A functional genomics approach reveals novel quantitative trait loci associated with platelet signalling pathways

Platelet response to activation varies widely between individuals but shows interindividual consistency and strong heritability. The genetic basis of this variation has not been properly explored. We therefore systematically measured the effect on function of sequence variation in 97 candidate genes in the collagen and adenosine-diphosphate (ADP) signaling pathways. Resequencing of the genes in 48 European DNA samples nearly doubled the number of known single nucleotide polymorphisms (SNPs) and informed the selection of 1327 SNPs for genotyping in 500 healthy Northern European subjects with known platelet responses to collagen-related peptide (CRP-XL) and ADP. This identified 17 novel associations with platelet function (P < .005) accounting for approximately 46% of the variation in response. Further investigations with platelets of known genotype explored the mechanisms behind some of the associations. SNPs in PEAR1 associated with increased platelet response to CRP-XL and increased PEAR1 protein expression after platelet degranulation. The minor allele of a 3' untranslated region (UTR) SNP (rs2769668) in VAV3 was associated with higher protein expression (P = .03) and increased P-selectin exposure after ADP activation (P = .004). Furthermore the minor allele of the intronic SNP rs17786144 in ITPR1 modified Ca2+ levels after activation with ADP (P < .004). These data provide novel insights into key hubs within platelet signaling networks.

A simple two-stage design for quantitative responses with application to a study in diabetic neuropathic pain

Two-stage designs offer substantial advantages for early phase II studies. The interim analysis following the first stage allows the study to he stopped for futility, or more positively, it might lead to early progression to the trials needed for late phase H and phase III. If the study is to continue to its second stage, then there is an opportunity for a revision of the total sample size. Two-stage designs have been implemented widely in oncology studies in which there is a single treatment arm and patient responses are binary. In this paper the case of two-arm comparative studies in which responses are quantitative is considered. This setting is common in therapeutic areas other than oncology. It will be assumed that observations are normally distributed, but that there is some doubt concerning their standard deviation, motivating the need for sample size review. The work reported has been motivated by a study in diabetic neuropathic pain, and the development of the design for that trial is described in detail. Copyright (C) 2008 John Wiley & Sons, Ltd.

Heat-shock response in Arabidopsis thaliana explored by multiplexed quantitative proteomics using differential metabolic labeling

We have developed a general method for multiplexed quantitative proteomics using differential metabolic stable isotope labeling and mass spectrometry. The method was successfully used to study the dynamics of heat-shock response in Arabidopsis thaliana. A number of known heat-shock proteins were confirmed, and some proteins not previously associated with heat shock were discovered. The method is applicable in stable isotope labeling and allows for high degrees of multiplexing.

Quantitative proteomics using uniform 15N-labeling, mascot and the trans-proteomic pipeline

Stable isotope labeling combined with MS is a powerful method for measuring relative protein abundances, for instance, by differential metabolic labeling of some or all amino acids with 14N and 15N in cell culture or hydroponic media. These and most other types of quantitative proteomics experiments using high-throughput technologies, such as LC-MS/MS, generate large amounts of raw MS data. This data needs to be processed efficiently and automatically, from the mass spectrometer to statistically evaluated protein identifications and abundance ratios. This paper describes in detail an approach to the automated analysis of uniformly 14N/15N-labeled proteins using MASCOT peptide identification in conjunction with the trans-proteomic pipeline (TPP) and a few scripts to integrate the analysis workflow. Two large proteomic datasets from uniformly labeled Arabidopsis thaliana were used to illustrate the analysis pipeline. The pipeline can be fully automated and uses only common or freely available software.

