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Objectives; The purpose of this study was to assess whether high-sensitivity C-reactive protein (hs-CRP), N-terminal pro-brain natriuretic peptide (NT-proBNP), and soluble urokinase plasminogen activator receptor (suPAR) differed in their ability to predict cardiovascular outcomes beyond traditional risk factors in younger and older men and women without known cardiovascular disease. Design. Prospective population-based cohort study of 1951 individuals from the MONItoring of trends and determinants in Cardiovascular disease (MONICA) study, examined 1993–1994. Participants were stratified into four groups based on sex and age. Subjects aged 41 or 51 years were classified as younger; those aged 61 or 71 years were classified as older. The principal endpoint was death from cardiovascular causes. Predictive capabilities of biomarkers were tested using Cox proportional-hazards regression, Harrell’s concordance-index, net reclassification improvement, and classification and regression tree (CART) analysis. Results. Median follow-up was 18.5 years, during which 19/597 younger men, 100/380 older men, 12/607 younger women, and 46/367 older women had died from a cardiovascular cause. NT-proBNP was independently associated with death from cardiovascular causes among all participants (p ≤ .02) except younger women (p = .70), whereas hs-CRP was associated with this endpoint in men (p ≤ .007), and suPAR in older men only (p Objectives: The purpose of this study was to assess whether high-sensitivity C-reactive protein (hs-CRP), N-terminal pro-brain natriuretic peptide (NT-proBNP), and soluble urokinase plasminogen activator receptor (suPAR) differed in their ability to predict cardiovascular outcomes beyond traditional risk factors in younger and older men and women without known cardiovascular disease. Design. Prospective population-based cohort study of 1951 individuals from the MONItoring of trends and determinants in Cardiovascular disease (MONICA) study, examined 1993–1994. Participants were stratified into four groups based on sex and age. Subjects aged 41 or 51 years were classified as younger; those aged 61 or 71 years were classified as older. The principal endpoint was death from cardiovascular causes. Predictive capabilities of biomarkers were tested using Cox proportional-hazards regression, Harrell’s concordance-index, net reclassification improvement, and classification and regression tree (CART) analysis. Results. Median follow-up was 18.5 years, during which 19/597 younger men, 100/380 older men, 12/607 younger women, and 46/367 older women had died from a cardiovascular cause. NT-proBNP was independently associated with death from cardiovascular causes among all participants (p ≤.02) except younger women (p =.70), whereas hs-CRP was associated with this endpoint in men (p ≤.007), and suPAR in older men only (p

Purpose: The objective of this study was to test if combining antecedent systolic blood pressure (SBP) with traditional risk factors and hypertension-mediated organ damage (HMOD) improves risk stratification for subsequent cardiovascular disease. Materials and methods: 1910 subjects participated in this study. Antecedent SBP was defined as the average of measurements obtained in 1982 and in 1987. Current SBP was obtained in 1993. HMOD were examined in 1993. HMOD was defined as either atherosclerotic plaque(s), increased pulse wave velocity, increased urine albumin creatinine ratio (above the 90th percentile) or left ventricular hypertrophy. Major adverse cardiovascular events (MACE) including myocardial infarction, cerebrovascular disease, heart failure and arrhythmia were obtained from national registries. Results: Subjects were divided into two age categories: a middle-aged group (aged 41 or 51) and an older group (aged 61 or 71). From 1993 to 2010, 425 events were observed. In multivariable analysis with both current and antecedent SBP adjusted for traditional risk factors, current SBP was associated with each measure of HMOD whilst antecedent SBP was not significantly associated with urine albumin creatinine ratio in the older group, LVMI in the middle-aged group, or the presence of plaque in any of the age groups (all p > 0.15). When current and antecedent SBP were evaluated together, current SBP was not associated with MACE in the middle-aged subgroup [HR = 1.09 (0.96–1.22), p = 0.18] but remained associated with MACE in the older subgroup [HR = 1.21 (1.10–1.34), p < 0.01]. Contrariwise, antecedent SBP was only associated with MACE in the middle-aged subgroup [HR = 1.24 (1.04–1.48), p = 0.02]. Adding antecedent SBP to traditional risk factors did not improve the predictive accuracy of the survival model. Conclusion: In healthy non-medicated middle-aged subjects, antecedent SBP is associated with cardiovascular outcome independently of current BP, traditional risk factors and HMOD. However, improvement in risk stratification seems to be limited.

The promoting effects of both high nitrogen (N) and exogenous gibberellin (GA) supply on regrowth of Lolium perenne have been widely reported. The mobilisation of carbohydrate reserves in response to N is a critical mechanism for promoting plant regrowth. However, our knowledge about GA regulation of carbohydrate metabolism remains limited. Here, we analysed the effects of both N and exogenous GA on the molecular mechanisms controlling perennial ryegrass regrowth and investigated the similarities and differences. Our analyses show that both high N and exogenous GA supply lead to a decline in the accumulation of carbohydrate reserves, but the regulatory mechanisms responsible for this decline varied between N and GA supply. The effects of elevated N were mainly through declining fructan biosynthesis which results in improving photosynthate use efficiency to promote plant regrowth, whereas the application of exogenous GA resulted in an increase in the hydrolytic activities of fructan exohydrolase and invertases capable of cleaving reserved carbohydrates to release energy sources for plant regrowth.

Aims The aim of this study was to examine whether high-sensitivity C-reactive protein (hs-CRP), N-terminal pro-brain natriuretic peptide (NT-proBNP), and soluble urokinase plasminogen activator receptor (suPAR) carried incremental prognostic value in predicting cardiovascular morbidity and mortality beyond traditional risk factors in apparently healthy individuals. Methods and results This was a prospective population-based cohort study comprising 1951 subjects included in the 10-year follow-up of the MONItoring of trends and determinants in CArdiovascular disease (MONICA) study, between 1993 and 1994. The principal endpoint was death from cardiovascular causes. Secondary endpoints were death from any cause, coronary artery disease, heart failure, and cerebrovascular disease. Predictive capabilities of each of the three biomarkers were tested using Cox proportional-hazards regression, Harrell’s concordance index (C-index), and net reclassification improvement (NRI). Study participants were aged 41, 51, 61, or 71 years, and equally distributed between the two sexes. During a median follow-up of 18.5 years (interquartile range: 18.1–19.0), 177 (9.1%) subjects died from a cardiovascular cause. Hs-CRP (adjusted standardized hazard ratio (HR): 1.37, 95% confidence interval (CI): 1.17–1.60), NT-proBNP (HR: 1.90, 95% CI: 1.58–2.29), and suPAR (HR: 1.35, 95% CI: 1.17–1.57) were all significantly associated with cardiovascular deaths after adjustment for age, sex, smoking status, systolic blood pressure, and total cholesterol ( p Conclusions Hs-CRP, suPAR, and particularly NT-proBNP predicted cardiovascular death and may enhance prognostication beyond traditional risk factors in apparently healthy individuals.

