We define a cost function that measures the differences between the unresolved drag inferred from observations (referred to here as the ‘observed’ GWD) and the GWD calculated with a parametrisation scheme. The geometry of the cost function presents some difficulties, including multiple minima and ill-conditioning because of the non-independence of the gravity wave parameters. To overcome these difficulties we propose a genetic algorithm to minimize the cost function, which provides a robust parameter estimation over a broad range of prescribed ‘true’ parameters. When real experiments using an independent estimate of the ‘observed’ GWD are performed, physically unrealistic values of the parameters can result due to the non-independence of the parameters. However, by constraining one of the parameters to lie within a physically realistic range, this degeneracy is broken and the other parameters are also found to lie within physically realistic ranges. This argues for the essential physical self-consistency of the gravity wave scheme. A much better fit to the observed GWD at high latitudes is obtained when the parameters are allowed to vary with latitude. However, a close fit can be obtained either in the upper or the lower part of the profiles, but not in both at the same time. This result is a consequence of assuming an isotropic launch spectrum. The changes of sign in theGWDfound in the tropical lower stratosphere, which are associated with part of the quasi-biennial oscillation forcing, cannot be captured by the parametrisation with optimal parameters.

Generic strategies and performance: evidence from manufacturing firms

Purpose – The purpose of this study is to examine the relationship between business-level strategy and organisational performance and to test the applicability of Porter's generic strategies in explaining differences in the performance of organisations. Design/methodology/approach – The study was focussed on manufacturing firms in the UK belonging to the electrical and mechanical engineering sectors. Data were collected through a postal survey using the survey instrument from 124 organisations and the respondents were all at CEO level. Both objective and subjective measures were used to assess performance. Non-response bias was assessed statistically and it was not found to be a major problem affecting this study. Appropriate measures were taken to ensure that common method variance (CMV) does not affect the results of this study. Statistical tests indicated that CMV problem does not affect the results of this study. Findings – The results of this study indicate that firms adopting one of the strategies, namely cost-leadership or differentiation, perform better than “stuck-in-the-middle” firms which do not have a dominant strategic orientation. The integrated strategy group has lower performance compared with cost-leaders and differentiators in terms of financial performance measures. This provides support for Porter's view that combination strategies are unlikely to be effective in organisations. However, the cost-leadership and differentiation strategies were not strongly correlated with the financial performance measures indicating the limitations of Porter's generic strategies in explaining performance heterogeneity in organisations. Originality/value – This study makes an important contribution to the literature by identifying some of the gaps in the literature through a systematic literature review and addressing those gaps.

Using QR codes to aid accessibility in a museum

Purpose – This paper describes visitors' reactions to using an Apple iPad or smartphone to follow trails in a museum by scanning QR codes and draws conclusions on the potential for this technology to help improve accessibility at low-cost. Design/methodology/approach – Activities were devised which involved visitors following trails around museum objects, each labelled with a QR code and symbolised text. Visitors scanned the QR codes using a mobile device which then showed more information about an object. Project-team members acted as participant-observers, engaging with visitors and noting how they used the system. Experiences from each activity fed into the design of the next. Findings – Some physical and technical problems with using QR codes can be overcome with the introduction of simple aids, particularly using movable object labels. A layered approach to information access is possible with the first layer comprising a label, the second a mobile-web enabled screen and the third choices of text, pictures, video and audio. Video was especially appealing to young people. The ability to repeatedly watch video or listen to audio seemed to be appreciated by visitors with learning disabilities. This approach can have low equipment-cost. However, maintaining the information behind labels and keeping-up with technological changes are on-going processes. Originality/value – Using QR codes on movable, symbolised object labels as part of a layered information system might help modestly-funded museums enhance their accessibility, particularly as visitors increasingly arrive with their own smartphones or tablets.

Comparative genomics suggests that an ancestral polyploidy event leads to enhanced root nodule symbiosis in the Papilionoideae

Root nodule symbiosis (RNS) is one of the most efficient biological systems for nitrogen fixation and it occurs in 90% of genera in the Papilionoideae, the largest subfamily of legumes. Most papilionoid species show evidence of a polyploidy event occurred approximately 58 million years ago. Although polyploidy is considered to be an important evolutionary force in plants, the role of this papilionoid polyploidy event, especially its association with RNS, is not understood. In this study, we explored this role using an integrated comparative genomic approach and conducted gene expression comparisons and gene ontology enrichment analyses. The results show the following: (1) approximately a quarter of the papilionoid-polyploidy-derived duplicate genes are retained; (2) there is a striking divergence in the level of expression of gene duplicate pairs derived from the polyploidy event; and (3) the retained duplicates are frequently involved in the processes crucial for RNS establishment, such as symbiotic signalling, nodule organogenesis, rhizobial infection and nutrient exchange and transport. Thus, we conclude that the papilionoid polyploidy event might have further refined RNS and induced a more robust and enhanced symbiotic system. This conclusion partly explains the widespread occurrence of the Papilionoideae.