Reproducibility of a short semi-quantitative food group questionnaire and its performance in estimating nutrient intake compared with a 7-day diet diary in the Million Women Study

Objectives: To assess the short- and long-term reproducibility of a short food group questionnaire, and to compare its performance for estimating nutrient intakes in comparison with a 7-day diet diary. Design: Participants for the reproducibility study completed the food group questionnaire at two time points, up to 2 years apart. Participants for the performance study completed both the food group questionnaire and a 7-day diet diary a few months apart. Reproducibility was assessed by kappa statistics and percentage change between the two questionnaires; performance was assessed by kappa statistics, rank correlations and percentages of participants classified into the same and opposite thirds of intake. Setting: A random sample of participants in the Million Women Study, a population-based prospective study in the UK. Subjects: In total, 12 221 women aged 50-64 years. Results: in the reproducibility study, 75% of the food group items showed at least moderate agreement for all four time-point comparisons. Items showing fair agreement or worse tended to be those where few respondents reported eating them more than once a week, those consumed in small amounts and those relating to types of fat consumed. Compared with the diet diary, the food group questionnaire showed consistently reasonable performance for the nutrients carbohydrate, saturated fat, cholesterol, total sugars, alcohol, fibre, calcium, riboflavin, folate and vitamin C. Conclusions: The short food group questionnaire used in this study has been shown to be reproducible over time and to perform reasonably well for the assessment of a number of dietary nutrients.

Quantitative and qualitative differences in morphological traits revealed between diploid Fragaria species

Background and Aims: The aims of this investigation were to highlight the qualitative and quantitative diversity apparent between nine diploid Fragaria species and produce interspecific populations segregating for a large number of morphological characters suitable for quantitative trait loci analysis. Methods: A qualitative comparison of eight described diploid Fragaria species was performed and measurements were taken of 23 morphological traits from 19 accessions including eight described species and one previously undescribed species. A principal components analysis was performed on 14 mathematically unrelated traits from these accessions, which partitioned the species accessions into distinct morphological groups. Interspecific crosses were performed with accessions of species that displayed significant quantitative divergence and, from these, populations that should segregate for a range of quantitative traits were raised. Key Results: Significant differences between species were observed for all 23 morphological traits quantified and three distinct groups of species accessions were observed after the principal components analysis. Interspecific crosses were performed between these groups, and F2 and backcross populations were raised that should segregate for a range of morphological characters. In addition, the study highlighted a number of distinctive morphological characters in many of the species studied. Conclusions: Diploid Fragaria species are morphologically diverse, yet remain highly interfertile, making the group an ideal model for the study of the genetic basis of phenotypic differences between species through map-based investigation using quantitative trait loci. The segregating interspecific populations raised will be ideal for such investigations and could also provide insights into the nature and extent of genome evolution within this group.

Mapping quantitative trait loci for flag leave senescence as a yield determinant in winter wheat under optimal and drought stressed environments

The timing of flag leaf senescence (FLS) is an important determinant of yield under stress and optimal environments. A doubled haploid population derived from crossing the photo period-sensitive variety Beaver,with the photo period-insensitive variety Soissons, varied significantly for this trait, measured as the percent green flag leaf area remaining at 14 days and 35 days after anthesis. This trait also showed a significantly positive correlation with yield under variable environmental regimes. QTL analysis based on a genetic map derived from 48 doubled haploid lines using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers, revealed the genetic control of this trait. The coincidence of QTL for senescence on chromosomes 2B and 2D under drought-stressed and optimal environments, respectively, indicate a complex genetic mechanism of this trait involving the re-mobilisation of resources from the source to the sink during senescence.

Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study

Hydroponic isotope labelling of entire plants (HILEP) is a cost-effective method enabling metabolic labelling of whole and mature plants with a stable isotope such as N-15. By utilising hydroponic media that contain N-15 inorganic salts as the sole nitrogen source, near to 100% N-15-labelling of proteins can be achieved. In this study, it is shown that HILEP, in combination with mass spectrometry, is suitable for relative protein quantitation of seven week-old Arabidopsis plants submitted to oxidative stress. Protein extracts from pooled N-14- and N-15-hydroponically grown plants were fractionated by SDS-PAGE, digested and analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Proteins were identified and the spectra of N-14/N-15 peptide pairs were extracted using their m/z chromatographic retention time, isotopic distributions, and the m/z difference between the N-14 and N-15 peptides. Relative amounts were calculated as the ratio of the sum of the peak areas of the two distinct N-14 and N-15 peptide isotope envelopes. Using Mascot and the open source trans-proteomic pipeline (TPP), the data processing was automated for global proteome quantitation down to the isoform level by extracting isoform specific peptides. With this combination of metabolic labelling and mass spectrometry it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress. Moreover, it was possible to discriminate between differentially expressed isoforms belonging to the same protein family, such as isoforms of xylanases and pathogen-related glucanases (PR 2). (C) 2008 Elsevier Ltd. All rights reserved.

