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.

DNA barcoding of a large genus, Aspalathus L. (Fabaceae)

The DNA barcode potential of three regions (the nuclear ribosomal ITS and the plastid psbA-trnH and trnT-trnL intergenic spacers) was investigated for the plant genus Aspalathus L. (Fabaceac: Crotalarieae). Aspalathus is a large genus (278 species) that revealed low levels of DNA variation in phylogenetic studies. In a 51-species dataset for the psbA-trnH and ITS regions, 45%, and 16% of sequences respectively were identical to the sequence of at least one other species, with two species undiscriminated even when the two regions were combined. In contrast, trnT-trnL, discriminated between all species in this dataset. In a larger ITS and trnT-trnL dataset. including a further 82 species. 7 species in five pairwise comparisons remained Undiscriminated when the two regions were combined. Four of the five pairs of species not discriminated by sequence data were readily distinguished using a combination of qualitative and quantitative morphological data. The difficulty of barcoding in this group is increased by the presence of intraspecific variation in all three regions studied. In the case of psbA-trnH, three intraspecific samples had a sequence identical to at least one other species. Overall, psbA-trnH. currently a candidate for plant barcoding, was the least discriminatory region in our study.

Chemical characterisation of wild populations of Thymus from different climatic regions in southeast Spain

Thymus is taxonomically a very complex genus with a high frequency of hybridisation and introgression among sympatric species. The variation in accumulation of leaf-surface flavonoids was investigated in 71 wild populations of Thymus front different putative hybrid swarm areas in Andalucia, Spain. Twenty-two flavones, five flavanones, two dihydroflavonols, a flavonol and two unknowns were detected by HPLC-DAD combined with LC-APCI-MS analysis. The majority of compounds were flavones with a lutelin-type substitution of the B-ring, in contrast to previous reports on Macedonian taxa, which predominantly accumulate flavones with apigenin-type substitution of the B-ring. Anatomical and morphometric studies, supported by cluster analysis, identified pure Thymus hyemalis and Thymus baeticus populations, and a large number of putative hybrids. Flavonoid variation was closely related to morphological variation in all populations and is suspected to be a result of genetic polymorphism. Principal component analysis identified the presence of species-specific and geographically linked chemotypes and putative hybrids with mixed morphological and chemical characteristics. Qualitative and quantitative flavonoid accumulation appears to be genetically regulated, while external factors play a secondary role. Flavonoid profiles can thus provide diagnostic markers for the taxonomy of Thymus and are also useful in detecting hybridising taxa. (C) 2007 Elsevier Ltd. All rights reserved.

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.

Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of post mortem miscoding lesions

Ancient DNA (aDNA) research has long depended on the power of PCR to amplify trace amounts of surviving genetic material from preserved specimens. While PCR permits specific loci to be targeted and amplified, in many ways it can be intrinsically unsuited to damaged and degraded aDNA templates. PCR amplification of aDNA can produce highly-skewed distributions with significant contributions from miscoding lesion damage and non-authentic sequence artefacts. As traditional PCR-based approaches have been unable to fully resolve the molecular nature of aDNA damage over many years, we have developed a novel single primer extension (SPEX)-based approach to generate more accurate sequence information. SPEX targets selected template strands at defined loci and can generate a quantifiable redundancy of coverage; providing new insights into the molecular nature of aDNA damage and fragmentation. SPEX sequence data reveals inherent limitations in both traditional and metagenomic PCR-based approaches to aDNA, which can make current damage analyses and correct genotyping of ancient specimens problematic. In contrast to previous aDNA studies, SPEX provides strong quantitative evidence that C U-type base modifications are the sole cause of authentic endogenous damage-derived miscoding lesions. This new approach could allow ancient specimens to be genotyped with unprecedented accuracy.

Attitudes of landscape professionals towards naturalistic versus formal urban landscapes in the UK

The last 30 years have seen a tide of interest sweeping across Europe in the development of nature in cities, and an increasing amount of landscape development in urban areas has involved the use of 'naturalistic' styles. This is an increasing attempt to find ways for urbanism and nature to co-exist. However, there have been considerable discussions among professionals regarding the advantages and disadvantages of 'naturalistic' styles in urban areas. This research examines professional attitudes to 'naturalistic' landscape styles in Britain, in contrast to more traditional, formal landscape styles, and aims to find out whether the interest in natural landscapes is really a fashion among landscape professionals. A self-administered postal survey was carried out using both quantitative and qualitative data collection techniques and analysis. The survey included 500 professionals from parks and recreation departments of local authorities, private landscape practices and conservation trusts, and resulted in a satisfactory response rate of 53 %. The results of this study suggested that professionals recognise most of the values attached to naturalistic landscapes in urban areas. However, possible benefits that natural areas may have for urban people are not attached to naturalistic landscapes alone. The study also revealed that the naturalistic style is highly popular among conservation trusts but is less so among professionals from local authorities and private landscape practices who seem to appreciate both styles and believe that these styles are not separable from each other and should co-exist in an urban environment. (C) 2006 Elsevier B.V. All rights reserved.

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.

