Selection of candidate coding DNA barcoding regions for use on land plants

An in silico screen of 41 of the 81 coding regions of the Nicotiana plastid genome generated a shortlist of 12 candidates as DNA barcoding loci for land plants. These loci were evaluated for amplification and sequence variation against a reference set of 98 land plant taxa. The deployment of multiple primers and a modified multiplexed tandem polymerase chain reaction yielded 85–94% amplification across taxa, and mean sequence differences between sister taxa of 6.1 from 156 bases of accD to 22 from 493 bases of matK. We conclude that loci should be combined for effective diagnosis, and recommend further investigation of the following six loci: matK, rpoB, rpoC1, ndhJ, ycf5 and accD.

A novel transcriptomic approach to identify candidate genes for grain quality traits in wheat

A novel methodology is described in which transcriptomics is combined with the measurement of bread-making quality and other agronomic traits for wheat genotypes grown in different environments (wet and cool or hot and dry conditions) to identify transcripts associated with these traits. Seven doubled haploid lines from the Spark x Rialto mapping population were selected to be matched for development and known alleles affecting quality. These were grown in polytunnels with different environments applied 14 days post-anthesis, and the whole experiment was repeated over 2 years. Transcriptomics using the wheat Affymetrix chip was carried out on whole caryopsis samples at two stages during grain filling. Transcript abundance was correlated with the traits for approximately 400 transcripts. About 30 of these were selected as being of most interest, and markers were derived from them and mapped using the population. Expression was identified as being under cis control for 11 of these and under trans control for 18. These transcripts are candidates for involvement in the biological processes which underlie genotypic variation in these traits.

Juvenile-mature wood transition in pine: correlation between wood properties and candidate gene expression profiles

There is a strong desire to exploit transcriptomics data from model species for the genetic improvement of non-model crops. Here, we use gene expression profiles from the commercial model Pinus taeda to identify candidate genes implicated in juvenile-mature wood transition in the non-model relative, P. sylvestris. Re-analysis of 'public domain' SAGE data from xylem tissues of P. taeda revealed 283 mature-abundant and 396 juvenile-abundant tags (P < 0.01), of which 70 and 137, respectively matched to genes with known function. Based on sequence similarity, we then isolated 16 putative homologues of genes that in P. taeda exhibited widest divergence in expression between juvenile and mature samples. Candidate expression levels in P. sylvestris were almost invariably differential between juvenile and mature woody tissue samples among two cohorts of five trees collected from the same seed source and selected for genetic uniformity by genetic distance analysis. However, the direction of differential expression was not always consistent with that described in the original P. taeda SAGE data. Correlation was observed between gene expression and juvenile-mature wood anatomical characteristics by OPLS analysis. Four candidates (alpha-tubulin, porin MIP1, lipid transfer protein and aquaporin like protein) apparently had greatest influence on the wood traits measured. Speculative function of these genes in relation to juvenile-mature wood transition is briefly explored. Thus, we demonstrate the feasibility of exploiting SAGE data from a model species to identify consistently differentially expressed candidates in a related non-model species.

Generation of candidate human influenza vaccine strains in cell culture: rehearsing the European response to an H7N1 pandemic threat

Background: Although H5N1 avian influenza viruses pose the most obvious imminent pandemic threat, there have been several recent zoonotic incidents involving transmission of H7 viruses to humans. Vaccines are the primary public health defense against pandemics, but reliance on embryonated chickens eggs to propagate vaccine and logistic problems posed by the use of new technology may slow our ability to respond rapidly in a pandemic situation. Objectives: We sought to generate an H7 candidate vaccine virus suitable for administration to humans whose generation and amplification avoided the use of eggs. Methods: We generated a suitable H7 vaccine virus by reverse genetics. This virus, known as RD3, comprises the internal genes of A/Puerto Rico/8/34 with surface antigens of the highly pathogenic avian strain A/Chicken/Italy/13474/99 (H7N1). The multi-basic amino acid site in the HA gene, associated with high pathogenicity in chickens, was removed. Results: The HA modification did not alter the antigenicity of the virus and the resultant single basic motif was stably retained following several passages in Vero and PER. C6 cells. RD3 was attenuated for growth in embryonated eggs, chickens, and ferrets. RD3 induced an antibody response in infected animals reactive against both the homologous virus and other H7 influenza viruses associated with recent infection by H7 viruses in humans. Conclusions: This is the first report of a candidate H7 vaccine virus for use in humans generated by reverse genetics and propagated entirely in mammalian tissue culture. The vaccine has potential use against a wide range of H7 strains.