Objective: To develop and test a simple medication-focused patient counseling intervention at hospital discharge, with the aim of improving patient satisfaction. Methods: The intervention was developed during a workshop and carried out by pharmaconomists. The final intervention comprised preparing information for the discharge counseling, medication reconciliation, discussion with physician, patient counseling at discharge, medication report to primary care physician, and phone follow-up three days after discharge. The intervention was tested against usual care in a gastrointestinal surgical unit in a feasibility study, using the setup of a randomized controlled trial, with patient satisfaction as the primary outcome. Results: A total of 85 patients were invited to participate in the study. Following refusals (n = 11) and exclusions (n = 10), 32 patients were included in each trial arm (median age of 66.5 years, 52% males, median admission length of seven days). Patient satisfaction was high in both groups, with 75% (intervention) and 91% (control) reporting being overall satisfied with the discharge process (p = 0.10). No other differences between the groups were identified. Conclusions: The intervention did not result in improved patient satisfaction. This is likely attributed to the low number of patients included, the high satisfaction at baseline, and the lack of a validated tool to measure patient satisfaction. The developed intervention and study findings can inform future studies.

INTRODUCTION: Psoriasis often sets on during childhood or adolescence, when parents have great importance for the young people's self-management, well-being and quality of life. The aim of the study was to understand parents' perspectives on young people's daily life with psoriasis in order to improve adolescents' self-management.METHOD: Adopting Interpretive Description methodology, focus group discussion and interviews were conducted with eight parents of adolescents with psoriasis. The analysis was inductive with an iterative comparative approach. Main themes conveying participants' perceptions were identified for constructing a coherent narrative of parents' perspectives on their young people's transition with psoriasis through adolescence.RESULTS: Parents initially perceived psoriasis mainly a physical and treatment-related burden and not until late realized its socio-emotional impact. They eventually found themselves balancing between declining treatment due to fear of side effects and acknowledging the impact on their young people's quality of life and their desire for effective treatment.CONCLUSION: Caring for young people with psoriasis is a stressful process involving experimental learning to understand and manage the complexity of psoriasis and its impact on adolescents' emotional and social life. Future research should consider integration of shared decision-making and self-management support interventions in routine daily care as focus points.

Objective: The aim of this study was to evaluate the impact of age on the associations between hemodynamic components derived from 24-h ambulatory blood pressure (24-h ABPM) and target organ damage, in apparently healthy, nonmedicated individuals. Methods: Twenty-four-hour ABPM and target organ damage (left ventricular mass index, pulse wave velocity, urine albumin : Creatinine ratio and carotid atherosclerotic plaques) were evaluated in 1408 individuals. Associations were examined in regression models, stratified for age [middle-aged (41 or 51 years) or elderly (61 or 71 years)], and adjusted for sex, smoking status, and total-cholesterol. Results: In middle-aged individuals, an increase of 10mmHg in 24-h SBP was independently associated with an increase of 3.8 (2.7-4.8) g/m2 in LVMI. The effect was nearly doubled in the elderly subgroup, where the same increase resulted in an increase in LVMI of 6.3 (5.0-7.6) g/ m2 (P for interaction 0.26). Conclusion: Age enhances the associations between hemodynamic components obtained from 24-h ABPM and measures of arterial stiffness, microvascular damage, and cardiac structure, but not atherosclerosis.

Markers of target-organ damage and 24-hour ambulatory blood pressure (BP) measurement improve cardiovascular risk stratification. The prevalence of target-organ damage and raised BP increases with aging. The study aim was to evaluate the impact of age and target-organ damage on the prognostic value of ambulatory BP. Markers of target-organ damage and ambulatory BP were measured in 1408 healthy people aged 41 or 51 (middle-aged group), and 61 or 71 (older group) years. The primary outcome was cardiovascular events after 16 years of follow-up, with data obtained from national registries. The prognostic value of BP was evaluated with Cox regression models, adjusted for traditional risk factors and target-organ damage, including left ventricular mass, pulse wave velocity, carotid plaques, and urine albumin/creatinine ratio. A total of 323 events were observed. In comparison with traditional risk factors, adding systolic BP and presence of target-organ damage improved risk stratification by increasing concordance index from 0.711 to 0.728 ( P =0.01). In middle-aged subjects with target-organ damage, increment in pulse pressure (hazard ratio, 1.70; 95% confidence interval, 1.31–2.21; P P =0.02) were associated with a greater risk of cardiovascular disease compared with subjects without target-organ damage: hazard ratio, 1.04 (95% confidence interval, 0.74–1.46; P =0.81); P for interaction, 0.02; and hazard ratio, 0.89 (95% confidence interval, 0.69–1.14; P =0.36); P for interaction, 0.01. Target-organ damage may be a marker of individual susceptibility to the harmful effects of pulse pressure and BP variability on the cardiovascular system in middle-aged individuals.

Baseret på feltarbejde i danske familier undersøger denne artikel håndtering og betydning af tid i familier, der lever med diagnosen Attention Deficit Hyperactivity Disorder (ADHD). Vi argumenterer for at se på et aspekt af ADHD som en tidsforstyrrelse, og undersøger hvordan dette håndteres i et moderne dansk hverdagsliv præget af mange strukturelle tidslige vilkår. Inspireret af den danske antropolog Mikka Nielsen (2016) fortolker vi hverdagslivet med ADHD ud fra et tidsperspektiv, hvor ADHD ses som en lidelse, der bliver særligt synlig i tidslige sammenstød med omgivelserne. Vi beskriver, hvordan væsentlige aspekter af ADHD kan forstås som forårsaget af en forstyrrelse i oplevelsen af tidens normale struktur af fortid-nutid-fremtid. Med sociologen Michael Flahertys begreb tidsarbejde (2011) belyser vi, hvordan brug af genstande, aktiviteter, medicin og støtte fra andre hjælper den enkelte med ADHD til at navigere i tiden. Vi kontekstualiserer analysen med strukturelle perspektiver på modernitetens kendetegn og belyser derved, at tidslige krav fra de institutionelle rammer påvirker familielivet med ADHD. Artiklen argumenterer således for et dobbelt-temporalt perspektiv på familielivet med ADHD. Undersøgelsen peger på, at feltarbejde i familier, der lever med ADHD, kan bidrage med vigtig viden, der kan opkvalificere vejledning der gives fra sundhedssystemet til patienter med ADHD og deres familier. Vi opfordrer til videre forskning om temporalitet i familier, der lever med ADHD.