Variation in pituitary expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and type-I and type-II activin receptors during the chicken ovulatory cycle

Secretion of LH and FSH from the anterior pituitary is regulated primarily by hypothalamic GnRH and ovarian steroid hormones. More recent evidence indicates regulatory roles for certain members of the transforming growth factor beta (TGF beta) superfamily including inhibin and activin. The aim of this study was to identify expression of mRNAs encoding key receptors and ligands of the inhibin/activin system in the hen pituitary gland and to monitor their expression throughout the 24-25-h ovulatory cycle. Hens maintained on long days (16 h light/8 h dark) were killed 20, 12, 6 and 2 h before predicted ovulation of a midsequence egg (n = 8 per group). Anterior pituitary glands were removed, RNA extracted and cDNA synthesized. Plasma concentrations of LH, FSH, progesterone and inhibin A were measured. Real-time quantitative PCR was used to quantify pituitary expression of mRNAs encoding betaglycan, activin receptor (ActR) subtypes (type I, IIA), GnRH receptor (GnP,H-R), LH beta subunit, FSH beta subunit and GAPDH. Levels of mRNA for inhibin/activin beta A and beta B subunits, inhibin alpha subunit, follistatin and ActRIIB mRNA in pituitary were undetectable by quantitative PCR (< 2 amol/reaction). Significant changes in expression (P < 0.05) of ActRIIA and betaglycan mRNA were found, both peaking 6 h before ovulation just prior to the preovulatory LH surge and reaching a nadir 2 h before ovulation, just after the LH surge. There were no significant changes in expression of ActRI mRNA throughout the cycle although values were correlated with mRNA levels for both ActRIIA (r=0.77; P < 0.001) and betaglycan (r=0.45; P < 0.01). Expression of GnRH-R mRNA was lowest 20 h before ovulation and highest (P < 0.05) 6 h before ovulation; values were weakly correlated with betaglycan (r=0.33; P=0.06) and ActRIIA (r=0.34; P=0.06) mRNA levels. Expression of mRNAs encoding LH beta and FSH beta subunit were both lowest (P < 0.05) after the LH surge, 2 h before ovulation. These results are consistent with an endocrine, but not a local intrapituitary, role of inhibin-related proteins in modulating gonadotroph function during the ovulatory cycle of the hen, potentially through interaction with betaglycan and ActRIIA. In contrast to mammals, intrapituitary expression of inhibin/activin subunits and follistatin appears to be extremely low or absent in the domestic fowl.

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.

The adrenal secretory serine protease AsP is a short secretory isoform of the transmembrane airway trypsin-like protease

To further elucidate the role of proteases capable of cleaving N-terminal proopiomelanocortin (N-POMC)-derived peptides, we have cloned two cDNAs encoding isoforms of the airway trypsin-like protease (AT) from mouse (MAT) and rat ( RAT), respectively. The open reading frames comprise 417 amino acids (aa) and 279 aa. The mouse AT gene was located at chromosome 5E1 and contains 10 exons. The longer isoform, which we designated MAT1 and RAT1, has a simple type II transmembrane protein structure, consisting of a short cytoplasmic domain, a transmembrane domain, a SEA (63-kDa sea urchin sperm protein, enteropeptidase, agrin) module, and a serine protease domain. The human homolog of MAT1 and RAT1 is the human AT ( HAT). The shorter isoform, designated MAT2 and RAT2, which contains an alternative N terminus, was formerly described in the rat as adrenal secretory serine protease (AsP) and has been shown to be involved in the processing of N-POMC-derived peptides. In contrast to the long isoform, neither MAT2 and RAT2 ( AsP) contain a transmembrane domain nor a SEA domain but an N-terminal signal peptide to direct the enzyme to the secretory pathway. The C terminus, covering the catalytic triad, is identical in both isoforms. Immunohistochemically, MAT/RAT was predominantly expressed in tissues of the upper gastrointestinal and the respiratory tract - but also in the adrenal gland. Moreover, isoform-specific RT-PCR and quantitative PCR analysis revealed a complex expression pattern of the two isoforms with differences between mice and rats. These findings indicate a multifunctional role of these proteases beyond adrenal proliferation.

Managing threatened species: the ecological toolbox, evolutionary theory and declining-population paradigm

1. The management of threatened species is an important practical way in which conservationists can intervene in the extinction process and reduce the loss of biodiversity. Understanding the causes of population declines (past, present and future) is pivotal to designing effective practical management. This is the declining-population paradigm identified by Caughley. 2. There are three broad classes of ecological tool used by conservationists to guide management decisions for threatened species: statistical models of habitat use, demographic models and behaviour-based models. Each of these is described here, illustrated with a case study and evaluated critically in terms of its practical application. 3. These tools are fundamentally different. Statistical models of habitat use and demographic models both use descriptions of patterns in abundance and demography, in relation to a range of factors, to inform management decisions. In contrast, behaviour-based models describe the evolutionary processes underlying these patterns, and derive such patterns from the strategies employed by individuals when competing for resources under a specific set of environmental conditions. 4. Statistical models of habitat use and demographic models have been used successfully to make management recommendations for declining populations. To do this, assumptions are made about population growth or vital rates that will apply when environmental conditions are restored, based on either past data collected under favourable environmental conditions or estimates of these parameters when the agent of decline is removed. As a result, they can only be used to make reliable quantitative predictions about future environments when a comparable environment has been experienced by the population of interest in the past. 5. Many future changes in the environment driven by management will not have been experienced by a population in the past. Under these circumstances, vital rates and their relationship with population density will change in the future in a way that is not predictable from past patterns. Reliable quantitative predictions about population-level responses then need to be based on an explicit consideration of the evolutionary processes operating at the individual level. 6. Synthesis and applications. It is argued that evolutionary theory underpins Caughley's declining-population paradigm, and that it needs to become much more widely used within mainstream conservation biology. This will help conservationists examine critically the reliability of the tools they have traditionally used to aid management decision-making. It will also give them access to alternative tools, particularly when predictions are required for changes in the environment that have not been experienced by a population in the past.

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.