Surface properties and locations of gluten proteins and lipids revealed using confocal scanning laser microscopy in bread dough

Surface properties of gluten proteins were measured in a dilation test and in compression and expansion tests. The results showed that monomeric gliadin was highly surface active, but polymer glutenin had almost no surface activity. The locations of those proteins in bread dough were investigated using confocal scanning laser microscopy and compared with polar and nonpolar lipids. Added gluten proteins participated in the formation of the film or the matrix, surrounding and separating individual gas cells in bread dough. Gliadin was found in the bulk of dough and gas 'cell walls'. Glutenin was found only in the bulk dough. Polar lipids were present in the protein matrix and in gas 'cell walls', as well as at the surface of some particles, which appeared to be starch granules. However, nonpolar lipid mainly occur-red on the surface of particles, which may be starch granules and small lipid droplets. It is suggested that the locations of gluten proteins in bread dough depends on their surface properties. Polar lipid participates the formation of gluten protein matrix and gas 'cell walls'. Nonpolar lipids may have an effect on the rheological properties by associating with starch granule surfaces and may form lipid droplets. (C) 2004 Published by Elsevier Ltd.

Genetic differentiation between hosts and locations in populations of latent Botrytis cinerea in southern England

This study tested the hypothesis that aggressive, localized infections and asymptomatic systemic infections were caused by distinct specialized groups of Botrytis cinerea, using microsatellite genotypes at nine loci of 243 isolates of B. cinerea obtained from four hosts (strawberry (Fragaria ´ananassa), blackberry (Rubus fruticosus agg.), dandelion, (Taraxacum of®- cinale agg.) and primrose (Primula vulgaris)) in three regions in southern England (in the vicinities of Brighton, Reading and Bath). The populations were extremely variable, with up to 20 alleles per locus and high genic diversity. Each host in each region had a population of B. cinerea with distinctive genetic features, and there were also consistent host and regional distinctions. The B. cinerea population from strawberry was distinguished from that on other hosts, including blackberry, most notably by a common 154-bp amplicon at locus 5 (present in 35 of 77 samples) that was rare in isolates from other hosts (9¤166), and by the rarity (3¤77) of a 112-bp allele at locus 7 that was common (58¤166) in isolates from other hosts. There was signi®cant linkage disequilibrium overall within the B. cinerea populations on blackberry and strawberry, but with quite different patterns of association among isolates from the two hosts. No evidence was found for differentiation between populations of B. cinerea from systemically infected hosts and those from locally infected fruits.

Measurement of paraben concentrations in human breast tissue at serial locations across the breast from axilla to sternum

The concentrations of five esters of p-hydroxybenzoic acid (parabens) were measured using HPLC-MS/MS at four serial locations across the human breast from axilla to sternum using human breast tissue collected from 40 mastectomies for primary breast cancer in England between 2005 and 2008. One or more paraben esters were quantifiable in 158/160 (99%) of the tissue samples and in 96/160 (60%) all five esters were measured. Variation was notable with respect to individual paraben esters, location within one breast and similar locations in different breasts. Overall median values in nanograms per gram tissue for the 160 tissue samples were highest for n-propylparaben [16.8 (range 0–2052.7)] and methylparaben [16.6 (range 0–5102.9)]; levels were lower for n-butylparaben [5.8 (range 0–95.4)], ethylparaben [3.4 (range 0–499.7)] and isobutylparaben 2.1 (range 0–802.9). The overall median value for total paraben was 85.5 ng g−1 tissue (range 0–5134.5). The source of the paraben cannot be identified, but paraben was measured in the 7/40 patients who reported never having used underarm cosmetics in their lifetime. No correlations were found between paraben concentrations and age of patient (37–91 years), length of breast feeding (0–23 months), tumour location or tumour oestrogen receptor content. In view of the disproportionate incidence of breast cancer in the upper outer quadrant, paraben concentrations were compared across the four regions of the breast: n-propylparaben was found at significantly higher levels in the axilla than mid (P = 0.004 Wilcoxon matched pairs) or medial (P = 0.021 Wilcoxon matched pairs) regions (P = 0.010 Friedman ANOVA).

Eight-plex iTRAQ analysis of variant metastatic human prostate cancer cells identifies candidate biomarkers of progression: an exploratory study.