Perennial ryegrass (Lolium perenne) is integral to temperate pastoral agriculture, which contributes most of the milk and meat production worldwide. Chemical profiles and diversity of ryegrass offer several opportunities to harness specific traits and elucidate underlying biological mechanisms for forage improvement. We conducted a large-scale metabolomics study of perennial ryegrass comprising 715 genotypes, representing 118 populations from 21 countries. Liquid/gas chromatography–mass spectrometry based targeted and non-targeted techniques were used to analyse fructan oligosaccharides, lipids, fatty acid methyl esters, polar and semi-polar compounds. Fructan diversity across all genotypes was evaluated, high- and low-sugar groups identified, and fructan accumulation mechanisms explored. Metabolites differentiating the two groups were characterised, modules and pathways they represent deduced, and finally, visualisation and interpretation provided in a biological context. We also demonstrate a workflow for large-scale metabolomics studies from raw data through to statistical and pathway analysis. Raw files and metadata are available at the MetaboLights database., Arvind Subbaraj et al. present the ryegrass metabolome, derived from 715 genotypes representing 118 populations of Lolium perenne from 21 countries. They analyze fructan diversity, identify high- and low-sugar groups, and explore biochemical modules and pathways that discriminate the phenotypes.

Psoriasis is a long-term condition with a possibly cumulative life course impairment. Young people struggle to minimize its effects on appearance and functioning. To date, the self-management needs of adolescents suffering from psoriasis have been underinvestigated. Using focus groups and individual interviews, we present an interpretive description of young people's experiences of living with psoriasis, the challenges they face, and the support they need to relieve suffering and come to terms with their condition. This process is characterized by loneliness, the self-imposition of limitations, and the lack of personalized knowledge and communication skills to manage the impact of disease and society's reactions. Our study provides insight into needs of early interventions tailored to address condition, role, and emotional management, involving parent education, peer support, storytelling, and roles for professionals. We argue that further research should involve young people, their parents, and professionals in the development and evaluation of interventions.

The ecological effects of novel grass–endophyte associations used in agriculture have not been widely studied. Previous studies of asexual Epichloe-infected Lolium perenne suggest that endophyte concentration is altered in high sugar grasses (HSGs) selectively bred to produce higher concentrations of water-soluble carbohydrates relative to conventional cultivars. We investigated whether differences are due to the effects of altered carbohydrates, or genetic background, by growing multiple cultivars in both high-sugar trait expression and non-expression conditions (using light/temperature treatments). Endophyte and alkaloid concentrations were measured in three HSG and three NSG (normal-sugar grass) cultivars infected with Lp19 or AR37 endophyte strains. Low molecular weight (LMW) carbohydrates had a small effect, explaining

Liquid chromatography coupled to mass spectrometry (LCMS) is widely used in metabolomics due to its sensitivity, reproducibility, speed and versatility. Metabolites are detected as peaks which are characterised by mass-over-charge ratio (m/z) and retention time (rt), and one of the most critical but also the most challenging tasks in metabolomics is to annotate the large number of peaks detected in biological samples. Accurate m/z measurements enable the prediction of molecular formulae which provide clues to the chemical identity of peaks, but often a number of metabolites have identical molecular formulae. Chromatographic behaviour, reflecting the physicochemical properties of metabolites, should also provide structural information. However, the variation in rt between analytical runs, and the complicating factors underlying the observed time shifts, make the use of such information for peak annotation a non-trivial task. To this end, we conducted Quantitative Structure–Retention Relationship (QSRR) modelling between the calculated molecular descriptors (MDs) and the experimental retention times (rts) of 93 authentic compounds analysed using hydrophilic interaction liquid chromatography (HILIC) coupled to high resolution MS. A predictive QSRR model based on Random Forests algorithm outperformed a Multiple Linear Regression based model, and achieved a high correlation between predicted rts and experimental rts (Pearson’s correlation coefficient = 0.97), with mean and median absolute error of 0.52 min and 0.34 min (corresponding to 5.1 and 3.2 % error), respectively. We demonstrate that rt prediction with the precision achieved enables the systematic utilisation of rts for annotating unknown peaks detected in a metabolomics study. The application of the QSRR model with the strategy we outlined enhanced the peak annotation process by reducing the number of false positives resulting from database queries by matching accurate mass alone, and enriching the reference library. The predicted rts were validated using either authentic compounds or ion fragmentation patterns. Electronic supplementary material The online version of this article (doi:10.1007/s11306-014-0727-x) contains supplementary material, which is available to authorized users.

Rising atmospheric CO2 has been shown to alter plant nitrogen metabolism, growth and secondary chemistry. We hypothesized that altered aphid performance under elevated CO2 is linked to phloem nitrogen chemistry. Rhopalosiphum padi performance on endophyte-infected or uninfected tall fescue was examined under three levels of CO2 (ambient, 800 and 1000 p.p.m.) and high and low nitrogen fertilization. Ethylenediaminetetracetic acid-facilitated exudation was used to sample phloem sap, followed by quantification of relative amino acid concentrations using reverse-phase high-performance liquid chromatography. Aphid abundance was reduced at 800 p.p.m. relative to ambient CO2 but returned to baseline at 1000 p.p.m. The density of aphids was reduced in both the elevated CO2 treatments. Aphids were unsuccessful at colonizing endophyte-infected plants, possibly as a result of the presence of loline alkaloids. Multivariate analysis showed that certain groups of phloem amino acids were altered by nitrogen fertilization and CO2. We found that four amino acids (valine, arginine, glutamine and aspartate) were correlated with aphid performance. These findings partially explained the effect of plant nitrogen fertilization and elevated CO2 on aphids. The present study represents a first step toward providing a mechanistic explanation of the aphid performance changes that may result from rising atmospheric CO2.

Tea is the second most consumed beverage in the world after water and there are numerous reported health benefits as a result of consuming tea, such as reducing the risk of cardiovascular disease and many types of cancer. Thus, there is much interest in the chemical composition of teas, for example; defining components responsible for contributing to reported health benefits; defining quality characteristics such as product flavor; and monitoring for pesticide residues to comply with food safety import/export requirements. Covered in this review are some of the latest developments in mass spectrometry-based analytical techniques for measuring and characterizing low molecular weight components of tea, in particular primary and secondary metabolites. The methodology; more specifically the chromatography and detection mechanisms used in both targeted and non-targeted studies, and their main advantages and disadvantages are discussed. Finally, we comment on the latest techniques that are likely to have significant benefit to analysts in the future, not merely in the area of tea research, but in the analytical chemistry of low molecular weight compounds in general.

