Quantification of physical and biological uncertainty in the simulation of the yield of a tropical crop using present-day and doubled CO2 climates

The impacts of climate change on crop productivity are often assessed using simulations from a numerical climate model as an input to a crop simulation model. The precision of these predictions reflects the uncertainty in both models. We examined how uncertainty in a climate (HadAM3) and crop General Large-Area Model (GLAM) for annual crops model affects the mean and standard deviation of crop yield simulations in present and doubled carbon dioxide (CO2) climates by perturbation of parameters in each model. The climate sensitivity parameter (lambda, the equilibrium response of global mean surface temperature to doubled CO2) was used to define the control climate. Observed 1966-1989 mean yields of groundnut (Arachis hypogaea L.) in India were simulated well by the crop model using the control climate and climates with values of lambda near the control value. The simulations were used to measure the contribution to uncertainty of key crop and climate model parameters. The standard deviation of yield was more affected by perturbation of climate parameters than crop model parameters in both the present-day and doubled CO2 climates. Climate uncertainty was higher in the doubled CO2 climate than in the present-day climate. Crop transpiration efficiency was key to crop model uncertainty in both present-day and doubled CO2 climates. The response of crop development to mean temperature contributed little uncertainty in the present-day simulations but was among the largest contributors under doubled CO2. The ensemble methods used here to quantify physical and biological uncertainty offer a method to improve model estimates of the impacts of climate change.

Quantification of ruminal Clostridium proteoclasticum by real-time PCR using a molecular beacon approach

Aims: All members of the ruminal Butyrivibrio group convert linoleic acid (cis-9,cis-12-18 : 2) via conjugated 18 : 2 metabolites (mainly cis-9,trans-11-18 : 2, conjugated linoleic acid) to vaccenic acid (trans-11-18 : 1), but only members of a small branch, which includes Clostridium proteoclasticum, of this heterogeneous group further reduce vaccenic acid to stearic acid (18 : 0, SA). The aims of this study were to develop a real-time polymerase chain reaction (PCR) assay that would detect and quantify these key SA producers and to use this method to detect diet-associated changes in their populations in ruminal digesta of lactating cows. Materials and Results: The use of primers targeting the 16S rRNA gene of Cl. proteoclasticum was not sufficiently specific when only binding dyes were used for detection in real-time PCR. Their sequences were too similar to some nonproducing strains. A molecular beacon probe was designed specifically to detect and quantify the 16S rRNA genes of the Cl. proteoclasticum subgroup. The probe was characterized by its melting curve and validated using five SA-producing and ten nonproducing Butyrivibrio-like strains and 13 other common ruminal bacteria. Analysis of ruminal digesta collected from dairy cows fed different proportions of starch and fibre indicated a Cl. proteoclasticum population of 2-9% of the eubacterial community. The influence of diet on numbers of these bacteria was less than variations between individual cows. Conclusion: A molecular beacon approach in qPCR enables the detection of Cl. proteoclasticum in ruminal digesta. Their numbers are highly variable between individual animals. Signifance and Impact of the Study: SA producers are fundamental to the flow of polyunsaturated fatty acid and vaccenic acid from the rumen. The method described here enabled preliminary information to be obtained about the size of this population. Further application of the method to digesta samples from cows fed diets of more variable composition should enable us to understand how to control these bacteria in order to enhance the nutritional characteristics of ruminant-derived foods, including milk and beef.

Inter-laboratory variation of in vitro cumulative gas production profiles of feeds using manual and automated methods