BACKGROUND: Due to the heterogeneity in the biological behavior of prostate cancer, biomarkers that can reliably distinguish indolent from aggressive disease are urgently needed to inform treatment choices. METHODS: We employed 8-plex isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to profile the proteomes of two distinct panels of isogenic prostate cancer cells with varying growth and metastatic potentials, in order to identify novel biomarkers associated with progression. The LNCaP, LNCaP-Pro5, and LNCaP-LN3 panel of cells represent a model of androgen-responsive prostate cancer, while the PC-3, PC-3M, and PC-3M-LN4 panel represent a model of androgen-insensitive disease. RESULTS: Of the 245 unique proteins identified and quantified (>or=95% confidence; >or=2 peptides/protein), 17 showed significant differential expression (>or=+/-1.5), in at least one of the variant LNCaP cells relative to parental cells. Similarly, comparisons within the PC-3 panel identified 45 proteins to show significant differential expression in at least one of the variant PC-3 cells compared with parental cells. Differential expression of selected candidates was verified by Western blotting or immunocytochemistry, and corresponding mRNA expression was determined by quantitative real-time PCR (qRT-PCR). Immunostaining of prostate tissue microarrays for ERp5, one of the candidates identified, showed a significant higher immunoexpression in pre-malignant lesions compared with non-malignant epithelium (P < 0.0001, Mann-Whitney U-test), and in high Gleason grade (4-5) versus low grade (2-3) cancers (P < 0.05). CONCLUSIONS: Our study provides proof of principle for the application of an 8-plex iTRAQ approach to uncover clinically relevant candidate biomarkers for prostate cancer progression.

Natal nest locations of the Asian house rat (Rattus tanezumi) in lowland rice–coconut cropping systems: a coconut penthouse or rice bunds with water frontage?

CONTEXT. Rattus tanezumi is a serious crop pest within the island of Luzon, Philippines. In intensive flood-irrigated rice field ecosystems of Luzon, female R. tanezumi are known to primarily nest within the tillers of ripening rice fields and along the banks of irrigation canals. The nesting habits of R. tanezumi in complex rice–coconut cropping systems are unknown. AIMS. To identify the natal nest locations of R. tanezumi females in rice–coconut systems of the Sierra Madre Biodiversity Corridor (SMBC), Luzon, during the main breeding season to develop a management strategy that specifically targets their nesting habitat. METHODS. When rice was at the booting to ripening stage, cage-traps were placed in rice fields adjacent to coconut habitat. Thirty breeding adult R. tanezumi females were fitted with radio-collars and successfully tracked to their nest sites. KEY RESULTS. Most R. tanezumi nests (66.7%) were located in coconut groves, five nests (16.7%) were located in rice fields and five nests (16.7%) were located on the rice field edge. All nests were located above ground level and seven nests were located in coconut tree crowns. The median distance of nest sites to the nearest rice field was 22.5m. Most nest site locations had good cover of ground vegetation and understorey vegetation, but low canopy cover. Only one nest location had an understorey vegetation height of less than 20 cm. CONCLUSIONS. In the coastal lowland rice–coconut cropping systems of the SMBC, female R. tanezumi showed a preference for nesting in adjacent coconut groves. This is contrary to previous studies in intensive flood-irrigated rice ecosystems of Luzon, where the species nests mainly in the banks of irrigation canals. It is important to understand rodent breeding ecology in a specific ecosystem before implementing appropriate management strategies. IMPLICATIONS. In lowland rice–coconut cropping systems, coconut groves adjacent to rice fields should be targeted for the 20 management of R. tanezumi nest sites during the main breeding season as part of an integrated ecologically based approach to rodent pest management.

Variation in the autism candidate gene GABRB3 modulates tactile sensitivity in typically developing children

Background: Autism spectrum conditions have a strong genetic component. Atypical sensory sensitivities are one of the core but neglected features of autism spectrum conditions. GABRB3 is a well-characterised candidate gene for autism spectrum conditions. In mice, heterozygous Gabrb3 deletion is associated with increased tactile sensitivity. However, no study has examined if tactile sensitivity is associated with GABRB3 genetic variation in humans. To test this, we conducted two pilot genetic association studies in the general population, analysing two phenotypic measures of tactile sensitivity (a parent-report and a behavioural measure) for association with 43 SNPs in GABRB3. Findings: Across both tactile sensitivity measures, three SNPs (rs11636966, rs8023959 and rs2162241) were nominally associated with both phenotypes, providing a measure of internal validation. Parent-report scores were nominally associated with six SNPs (P <0.05). Behaviourally measured tactile sensitivity was nominally associated with 10 SNPs (three after Bonferroni correction). Conclusions: This is the first human study to show an association between GABRB3 variation and tactile sensitivity. This provides support for the evidence from animal models implicating the role of GABRB3 variation in the atypical sensory sensitivity in autism spectrum conditions. Future research is underway to directly test this association in cases of autism spectrum conditions.