One of the most expanded upper Campanian–Maastrichtian successions worldwide has been cored in a series of boreholes in eastern Denmark. A high-resolution holostratigraphic analysis of this part of the Chalk Group has been undertaken on these cores, notably Stevns-1, in order to provide a record of changes in chalk facies, water depths and sea-water temperatures. Combined lithological data, a suite of petrophysical logs including gamma ray (GR) logs, nannofossil and dinoflagellate palaeontology, stable carbon isotopes, seismic reflection and refraction sections form the basis for the definition of two new formations and six members, three of which are new, and for recognition of Boreal nannofossil subzones UC15e BP to UC20d BP . The upper Campanian–lowermost Maastrichtian Mandehoved Formation is subdivided into the Flagbanke and Boesdal Members and the Maastrichtian Mons Klint Formation is subdivided into the Hvidskud, Rordal, Sigerslev, Kjolby Gaard Marl and Hojerup Members. The Boesdal and Rordal Members show high GR values and a pronounced chalk-marl cyclicity. The Rordal and the thin Kjolby Gaard Marl Members have a regional distribution and can be traced over most of the Danish Basin, whereas the Hojerup Member is restricted to the easternmost part of Sjaelland. The other members consist of rather featureless white chalk.

Mass spectrometry coupled with chromatography has become the major technical platform in metabolomics. Aided by peak detection algorithms, the detected signals are characterized by mass-over-charge ratio (m/z) and retention time. Chemical identities often remain elusive for the majority of the signals. Multi-stage mass spectrometry based on electrospray ionization (ESI) allows collision-induced dissociation (CID) fragmentation of selected precursor ions. These fragment ions can assist in structural inference for metabolites of low molecular weight. Computational investigations of fragmentation spectra have increasingly received attention in metabolomics and various public databases house such data. We have developed an R package “iontree” that can capture, store and analyze MS2 and MS3 mass spectral data from high throughput metabolomics experiments. The package includes functions for ion tree construction, an algorithm (distMS2) for MS2 spectral comparison, and tools for building platform-independent ion tree (MS2/MS3) libraries. We have demonstrated the utilization of the package for the systematic analysis and annotation of fragmentation spectra collected in various metabolomics platforms, including direct infusion mass spectrometry, and liquid chromatography coupled with either low resolution or high resolution mass spectrometry. Assisted by the developed computational tools, we have demonstrated that spectral trees can provide informative evidence complementary to retention time and accurate mass to aid with annotating unknown peaks. These experimental spectral trees once subjected to a quality control process, can be used for querying public MS2 databases or de novo interpretation. The putatively annotated spectral trees can be readily incorporated into reference libraries for routine identification of metabolites.

Tea is an infusion made from the dried leaves of Camellia sinensis L. and is the second most consumed beverage in the world. It has been shown that factors such as fermentation methods, cultivar, geographical origin and season can affect the biochemical composition of tea. In this study, the biochemical composition of green, oolong and black commercial tea samples from around the world was studied using a non-targeted method utilising reversed phase ultra high pressure liquid chromatography (UHPLC) and high resolution mass spectrometry. Principal component analysis of green, oolong and black tea extracts clearly showed that fermented tea can be resolved from non-fermented tea. When the non-targeted data were combined with the supervised multivariate technique, partial least squares discriminant analysis, the method was able to clearly distinguish ‘country of origin’ within green tea and to a lesser extent within a black tea sample set, plus provide indicative marker ions for the country of origin. Many of the significant components detected in this study are unknowns, emphasising the importance of un-biased non-targeted analytical techniques. This study highlights the potential efficacy of non-targeted UHPLC–mass spectrometry when combined with multivariate statistics to differentiate fermented from non-fermented tea and provide potential indicators of provenance of tea samples for further examination.

GR was supported by a fellowship from the Natural Science and Engineering Research Council (NSERC) Canada. Funding to support this research was provided by grants from NSERC and the Ontario Ministry of Agriculture, Food and Rural Affairs, to JN.

The majority of clear-cell renal cell carcinomas (ccRCC) show high and homogeneous expression levels of the tumor associated antigen (TAA) carbonic anhydrase IX (CAIX), and treatment with interleukin-2 (IL-2) based immunotherapy can lead to cure in patients with metastatic renal cell carcinoma (mRCC). However, the involvement of CAIX specific CD8+ T cells and/or NK cells in the tumor eradication is unknown. We investigated T cell and antibody reactivity against overlapping 15-mer CAIX-peptides as well as HLA haplotype frequency and NK cell cytotoxicity in 11 patients with no evidence of disease (NED) following treatment with IL-2 based immunotherapy, and thus potentially cured. Immune reactivity in these patients was compared with samples from patients with dramatic tumor response obtained immediately at the cessation of therapy, samples from patients that experienced progressive disease during treatment and samples from healthy controls. We observed more focused but only weak and not consistent CAIX specific T-cells in the late observation and early observation response groups compared with the healthy control group. An increased frequency of the class II alleles HLA-DRB4 01:01, HLA-DPB 01:01 and HLA-DPB 03:01 was noted in the NED patients. In contrast, NK cytotoxicity was low even in the late observation response group as compared with controls. In particular, a HLA-B*40:01 restricted CD8+ T cell response recognizing the CAIX- derived peptide SEEEGSLKL was identified. This may have interest in future cancer vaccines, but more studies are needed to elucidate the immunological mechanisms of action in potentially cured patients treated with an immunotherapeutic agent.

Alkaline phosphatases (ALP) in arbuscular mycorrhizal (AM) fungi have been suggested to be involved in transfer of phosphate from the mycorrhizal fungus to the host plant, but exact mechanisms are still unknown, partially due to the lack of molecular information. We isolated a full-length cDNA (FmALP) from the AM fungus Funneliformis mosseae (syn. Glomus mosseae) showing similarity with putative ALP genes from Rhizophagus intraradices (syn. Glomus intraradices) and Gigaspora margarita. For functional characterisation FmALP was expressed heterologously in the yeast Pichia pastoris. The recombinant FmALP protein had a pH optimum of 9.5, and catalysed the hydrolysis of glycerolphosphate and, to a lesser extent of glucose-1- and 6-phosphate, confirming it to be an alkaline phosphatase belonging to the family of alkaline phosphomonoesterases (EC 3.1.3.1). FmALP did not catalyse the hydrolysis of ATP or polyP. Relative FmALP transcript levels were analysed in intra- and extraradical hyphae isolated from F. mosseae infected ryegrass (Lolium perenne) using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). FmALP was highly expressed in intraradical hyphae at low Pi supply, and its expression was repressed by high Pi supply. Taken together this study provides evidence for mycorrhizal alkaline phosphatases playing a role in P mobilisation from organic substrates under P starvation conditions.