The adsorption geometry of sulphur on Ir{100}: a quantitative LEED study

A quantitative low energy electron diffraction (LEED) analysis has been performed for the p(2 x 2)-S and c(2 x 2)-S surface structures formed by exposing the (1 x 1) phase of Ir{100} to H2S at 750 K. S is found to adsorb on the fourfold hollow sites in both structures leading to Pendry R-factor values of 0.17 for the p(2 x 2)-S and 0.16 for the c(2 x 2)-S structures. The distances between S and the nearest and next-nearest Ir atoms were found to be similar in both structures: 2.36 +/- 0.01 angstrom and 3.33 +/- 0.01 angstrom, respectively. The buckling in the second substrate layer is consistent with other structural studies for S adsorption on fcc{100} transition metal surfaces: 0.09 angstrom for p(2 x 2)-S and 0.02 angstrom for c(2 x 2)-S structures. The (1 x 5) reconstruction, which is the most stable phase for clean Ir{100}, is completely lifted and a c(2 x 2)-S overlayer is formed after exposure to H,S at 300 K followed by annealing to 520 K. CO temperature-programmed desorption (TPD) experiments indicate that the major factor in the poisoning of Ir by S is site blocking. (c) 2005 Elsevier B.V. All rights reserved.

Quantitative proteomics using uniform N-15-labeling, MASCOT, and the trans-proteomic pipeline

Stable isotope labeling combined with MS is a powerful method for measuring relative protein abundances, for instance, by differential metabolic labeling of some or all amino acids with N-14 and N-15 in cell culture or hydroponic media. These and most other types of quantitative proteomics experiments using high-throughput technologies, such as LC-MS/MS, generate large amounts of raw MS data. This data needs to be processed efficiently and automatically, from the mass spectrometer to statistically evaluated protein identifications and abundance ratios. This paper describes in detail an approach to the automated analysis of Uniformly N-14/N-15-labeled proteins using MASCOT peptide identification in conjunction with the trans-proteomic pipeline (TPP) and a few scripts to integrate the analysis workflow. Two large proteomic datasets from uniformly labeled Arabidopsis thaliana were used to illustrate the analysis pipeline. The pipeline can be fully automated and uses only common or freely available software.

Quantitative model for the kinetics of lyotropic phase transitions involving changes in monolayer curvature

We present a kinetic model for transformations between different self-assembled lipid structures. The model shows how data on the rates of phase transitions between mesophases of different geometries can be used to provide information on the mechanisms of the transformations and the transition states involved. This can be used, for example, to gain an insight into intermediate structures in cell membrane fission or fusion. In cases where the monolayer curvature changes on going from the initial to the final mesophase, we consider the phase transition to be driven primarily by the change in the relaxed curvature with pressure or temperature, which alters the relative curvature elastic energies of the two mesophase structures. Using this model, we have analyzed previously published kinetic data on the inter-conversion of inverse bicontinuous cubic phases in the 1-monoolein-30 wt% water system. The data are for a transition between QII(G) and QII(D) phases, and our analysis indicates that the transition state more closely resembles the QII(D) than the QII(G) phase. Using estimated values for the monolayer mean curvatures of the QII(G) and QII(D) phases of -0.123 nm(-1) and -0.133 nm(-1), respectively, gives values for the monolayer mean curvature of the transition state of between -0.131 nm(-1) and -0.132 nm(-1). Furthermore, we estimate that several thousand molecules undergo the phase transition cooperatively within one "cooperative unit", equivalent to 1-2 unit cells of QII(G) or 4-10 unit cells of QII(D).