A study was conducted to estimate variation among laboratories and between manual and automated techniques of measuring pressure on the resulting gas production profiles (GPP). Eight feeds (molassed sugarbeet feed, grass silage, maize silage, soyabean hulls, maize gluten feed, whole crop wheat silage, wheat, glucose) were milled to pass a I mm screen and sent to three laboratories (ADAS Nutritional Sciences Research Unit, UK; Institute of Grassland and Environmental Research (IGER), UK; Wageningen University, The Netherlands). Each laboratory measured GPP over 144 h using standardised procedures with manual pressure transducers (MPT) and automated pressure systems (APS). The APS at ADAS used a pressure transducer and bottles in a shaking water bath, while the APS at Wageningen and IGER used a pressure sensor and bottles held in a stationary rack. Apparent dry matter degradability (ADDM) was estimated at the end of the incubation. GPP were fitted to a modified Michaelis-Menten model assuming a single phase of gas production, and GPP were described in terms of the asymptotic volume of gas produced (A), the time to half A (B), the time of maximum gas production rate (t(RM) (gas)) and maximum gas production rate (R-M (gas)). There were effects (P<0.001) of substrate on all parameters. However, MPT produced more (P<0.001) gas, but with longer (P<0.001) B and t(RM gas) (P<0.05) and lower (P<0.001) R-M gas compared to APS. There was no difference between apparatus in ADDM estimates. Interactions occurred between substrate and apparatus, substrate and laboratory, and laboratory and apparatus. However, when mean values for MPT were regressed from the individual laboratories, relationships were good (i.e., adjusted R-2 = 0.827 or higher). Good relationships were also observed with APS, although they were weaker than for MPT (i.e., adjusted R-2 = 0.723 or higher). The relationships between mean MPT and mean APS data were also good (i.e., adjusted R 2 = 0. 844 or higher). Data suggest that, although laboratory and method of measuring pressure are sources of variation in GPP estimation, it should be possible using appropriate mathematical models to standardise data among laboratories so that data from one laboratory could be extrapolated to others. This would allow development of a database of GPP data from many diverse feeds. (c) 2005 Published by Elsevier B.V.

Agonist-dependent internalization of D2 receptors: imaging quantification by confocal microscopy

In positron emission tomography and single photon emission computed tomography studies using D2 dopamine (DA) receptor radiotracers, a decrease in radiotracer binding potential (BP) is usually interpreted in terms of increased competition with synaptic DA. However, some data suggest that this signal may also reflect agonist (DA)-induced increases in D2 receptor (D2R) internalization, a process which would presumably also decrease the population of receptors available for binding to hydrophilic radioligands. To advance interpretation of alterations in D2 radiotracer BP, direct methods of assessment of D2R internalization are required. Here, we describe a confocal microscopy-based approach for the quantification of agonist-dependent receptor internalization. The method relies upon double-labeling of the receptors with antibodies directed against intracellular as well as extracellular epitopes. Following agonist stimulation, DA D2R internalization was quantified by differentiating, in optical cell sections, the signal due to the staining of the extracellular from intracellular epitopes of D2Rs. Receptor internalization was increased in the presence of the D2 agonists DA and bromocriptine, but not the D1 agonist SKF38393. Pretreatment with either the D2 antagonist sulpiride, or inhibitors of internalization (phenylarsine oxide and high molarity sucrose), blocked D2-agonist induced receptor internalization, thus validating this method in vitro. This approach therefore provides a direct and streamlined methodology for investigating the pharmacological and mechanistic aspects of D2R internalization, and should inform the interpretation of results from in vivo receptor imaging studies.

An extensible automated protein annotation tool: standardizing input and output using validated XML

Motivation: There is a frequent need to apply a large range of local or remote prediction and annotation tools to one or more sequences. We have created a tool able to dispatch one or more sequences to assorted services by defining a consistent XML format for data and annotations. Results: By analyzing annotation tools, we have determined that annotations can be described using one or more of the six forms of data: numeric or textual annotation of residues, domains (residue ranges) or whole sequences. With this in mind, XML DTDs have been designed to store the input and output of any server. Plug-in wrappers to a number of services have been written which are called from a master script. The resulting APATML is then formatted for display in HTML. Alternatively further tools may be written to perform post-analysis.

Liquid ultraviolet matrix-assisted laser desorption/ionization - mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet -- matrix-assisted laser desorption/ionisation -- mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. The low-femtomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydroxybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and low-mass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.

Seasonal 'fall out' of sessile macro-fauna from submarine cliffs: quantification, causes and implications