We used exogenous application of a phytohormone (gibberellin GA3, ‘GA’) to test the hypothesis that common perennial grasses may not be growing at all times to the limit of resource availability. Plants were taken from the field in winter, and again in summer and their responses to GA assessed under standard conditions, indoors, to reveal their ‘potential’ for growth at different times. Time of year, and associated developmental state, had a major impact on the capacity of plants to respond to exogenous GA, and less so their current growing conditions, temperature and N availability, during measurement. A major increase in dry matter (DM) production in winter-derived plants took place at both low and high N, with no evidence of a reduction in N content in tissues. That ryegrass plant growth can be stimulated, without externally adding resources, supports the hypothesis there is an element of internal control in how plants respond to ‘signals’ in their environment, that might be manipulated. This offers prospects for reducing environmental impacts (leaching, N2O) compared with obtaining the same yield increase by adding fertilizer N in early season. Responses to exogenous GA were detected (as significant) but far smaller in summer-derived plants. Molecular mechanisms of detection of N resource signals, developmental triggers and the role of endogenous gibberellin need to be unravelled to assess scope for breeding ryegrass germplasm to better match demands for increased production with greater resource-use efficiency.

Isoflavones are a subgroup of isoflavonoids, a group of flavonoids found almost exclusively in Leguminosae. The main isoflavones accumulating in legumes are daidzein and genistein and 4-0-methylated formononetin and biochanin A. Isoflavones and derived isoflavonoids play important roles in plant protection against pathogens, as signalling molecules in nitrogen fixing legume-rhizobia associations and can be beneficial for human health. Considerable research efforts aim to introduce isoflavones into non-leguminous plants through genetic transformation. The biosynthesis of isoflavones requires the activity of isoflavone synthase (IFS) and transformation of non-leguminous plants with IFS demonstrated that it is, in principle, possible to introduce the biosynthesis of genistein into plants not normally synthesizing this compound. However, isoflavone concentrations are typically at least an order of magnitude lower than those found in soybean seeds or red clover leaves. Major limiting factors are insufficient flux through naringenin, the general precursor of (iso)flavonoids and in particular competition between flavonoid specific enzymes and isoflavonoid specific IFS. Attempts to increase isoflavone concentrations beyond the natural variation in soybean seeds through overexpression of IFS have also been met with limited success and recently identified transcriptional regulators of the isoflavonoid biosynthetic pathway might present a more promising option. All reviewed studies focused on the manipulation of genes encoding proteins directly involved in the (iso)flavonoid pathway and we propose that engineering strategies introducing or enhancing the biosynthesis of isoflavones might have to consider wider metabolic network interactions such as the link of (iso)flavonoid precursors with protein, lipid and carotenoid biosynthetic pathways.

BACKGROUND: Carotid-femoral pulse wave velocity (cfPWV) adds significantly to traditional cardiovascular risk prediction, but is not widely available. Therefore, it would be helpful if cfPWV could be replaced by an estimated carotid-femoral pulse wave velocity (ePWV) using age and mean blood pressure, and previously published equations. The aim of this study was to investigate whether ePWV could predict cardiovascular events independently of traditional cardiovascular risk factors and/or cfPWV.METHOD: cfPWV was measured and ePWV was calculated in 2366 patients from four age groups of the Danish MONICA10 cohort. Additionally, the patients were divided into four cardiovascular risk groups based on Systematic COronary Risk Evaluation (SCORE) or Framingham risk score (FRS). In 2006, the combined cardiovascular endpoint of cardiovascular death, nonfatal myocardial infarction, stroke and hospitalization for ischemic heart disease was registered.RESULTS: Most results were retested in 1045 hypertensive patients from a Paris cohort. Bland-Altman plot demonstrated a relative difference of -0.3% [95% confidence interval (CI) -15 to 17%] between ePWV and cfPWV. In Cox regression models in apparently healthy patients, ePWV and cfPWV (per SD) added independently to SCORE in prediction of combined endpoint [hazard ratio (95%CI) = 1.38(1.09-1.76) and hazard ratio (95%CI) = 1.18(1.01-1.38)] and to FRS [hazard ratio (95%CI) = 1.33(1.06-1.66) and hazard ratio (95%CI) = 1.16(0.99-1.37)]. If healthy patients with ePWV and/or cfPWV at least 10 m/s were reclassified to a higher SCORE risk category, net reclassification index was 10.8%, P less than 0.01. These results were reproduced in the Paris cohort.CONCLUSION: ePWV predicted major cardiovascular events independently of SCORE, FRS and cfPWV indicating that these traditional risk scores have underestimated the complicated impact of age and blood pressure on arterial stiffness and cardiovascular risk.

BACKGROUND: Arterial age can be estimated from equations relating arterial stiffness to age and blood pressure in large cohorts. We investigated whether estimated arterial age (eAA) was elevated in patients with the metabolic syndrome and/or known cardiovascular disease (CVD), which factors were associated with eAA and whether eAA added prognostic information.METHODS: In 1993, 2366 study participants, 41, 51, 61, and 71 years old, had traditional cardiovascular risk factors and carotid-femoral pulse wave velocity (cfPWV) measured. Risk groups were identified based on known CVD and components of metabolic syndrome, Systematic COronary Risk Evaluation, or Framingham risk score. From age, mean blood pressure, and cfPWV, eAA and estimated cfPWV (ePWV) were calculated. In 2006, the combined cardiovascular endpoint (CEP) of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, and hospitalization for ischemic heart disease was registered.RESULTS: cfPWV and ePWV increased with ageing and cardiovascular risk (all P CONCLUSION: Elevated eAA was associated with male sex, higher plasma glucose, and soluble urokinase plasminogen activator receptor and known CVD independently of age, SBP, and heart rate.

Tea is the second most consumed beverage in the world and its consumption has been associated with numerous potential health benefits. Factors such as fermentation methods, geographical origin and season can affect the primary and secondary metabolite composition of tea. In this study, a hydrophilic interaction liquid chromatography (HILIC) method coupled to high resolution mass spectrometry in both positive and negative ionisation modes was developed and optimised. The method when combined with principal component analysis to analyse three different types of tea, successfully distinguished samples into different categories, and provided evidence of the metabolites which differed between them. The accurate mass and high resolution attributes of the mass spectrometric data were utilised and relative quantification data were extracted post-data acquisition on 18 amino acids, showing significant differences in amino acid concentrations between tea types and countries. This study highlights the potential of HILIC chromatography combined with non-targeted mass spectrometric methods to provide a comprehensive understanding of polar metabolites in plant extracts.