Genome-wide association mapping to candidate polymorphism resolution in the unsequenced barley genome

Although commonplace in human disease genetics, genome-wide association (GWA) studies have only relatively recently been applied to plants. Using 32 phenotypes in the inbreeding crop barley, we report GWA mapping of 15 morphological traits across ∼500 cultivars genotyped with 1,536 SNPs. In contrast to the majority of human GWA studies, we observe high levels of linkage disequilibrium within and between chromosomes. Despite this, GWA analysis readily detected common alleles of high penetrance. To investigate the potential of combining GWA mapping with comparative analysis to resolve traits to candidate polymorphism level in unsequenced genomes, we fine-mapped a selected phenotype (anthocyanin pigmentation) within a 140-kb interval containing three genes. Of these, resequencing the putative anthocyanin pathway gene HvbHLH1 identified a deletion resulting in a premature stop codon upstream of the basic helix-loop-helix domain, which was diagnostic for lack of anthocyanin in our association and biparental mapping populations. The methodology described here is transferable to species with limited genomic resources, providing a paradigm for reducing the threshold of map-based cloning in unsequenced crops.

Influence of SNPs in nutrient-sensitive candidate genes and gene–diet interactions on blood lipids: the DiOGenes study

Blood lipid response to a given dietary intervention could be determined by the effect of diet, gene variants or gene–diet interactions. The objective of the present study was to investigate whether variants in presumed nutrient-sensitive genes involved in lipid metabolism modified lipid profile after weight loss and in response to a given diet, among overweight European adults participating in the Diet Obesity and Genes study. By multiple linear regressions, 240 SNPs in twenty-four candidate genes were investigated for SNP main and SNP–diet interaction effects on total cholesterol, LDL-cholesterol, HDL-cholesterol and TAG after an 8-week low-energy diet (only main effect), and a 6-month ad libitum weight maintenance diet, with different contents of dietary protein or glycaemic index. After adjusting for multiple testing, a SNP–dietary protein interaction effect on TAG was identified for lipin 1 (LPIN1) rs4315495, with a decrease in TAG of − 0·26 mmol/l per A-allele/protein unit (95 % CI − 0·38, − 0·14, P= 0·000043). In conclusion, we investigated SNP–diet interactions for blood lipid profiles for 240 SNPs in twenty-four candidate genes, selected for their involvement in lipid metabolism pathways, and identified one significant interaction between LPIN1 rs4315495 and dietary protein for TAG concentration.

Candidate genes for obesity-susceptibility show enriched association within a large genome-wide association study for BMI

Before the advent of genome-wide association studies (GWASs), hundreds of candidate genes for obesity-susceptibility had been identified through a variety of approaches. We examined whether those obesity candidate genes are enriched for associations with body mass index (BMI) compared with non-candidate genes by using data from a large-scale GWAS. A thorough literature search identified 547 candidate genes for obesity-susceptibility based on evidence from animal studies, Mendelian syndromes, linkage studies, genetic association studies and expression studies. Genomic regions were defined to include the genes ±10 kb of flanking sequence around candidate and non-candidate genes. We used summary statistics publicly available from the discovery stage of the genome-wide meta-analysis for BMI performed by the genetic investigation of anthropometric traits consortium in 123 564 individuals. Hypergeometric, rank tail-strength and gene-set enrichment analysis tests were used to test for the enrichment of association in candidate compared with non-candidate genes. The hypergeometric test of enrichment was not significant at the 5% P-value quantile (P = 0.35), but was nominally significant at the 25% quantile (P = 0.015). The rank tail-strength and gene-set enrichment tests were nominally significant for the full set of genes and borderline significant for the subset without SNPs at P < 10(-7). Taken together, the observed evidence for enrichment suggests that the candidate gene approach retains some value. However, the degree of enrichment is small despite the extensive number of candidate genes and the large sample size. Studies that focus on candidate genes have only slightly increased chances of detecting associations, and are likely to miss many true effects in non-candidate genes, at least for obesity-related traits.