Submarine cliffs are typically crowded with sessile organisms, most of which are ultimately exported downwards. Here we report a 24 month study of benthic fauna dropping from such cliffs at sites of differing cliff angle and flow rates at Lough Hyne Marine Nature Reserve, Co. Cork, Ireland. The magnitude of 'fall out' material collected in capture nets was highly seasonal and composed of sessile and mobile elements. Sponges, ascidians, cnidarians, polychaetes, bryozoans and barnacles dominated the sessile forms. The remainder (mobile fauna) were scavengers and predators such as asteroid echinoderms, gastropod molluscs and malacostracan crustaceans. These were probably migrants targeting fallen sessile organisms. 'Fall out' material (including mobile forms) increased between May and August in both years. This increase in 'fall out' material was correlated with wrasse abundance at the cliffs (with a one month lag period). The activities of the wrasse on the cliffs (feeding, nest building and territory defence) were considered responsible for the majority of 'fall out' material, with natural mortality and the activity of other large mobile organisms (e.g. crustaceans) also being triplicated. Current flow rate and cliff profile were important in amount of 'fall out' material collected. In low current situations export of fallen material was vertical, while both horizontal and vertical export was associated with moderate to high current environments. Higher 'fall out' was associated with overhanging than vertical cliff surfaces. The 'fall out' of marine organisms in low current situations is likely to provide ail important source of nutrition in close proximity to the cliff, in an otherwise impoverished soft sediment habitat. However, in high current areas material will be exported some distance from the source, with final settlement again occurring in soft sediment habitats (as current speed decreases).

Liquid ultraviolet matrix-assisted laser desorption/ionization mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet - matrix-assisted laser desorption/ ionisation - mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. U. Am. Soc. Mass Spectrom. 1998, 9, 166-174). The low-ferntomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydrox-ybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and lowmass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.

Proteomic method for the quantification of methionine sulfoxide

Glycoxidation and lipoxidation reactions contribute to the chemical modification of proteins during the Maillard reaction. Reactive oxygen species, produced during the oxidation of sugars and lipids in these processes, irreversibly oxidize proteins. Methionine is particularly susceptible to oxidation, yielding the oxidation product methionine sulfoxide (MetSO). Here we describe a method for the analysis of MetSO using proteomic techniques. Using these techniques, we measured MetSO formation on the model protein RNase during aerobic incubations with glucose and arachidonate. We also evaluated the susceptibility of MetSO to reduction by NaBH4, a commonly used reductant in the analysis of Maillard reaction products.

Agonist-dependent internalization of D2 receptors: Imaging quantification by confocal microscopy

In positron emission tomography and single photon emission computed tomography studies using D2 dopamine (DA) receptor radiotracers, a decrease in radiotracer binding potential (BP) is usually interpreted in terms of increased competition with synaptic DA. However, some data suggest that this signal may also reflect agonist (DA)-induced increases in D2 receptor (D2R) internalization, a process which would presumably also decrease the population of receptors available for binding to hydrophilic radioligands. To advance interpretation of alterations in D2 radiotracer BP, direct methods of assessment of D2R internalization are required. Here, we describe a confocal microscopy-based approach for the quantification of agonist-dependent receptor internalization. The method relies upon double-labeling of the receptors with antibodies directed against intracellular as well as extracellular epitopes. Following agonist stimulation, DA D2R internalization was quantified by differentiating, in optical cell sections, the signal due to the staining of the extracellular from intracellular epitopes of D2Rs. Receptor internalization was increased in the presence of the D2 agonists DA and bromocriptine, but not the D1 agonist SKF38393. Pretreatment with either the D2 antagonist sulpiride, or inhibitors of internalization (phenylarsine oxide and high molarity sucrose), blocked D2-agonist induced receptor internalization, thus validating this method in vitro. This approach therefore provides a direct and streamlined methodology for investigating the pharmacological and mechanistic aspects of D2R internalization, and should inform the interpretation of results from in vivo receptor imaging studies.

An automated calibration method for non-see-through head mounted displays

Accurate calibration of a head mounted display (HMD) is essential both for research on the visual system and for realistic interaction with virtual objects. Yet, existing calibration methods are time consuming and depend on human judgements, making them error prone. The methods are also limited to optical see-through HMDs. Building on our existing HMD calibration method [1], we show here how it is possible to calibrate a non-see-through HMD. A camera is placed inside an HMD displaying an image of a regular grid, which is captured by the camera. The HMD is then removed and the camera, which remains fixed in position, is used to capture images of a tracked calibration object in various positions. The locations of image features on the calibration object are then re-expressed in relation to the HMD grid. This allows established camera calibration techniques to be used to recover estimates of the display’s intrinsic parameters (width, height, focal length) and extrinsic parameters (optic centre and orientation of the principal ray). We calibrated a HMD in this manner in both see-through and in non-see-through modes and report the magnitude of the errors between real image features and reprojected features. Our calibration method produces low reprojection errors and involves no error-prone human measurements.