Markers of subclinical target organ damage (TOD) increase cardiovascular (CV) risk prediction beyond traditional risk factors. We wanted to establish thresholds for three markers of TOD based on absolute CV risk in different risk chart categories.In a cohort of 1968 healthy patients, we measured urine albumin creatine ratio (UACR), pulse wave velocity (PWV), left ventricular mass index (LVMI) and traditional risk factors. Patients were categorized according to Systemic Coronary Evaluation (SCORE), European Society of Hypertension/European Society of Cardiology (ESH/ESC) risk chart and Framingham risk score (FRS) and three corresponding endpoints were recorded: CV death (SCORE-endpoint), a composite of CV death and nonfatal myocardial infarction and stroke (ESH/ESC-endpoint), and a composite that also included hospital admissions for ischemic heart disease, heart failure, peripheral arterial disease and transient cerebral ischemic attack (FRS-endpoint). During a median follow of 12.8 years events totaled 81 SCORE-, 153 ESH/ESC-endpoints and 280 FRS-endpoints. Thresholds for UACR, PWV and LVMI are presented using 10-year risk threshold of more than 5% (SCORE-endpoint), more than 10%(ESH/ESC-endpoint) and more than 20%(FRS-endpoint), which indicated high risk and eligibility for primary prevention. As an example, the threshold was 0.83 mg/mmol, 13.7 m/s and 119 g/m for UACR, PWV and LVMI, respectively, for patients at moderate added risk according to ESH/ESC risk chart.Thresholds for UACR, PVW and LVMI based on absolute risk have primarily impact on risk stratification in patients with intermediate risk. The thresholds for PWV and LVMI in patients with moderate risk according to the ESH/ESC risk chart were similar to currently applied thresholds whereas the threshold for UACR was considerable lower than the threshold for microalbuminuria.

Background and Aims Trifolium repens (white clover) is a valuable component of pastures due to its ability to fix nitrogen. Productivity of T. repens is sometimes threatened by insect pests, and it has been suggested that phenylpropanoid-derived isoflavonoids such as formononetin can protect white clover from insect damage. The aim of this study was to isolate and functionally characterize an isoflavone synthase (IFS2_12) from T. repens by expressing it in Nicotiana tabacum (tobacco), a plant which does not naturally produce isoflavonoids. Methods To induce anthocyanin production and increase isoflavonoid precursors in tobacco, the tomato R2R3 MYB transcription factor ANT1 was expressed in tobacco (Nt-ANT1 plants). IFS2_12 was heterologously expressed in tobacco both transiently and stably, and isoflavonoids in leaf extracts were analysed by liquid chromatography (LC) coupled to mass spectrometry (MSn). As a positive control, a double construct of soybean IFS and alfalfa chalcone isomerase (IFS/CHI), which had been previously shown to induce isoflavonoid production in tobacco, was also expressed. Stable transformants expressing IFS2_12, soybean/alfalfa IFS/CHI and ANT1 were crossed and the resulting plants were analysed for isoflavonoid production. Key results Leaves of tobacco plants expressing ANT1 had a range of phenotypes from mainly green to uniformly bronze coloured. Both transient and stable expression of the IFS2_12 or IFS/CHI constructs resulted in the production of the isoflavonoid genistein and its conjugates. The highest levels (up to 19·2 mg g−1 d. wt) accumulated in a progeny of a cross between a purple ANT1 and a IFS/ CHI transformant, while the second highest concentration was found in a plant derived from a selfed IFS2-12 transformant. Conclusions It is concluded that the gene IFS2_12 isolated from T. repens encodes an isoflavone synthase. This study paves the way for engineering white clover plants with higher levels of isoflavonoids than naturally found in this species for sufficient insect protection.

Proanthocyanidins (PAs) are oligomeric flavonoids and one group of end products of the phenylpropanoid pathway. PAs have been reported to be beneficial for human and animal health and are particularly important in pastoral agricultural systems for improved animal production and reduced greenhouse gas emissions. However, the main forage legumes grown in these systems, such as Trifolium repens and Medicago sativa, do not contain any substantial amounts of PAs in leaves. We have identified from the foliar PA-accumulating legume Trifolium arvense an R2R3-MYB transcription factor, TaMYB14, and provide evidence that this transcription factor is involved in the regulation of PA biosynthesis in legumes. TaMYB14 expression is necessary and sufficient to up-regulate late steps of the phenylpropanoid pathway and to induce PA biosynthesis. RNA interference silencing of TaMYB14 resulted in almost complete cessation of PA biosynthesis in T. arvense, whereas Nicotiana tabacum, M. sativa, and T. repens plants constitutively expressing TaMYB14 synthesized and accumulated PAs in leaves up to 1.8% dry matter. Targeted liquid chromatography-multistage tandem mass spectrometry analysis identified foliar PAs up to degree of polymerization 6 in leaf extracts. Hence, genetically modified M. sativa and T. repens plants expressing TaMYB14 provide a viable option for improving animal health and mitigating the negative environmental impacts of pastoral animal production systems.

Polyphenolics are an important class of biologically active compounds found in plants and are purported to have benefits for human health. To assess their significance in foods, improved methods are required for their detection and classification. In this paper, we describe a targeted MS2 approach using an ion-trap mass spectrometer for the analysis and classification of proanthocyanidins (PAs) in crude plant-derived extracts, which has been applied to compare green, oolong and black tea crude extracts and a grape seed extract. The method could separate, detect and provide qualitative information (full scan MS2 spectra) to enable chromatographic and mass spectral confirmation of PAs in the crude tea extracts. Using the developed methodology we report here for the first time the detection of PA tetramers in black tea, and PA trimers and tetramers in oolong tea.