Modelling metal cutting using modern ductile fracture mechanics: Quantitative explanations for some longstanding problems

The assumption that negligible work is involved in the formation of new surfaces in the machining of ductile metals, is re-examined in the light of both current Finite Element Method (FEM) simulations of cutting and modern ductile fracture mechanics. The work associated with separation criteria in FEM models is shown to be in the kJ/m2 range rather than the few J/m2 of the surface energy (surface tension) employed by Shaw in his pioneering study of 1954 following which consideration of surface work has been omitted from analyses of metal cutting. The much greater values of surface specific work are not surprising in terms of ductile fracture mechanics where kJ/m2 values of fracture toughness are typical of the ductile metals involved in machining studies. This paper shows that when even the simple Ernst–Merchant analysis is generalised to include significant surface work, many of the experimental observations for which traditional ‘plasticity and friction only’ analyses seem to have no quantitative explanation, are now given meaning. In particular, the primary shear plane angle φ becomes material-dependent. The experimental increase of φ up to a saturated level, as the uncut chip thickness is increased, is predicted. The positive intercepts found in plots of cutting force vs. depth of cut, and in plots of force resolved along the primary shear plane vs. area of shear plane, are shown to be measures of the specific surface work. It is demonstrated that neglect of these intercepts in cutting analyses is the reason why anomalously high values of shear yield stress are derived at those very small uncut chip thicknesses at which the so-called size effect becomes evident. The material toughness/strength ratio, combined with the depth of cut to form a non-dimensional parameter, is shown to control ductile cutting mechanics. The toughness/strength ratio of a given material will change with rate, temperature, and thermomechanical treatment and the influence of such changes, together with changes in depth of cut, on the character of machining is discussed. Strength or hardness alone is insufficient to describe machining. The failure of the Ernst–Merchant theory seems less to do with problems of uniqueness and the validity of minimum work, and more to do with the problem not being properly posed. The new analysis compares favourably and consistently with the wide body of experimental results available in the literature. Why considerable progress in the understanding of metal cutting has been achieved without reference to significant surface work is also discussed.

Quantitative thermal imaging analysis of aircraft materials: through skin sensing (Invited Paper)

Thermal non-destructive testing (NDT) is commonly used for assessing aircraft structures. This research work evaluates the potential of pulsed -- transient thermography for locating fixtures beneath aircraft skins in order to facilitate accurate automated assembly operations. Representative aluminium and carbon fibre aircraft skin-fixture assemblies were modelled using thermal modelling software. The assemblies were also experimentally investigated with an integrated pulsed thermographic evaluation system, as well as using a custom built system incorporating a miniature un-cooled camera. Modelling showed that the presence of an air gap between skin and fixture significantly reduced the thermal contrast developed, especially in aluminium. Experimental results show that fixtures can be located to accuracies of 0.5 mm.

Quantitative comparison between the use of 3D vs 2D visualization tools to present building design proposals to non-spatial skilled end users

Information technology in construction (ITC) has been gaining wide acceptance and is being implemented in the construction research domains as a tool to assist decision makers. Most of the research into visualization technologies (VT) has been on the wide range of 3D and simulation applications suitable for construction processes. Despite its development with interoperability and standardization of products, VT usage has remained very low when it comes to communicating and addressing the needs of building end-users (BEU). This paper argues that building end users are a source of experience and expertise that can be brought into the briefing stage for the evaluation of design proposals. It also suggests that the end user is a source of new ideas promoting innovation. In this research a positivistic methodology that includes the comparison of 3D models and the traditional 2D methods is proposed. It will help to identify "how much", if anything, a non-spatial specialist can gain in terms Of "understanding" of a particular design proposal presented, using both methods.