An easily implemented agro-hydrological procedure with dynamic root simulation for water transfer in the crop-soil system: validation and application

Models for water transfer in the crop-soil system are key components of agro-hydrological models for irrigation, fertilizer and pesticide practices. Many of the hydrological models for water transfer in the crop-soil system are either too approximate due to oversimplified algorithms or employ complex numerical schemes. In this paper we developed a simple and sufficiently accurate algorithm which can be easily adopted in agro-hydrological models for the simulation of water dynamics. We used a dual crop coefficient approach proposed by the FAO for estimating potential evaporation and transpiration, and a dynamic model for calculating relative root length distribution on a daily basis. In a small time step of 0.001 d, we implemented algorithms separately for actual evaporation, root water uptake and soil water content redistribution by decoupling these processes. The Richards equation describing soil water movement was solved using an integration strategy over the soil layers instead of complex numerical schemes. This drastically simplified the procedures of modeling soil water and led to much shorter computer codes. The validity of the proposed model was tested against data from field experiments on two contrasting soils cropped with wheat. Good agreement was achieved between measurement and simulation of soil water content in various depths collected at intervals during crop growth. This indicates that the model is satisfactory in simulating water transfer in the crop-soil system, and therefore can reliably be adopted in agro-hydrological models. Finally we demonstrated how the developed model could be used to study the effect of changes in the environment such as lowering the groundwater table caused by the construction of a motorway on crop transpiration. (c) 2009 Elsevier B.V. All rights reserved.

First human hNT neurons patterned on parylene-C/silicon dioxide substrates: Combining an accessible cell line and robust patterning technology for the study of the pathological adult human brain

In this communication, we describe a new method which has enabled the first patterning of human neurons (derived from the human teratocarcinoma cell line (hNT)) on parylene-C/silicon dioxide substrates. We reveal the details of the nanofabrication processes, cell differentiation and culturing protocols necessary to successfully pattern hNT neurons which are each key aspects of this new method. The benefits in patterning human neurons on silicon chip using an accessible cell line and robust patterning technology are of widespread value. Thus, using a combined technology such as this will facilitate the detailed study of the pathological human brain at both the single cell and network level.

Attempts to control pain prioritize attention to signals of pain: an experimental study

Clinical evidence suggests that a persistent search for solutions for chronic pain may bring along costs at the cognitive, affective, and behavioral level. Specifically, attempts to control pain may fuel hypervigilance and prioritize attention towards pain-related information. This hypothesis was investigated in an experiment with 41 healthy volunteers. Prioritization of attention towards a signal for pain was measured using an adaptation of a visual search paradigm in which participants had to search for a target presented in a varying number of colored circles. One of these colors (Conditioned Stimulus) became a signal for pain (Unconditioned Stimulus: electrocutaneous stimulus at tolerance level) using a classical conditioning procedure. Intermixed with the visual search task, participants also performed another task. In the pain-control group, participants were informed that correct and fast responses on trials of this second task would result in an avoidance of the Unconditioned Stimulus. In the comparison group, performance on the second task was not instrumental in controlling pain. Results showed that in the pain-control group, attention was more prioritized towards the Conditioned Stimulus than in the comparison group. The theoretical and clinical implications of these results are discussed.

Building the niche through time: using 13,000 years of data to predict the effects of climate change on three tree species in Europe

Aim  Species distribution models (SDMs) based on current species ranges underestimate the potential distribution when projected in time and/or space. A multi-temporal model calibration approach has been suggested as an alternative, and we evaluate this using 13,000 years of data. Location  Europe. Methods  We used fossil-based records of presence for Picea abies, Abies alba and Fagus sylvatica and six climatic variables for the period 13,000 to 1000 yr bp. To measure the contribution of each 1000-year time step to the total niche of each species (the niche measured by pooling all the data), we employed a principal components analysis (PCA) calibrated with data over the entire range of possible climates. Then we projected both the total niche and the partial niches from single time frames into the PCA space, and tested if the partial niches were more similar to the total niche than random. Using an ensemble forecasting approach, we calibrated SDMs for each time frame and for the pooled database. We projected each model to current climate and evaluated the results against current pollen data. We also projected all models into the future. Results  Niche similarity between the partial and the total-SDMs was almost always statistically significant and increased through time. SDMs calibrated from single time frames gave different results when projected to current climate, providing evidence of a change in the species realized niches through time. Moreover, they predicted limited climate suitability when compared with the total-SDMs. The same results were obtained when projected to future climates. Main conclusions  The realized climatic niche of species differed for current and future climates when SDMs were calibrated considering different past climates. Building the niche as an ensemble through time represents a way forward to a better understanding of a species' range and its ecology in a changing climate.

Towards radiocarbon calibration beyond 28ka using speleothems from the Bahamas

We present a new speleothem record of atmospheric Δ14C between 28 and 44 ka that offers considerable promise for resolving some of the uncertainty associated with existing radiocarbon calibration curves for this time period. The record is based on a comprehensive suite of AMS 14C ages, using new low-blank protocols, and U–Th ages using high precision MC-ICPMS procedures. Atmospheric Δ14C was calculated by correcting 14C ages with a constant dead carbon fraction (DCF) of 22.7 ± 5.9%, based on a comparison of stalagmite 14C ages with the IntCal04 (Reimer et al., 2004) calibration curve between 15 and 11 ka. The new Δ14C speleothem record shows similar structure and amplitude to that derived from Cariaco Basin foraminifera (Hughen et al., 2004, 2006), and the match is further improved if the latter is tied to the most recent Greenland ice core chronology (Svensson et al., 2008). These data are however in conflict with a previously published 14C data set for a stalagmite record from the Bahamas — GB-89-24-1 (Beck et al., 2001), which likely suffered from 14C analytical blank subtraction issues in the older part of the record. The new Bahamas speleothem ∆14C data do not show the extreme shifts between 44 and 40 ka reported in the previous study (Beck et al., 2001). Causes for the observed structure in derived atmospheric Δ14C variation based on the new speleothem data are investigated with a suite of simulations using an earth system model of intermediate complexity. Data-model comparison indicates that major fluctuations in atmospheric ∆14C during marine isotope stage 3 is primarily a function of changes in geomagnetic field intensity, although ocean–atmosphere system reorganisation also played a supporting role.