Objective: We have previously demonstrated that markers of subclinical organ damage (SOD) improve cardiovascular risk prediction in healthy individuals. We wanted to investigate whether this additive effect of SOD was due to inaccurate blood pressure (BP) measurement or whether ambulatory BP (AMBP) added further to risk prediction. Methods: In a population cohort of 1385 Danish individuals free of cardiovascular disease and diabetes, we recorded traditional risk factors, AMBP and pulse wave velocity (PWV), urine albumin/creatinine ratio (UACR), left ventricular mass index (LVMI) and carotid atherosclerotic plaques at baseline. A composite cardiovascular endpoint (CEP) consisting of cardiovascular death and nonfatal myocardial infarction and stroke was recorded in national registries. Results: During a median follow-up of 12.8 years, a total of 119 CEPs occurred. In categorical analysis, presence of SOD as well as masked hypertension increased sensitivity of Systemic Coronary Risk Estimation from 73.9 to 89.1% (P

Graphical abstract Epichloënin A (1) has been isolated from fungal cultures together with a minor variant epichloënin B, and ferriepichloënin A and a related peptide epichloëamide have been found in endophyte-infected plants. Highlights ► Epichloënin A has been characterized as a ferrichrome octapeptide by HRMSn and NMR. ► The minor heptapeptide variant epichloënin B has been characterized by HRMSn. ► Ferriepichloënin A was detected in guttation fluid of endophyte-infected plants. ► The related peptide epichloëamide was also detected in guttation fluid. ► Epichloëamide has been characterized as an octapeptide by HRMSn., A number of genes encoding non-ribosomal peptide synthetases (NRPSs) have been identified in fungi of Epichloë/Neotyphodium species, endophytes of Pooid grasses, including sidN, putatively encoding a ferrichrome siderophore-synthesizing NRPS. Targeted gene replacement and complementation of sidN in Epichloë festucae has established that extracellular siderophore epichloënin A is the major product of the SidN enzyme complex (Johnson et al., 2007a). We report here high resolution mass spectrometric fragmentation experiments and NMR analysis of an isolated fraction establishing that epichloënin A is a siderophore of the ferrichrome family, comprising a cyclic sequence of four glycines, a glutamine and three Nδ-trans-anhydromevalonyl–Nδ-hydroxyornithine (AMHO) moieties. Epichloënin A is unusual among ferrichrome siderophores in comprising an octapeptide rather than hexapeptide sequence, and in incorporating a glutamine residue. During this investigation we have established that desferrichrome siderophores with pendant trans-AMHO groups can be distinguished from those with pendant cis-AMHO groups by the characteristic neutral loss of an hydroxyornithine moiety in the MS/MS spectrum. A minor component, epichloënin B, has been characterized as the triglycine variant by mass spectrometry. A peptide characterized by mass spectrometry as the putative deoxygenation product, epichloëamide has been detected together with ferriepichloënin A in guttation fluid from ryegrass (Lolium perenne) plants infected with wild-type E. festucae, but not in plants infected with the ΔsidN mutant strain, and also detected at trace levels in wild-type E. festucae fungal culture.

Background Forage plant breeding is under increasing pressure to deliver new cultivars with improved yield, quality and persistence to the pastoral industry. New innovations in DNA sequencing technologies mean that quantitative trait loci analysis and marker-assisted selection approaches are becoming faster and cheaper, and are increasingly used in the breeding process with the aim to speed it up and improve its precision. High-throughput phenotyping is currently a major bottle neck and emerging technologies such as metabolomics are being developed to bridge the gap between genotype and phenotype; metabolomics studies on forages are reviewed in this article. Scope Major challenges for pasture production arise from the reduced availability of resources, mainly water, nitrogen and phosphorus, and metabolomics studies on metabolic responses to these abiotic stresses in Lolium perenne and Lotus species will be discussed here. Many forage plants can be associated with symbiotic microorganisms such as legumes with nitrogen fixing rhizobia, grasses and legumes with phosphorus-solubilizing arbuscular mycorrhizal fungi, and cool temperate grasses with fungal anti-herbivorous alkaloid-producing Neotyphodium endophytes and metabolomics studies have shown that these associations can significantly affect the metabolic composition of forage plants. The combination of genetics and metabolomics, also known as genetical metabolomics can be a powerful tool to identify genetic regions related to specific metabolites or metabolic profiles, but this approach has not been widely adopted for forages yet, and we argue here that more studies are needed to improve our chances of success in forage breeding. Conclusions Metabolomics combined with other ‘-omics’ technologies and genome sequencing can be invaluable tools for large-scale geno- and phenotyping of breeding populations, although the implementation of these approaches in forage breeding programmes still lags behind. The majority of studies using metabolomics approaches have been performed with model species or cereals and findings from these studies are not easily translated to forage species. To be most effective these approaches should be accompanied by whole-plant physiology and proof of concept (modelling) studies. Wider considerations of possible consequences of novel traits on the fitness of new cultivars and symbiotic associations need also to be taken into account.

Compound identification is a major issue for many scientists but especially practitioners of metabolomics and phytochemists. The methods used, however, only highlight a mass-to-charge ratio and a retention time for identification purposes. This level of information is usually insufficient to identify a metabolite categorically, even when accurate mass systems have been used. Achieving full characterisation of the analyte requires the use of labour-intensive methods for isolation and subsequent analysis by orthogonal analytical techniques such as nuclear magnetic resonance spectroscopy to enable the identification of compounds or for further confirmation of activity by techniques such as bioassays. This paper reports on an automated and rapid method for the monitoring of preparative separation techniques. The described method is based on sequential flow injection analysis with mass spectral monitoring, using either single-stage or mass spectrometry to the nth power (MS n ) techniques. The techniqu...

Fructans are polymeric carbohydrates, which play important roles as plant reserve carbohydrates and stress protectants, and are beneficial for human health and animal production. Fructans are formed by the addition of β-d-fructofuranosyl units to sucrose, leading to very complex mixtures of 1-kestose based inulins, 6-kestose linked levans, and 6G-kestose derived neoseries inulins and levans in cool season grasses such as Lolium perenne. The identification of isomeric fructan oligomers in chromatographic analysis of crude plant extracts is often hampered by the lack of authentic standards, and unambiguous peak assignment usually requires time-consuming analyses of purified fructan oligomers. We have developed a LC-MS(n) method for the separation and detection of fructan isomers and present here evidence for specific MS(n) fragmentation patterns associated with β 1-2 (inulins) and β 2-6 (levans) fructans. LC-MS(n) analysis of (13)C labeled fructan oligomers produced by L. perenne fructosyltransferases expressed in yeast has enabled us to account for the observed fragmentation patterns in terms of preferential cleavage of the glycosidic bond between O- and fructose C2 in both inulins and levans and to differentiate reducing-end from nonreducing end cross ring cleavages in levans. We propose that higher order MS fragmentation patterns can be used to distinguish between the two major classes of fructan, i.e., inulins and levans, without the need for authentic standards.

The roles of the c-Jun N-terminal kinases (JNKs) in inflammatory arthritis have been investigated; however, the roles of each isotype (ie, JNK1 and JNK2) in rheumatoid arthritis and conclusions about whether inhibition of one or both is necessary for amelioration of disease are unclear. By using JNK1- or JNK2-deficient mice in the collagen-induced arthritis and the KRN T-cell receptor transgenic mouse on C57BL/6 nonobese diabetic (K/BxN) serum transfer arthritis models, we demonstrate that JNK1 deficiency results in protection from arthritis, as judged by clinical score and histological evaluation in both models of inflammatory arthritis. In contrast, abrogation of JNK2 exacerbates disease. In collagen-induced arthritis, the distinct roles of the JNK isotypes can, at least in part, be explained by altered regulation of CD86 expression in JNK1- or JNK2-deficient macrophages in response to microbial products, thereby affecting T-cell-mediated immunity. The protection from K/BxN serum-induced arthritis in Jnk1(-/-) mice can also be explained by inept macrophage function because adoptive transfer of wild-type macrophages to Jnk1(-/-) mice restored disease susceptibility. Thus, our results provide a possible explanation for the modest therapeutic effects of broad JNK inhibitors and suggest that future therapies should selectively target the JNK1 isoform.

High-sugar grass varieties have received considerable attention for their potential to reduce nitrogen (N) excretion and increase milk yield in cattle. However, considerable variation exists in the magnitude of response in published results. The purpose of this study is to explain the variation in response using a dynamic mechanistic model to predict observed N and milk yield results from the literature, and from simulated data. Examined effects were (1) water-soluble carbohydrate [WSC; g/kg of dry matter (DM)] increase; (2) change in crude protein (CP) and neutral detergent fiber (NDF) content of the plant with WSC increase; and (3) the level of N fertilization. The database for evaluation of model N and milk yield predictions consisted of 4 published studies with 28 treatment means for which high-sugar grasses were being evaluated. Water-soluble carbohydrate content of the diets ranged from 95 to 248 g/kg of DM, CP content ranged from 115 to 263 g/kg of DM, and the NDF content ranged from 400 to 568 g/kg of DM. Urine N, milk N, and total N excretion were predicted well by the model and followed the directional pattern of observed values within each study. Simulation results showed that the N utilization ratio increased as the WSC content of the diet increased, but to varying degrees depending on the grass scenario examined. The greatest benefit in terms of N utilization ratio and urine N levels were seen when the WSC content of grass increased at the expense of CP, followed by a 50:50 CP and NDF mix, followed by a trade for NDF. Simulated milk yield decreased slightly when WSC increased at the expense of CP, increased slightly when it increased at the expense of a CP and NDF mix, and increased most when WSC increased at the expense of NDF. Results were amplified slightly under conditions of low-N fertilization and in the absence of grain feeding. Overall, modeling is useful as an explanatory tool. The variation from results in the literature with high-WSC grass feeding may be, at least in part, the result of the level of WSC (g/kg of DM) increase, concurrent changes occurring within the CP and NDF fractions of the plant, and the plane of nutrition of the diet (grain feeding and N fertilization levels).

Summary 1. Cool-season grasses can be simultaneously infected by foliar fungal endophytes and colonised by mycorrhizal fungi, the integrated functions of which are strong predictors of plant fitness within grassland ecosystems. Evidence has been presented previously that infection of grass species with foliar endophytes can negatively affect mycorrhizal colonisation. Here, we tested the hypothesis that mycorrhizal colonisation in turn adversely affects Neotyphodium endophyte concentrations and that the competitive interaction between the two endosymbionts is affected by resource supply. 2. Specifically, we report how competition between Glomus (G. mosseae– GM, G. intraradices– GI) mycorrhizal fungi and N. lolii (common strain (CS) and AR1) foliar endophytic strains is affected by P supply and water-soluble carbohydrate (WSC) content in two Lolium perenne (perennial ryegrass) cultivars: a high sugar grass, AberDart, and a conventional (control) grass, Fennema. 3. The presence of Glomus mycorrhizae reduced the concentrations of endophytes and alkaloids in leaf blades and pseudostems. The reduction depended on P supply, ryegrass cultivar (notably WSC content) and endophyte strain. Conversely, foliar endophyte infection reduced mycorrhizal colonisation rates and concentrations in the roots of the control cultivar Fennema, although not in the high sugar cultivar, AberDart. 4. Neither GM nor N. lolii infection had an effect per se on the yield of root or blade compared with mycorrhiza-free (M-) and endophyte-free (E-) plants, respectively; though, yield of roots and blades was reduced by GI infection and at low P. 5. Competitive interactions between ecologically widespread foliar endophytes (valuable for plant protection) and mycorrhizal endosymbionts (valuable for P acquisition) as seen in this study are of critical importance especially in areas of high pest prevalence and low P availability. Our work stresses the need for elucidating the physiological/metabolic basis for such interactions between endosymbionts to understand how these processes contribute to plant performance and fitness in grassland ecosystems.

Pastoral-based animal production systems are under increasing pressure to provide the high quantity and quality of feed needed for optimal ruminant performance. The capacity of farmers to increase forage yield further, solely by increasing fertilizer inputs or through improved pasture management, is limited. Emerging requirements to balance industry production targets against the need to reduce greenhouse gas emissions and N losses pose further challenges. Plant breeding is being asked to deliver results more urgently than at any time previously, and this review attempts to highlight issues that might limit the prospects for future progress by seeking lessons from four past examples: (i) white clover breeding gains and the need to consider the complexity of the grazed grass-clover mixed sward, with its tendency for cycling in plant species composition; (ii) a systems field trial of new and old grass ⁄ clover cultivars, and how the complexity of growth of perennial forage crops, and the dynamic optimality required for sustainable harvesting might limit our ability to breed for ‘yield’ per se; (iii) the manipulation of a physiological trait (low ‘maintenance’ respiration) and the implications of such changes for plant fitness and G · E interactions; and (iv) an hypothesis-driven development of a trait (high-sugar grasses) and the value of ‘proof of concept’ studies, the requirement of scientific understanding of the mechanisms of trait expression, and how one might in future go about assessing breeding achievements. We discuss the general ecological considerations around shifts in the frequency distribution of traits in new populations, whether altered conventionally or by genetic modification, and how selection for a particular trait might inadvertently reduce both fitness and persistence. A major priority for breeding, we propose, might be to revisit previously abandoned traits that affected the physiological performance of forage species, armed now with a capacity to monitor gene expression at the molecular level, and so unravel ⁄ control the G · E interactions that limited their benefits. We also discuss how a ‘loss of yield advantage’ of new cultivars, seen when tested several years after sowing, requires urgent investigation and propose this might be associated with fitness costs of perenniality. Finally, we argue for a careful reconsideration of what are realistic expectations for systems field trials and that focus on forage breeding might be shifted more to ‘proof of concept’ studies, critical experimental design, comparing ‘traits’ rather than ‘cultivars’, and the wider ecological assessment of fitness and function of traits in the plant, community and ecosystem.