G-TwYST harmonisation of statistical methods for use of omics data in food safety assessment

The G-TwYST project mainly focused on the statistical analysis of results from animal studies where a limited number of variables based on OECD guidance was measured. In the statistical analysis of these measurements, emphasis was placed on the possibilities to test the equivalence of the GM groups relative to historical non-GM groups from the GRACE study. A new univariate statistical approach was proposed for this purpose. In G-TwYST, only limited attention was paid to food safety assessment using the plant material that was used as feed in the animal studies, even though such material can be easily obtained. Especially high-throughput untargeted omics measurements (e.g. metabolomics or transcriptomics) of this material could be an excellent approach for checks on unintended effects due to the larger number of compounds (e.g. metabolites, transcripts) that are simultaneously measured. In G-TwYST a limited number of samples from the maize harvests has been analysed by transcriptomics and metabolomics. Subsequently, an already available multivariate one-class model was used to compare the G-TwYST samples to a reference set of measurements, as had already been done before in the GRACE project. The pilot studies in GRACE and G-TwYST highlight the potential of omics-based for food safety assessment. However, there is a need to study the statistical properties of the applied multivariate one-class model and among a range of alternative models it is unclear whether it is the most appropriate method for omics-based food safety assessment. Additionally, the applied approach is distinctly different from the univariate methodology that was developed in G-TwYST for animal studies. There is no reason why statistical methodology should be different for similar data from plant or animal studies. From a regulator’s point of view the statistical criteria for evaluating food safety data should as much as possible be the same. However, statistical methods will need to be different for highly multivariate omics data and for the more traditional univariate measurements. The main purpose of this short report is therefore to identify the differences between statistical approaches for food safety assessment as were applied in the context of the G-TwYST project, and to suggest directions for future research to improve the harmonisation of statistical methodology for analysing omics data

Metabolomics of photosynthetically active tissues in white grapes: effects of light microclimate and stress mitigation strategies

The effects of climate change are becoming a real concern for the viticulture sector, with impacts on both grapevine physiology and the quality of the fresh berries and wine. Short-term mitigation strategies, like foliar kaolin application and smart irrigation regimes, have been implemented to overcome these problems. We previously showed that these strategies also influence the photosynthetic activity of the berries themselves, specifically in the exocarp and seed. In the present work, we assessed the modulating effects of both canopy-light microclimate, kaolin and irrigation treatments on the metabolic profiles of the exocarp and seed, as well as the potential role of berry photosynthesis herein. Berries from the white variety Alvarinho were collected at two contrasting light microclimate positions within the vine canopy (HLhigh light and LLlow light) from both irrigated and kaolin-treated plants, and their respective controls, at three fruit developmental stages (green, véraison and mature). Untargeted liquid chromatography mass spectrometry (LCMS) profiling of semi-polar extracts followed by multivariate statistical analysis indicate that both the light microclimate and irrigation influenced the level of a series of phenolic compounds, depending on the ripening stage of the berries. Moreover, untargeted gas chromatography mass spectrometry (GCMS) profiling of polar extracts show that amino acid and sugar levels were influenced mainly by the interaction of irrigation and kaolin treatments. The results reveal that both photosynthetically active berry tissues had a distinct metabolic profile in response to the local light microclimate, which suggests a specific role of photosynthesis in these tissues. A higher light intensity within the canopy mainly increased the supply of carbon precursors to the phenylpropanoid/flavonoid pathway, resulting in increased levels of phenolic compounds in the exocarp, while in seeds, light mostly influenced compounds related to carbon storage and seed development. In addition, our work provides new insights into the influence of abiotic stress mitigation strategies on the composition of exocarps and seeds, which are both important tissues for the quality of grape-derived products.This research and APC were funded by the FCT-Portuguese Foundation for Science and Technology, grant provided to Andreia Garrido (PD/BD/128275/2017), under the Doctoral Program "Agricultural Production Chains-from fork to farm" (PD/00122/2012).info:eu-repo/semantics/publishedVersio

Space and patchiness affects diversity–function relationships in fungal decay communities

The space in which organisms live determines health and physicality, shaping the way in which they interact with their peers. Space, therefore, is critically important for species diversity and the function performed by individuals within mixed communities. The biotic and abiotic factors defined by the space that organisms occupy are ecologically significant and the difficulty in quantifying space-defined parameters within complex systems limits the study of ecological processes. Here, we overcome this problem using a tractable system whereby spatial heterogeneity in interacting fungal wood decay communities demonstrates that scale and patchiness of territory directly influence coexistence dynamics. Spatial arrangement in 2- and 3-dimensions resulted in measurable metabolic differences that provide evidence of a clear biological response to changing landscape architecture. This is of vital importance to microbial systems in all ecosystems globally, as our results demonstrate that community function is driven by the effects of spatial dynamics

Intragraft heme oxygenase-1 and coronary artery disease after heart transplantation

Peri-operative tissue injury triggers the development of Transplant Coronary Artery Disease (TCAD). Animal studies have shown that induction of heme oxygenase (HO)-1 protects the donor organ from the development of TCAD. To investigate the role of HO-1 in TCAD after clinical heart transplantation, we measured intragraft mRNA expression of HO-1, HIF-1α, TGF-β, FLIP, and the Bcl-2/Bax balance. Immunohistochemical staining of HO-1 was performed to determine its origin. Myocardial biopsies taken at the end of the transplantation procedure (time 0), at 1 week and at 10 months after transplantation were studied from recipients with or without angiographic signs of accelerated TCAD, diagnosed after 1 year. At time 0, no differences in mRNA expression for any of the measured parameters were found between TCAD positive and negative patients. At 1 week, mRNA expression of HO-1 and TGF-β was higher in grafts that developed accelerated TCAD (p=0.001 and p=0.0002). These higher mRNA levels were accompanied by a pro-apoptotic shift in Bcl-2/Bax (p=0.02), suggesting proneness for apoptosis via the mitochondrial pathway. Immunohistochemical staining showed that HO-1 was mainly produced by infiltrating macrophages. At 10 months, again HO-1 and TGF-β levels were high in TCAD positive patients (p=0.02 and p=0.05), but the expression of apoptotic markers was comparable at this time point. Our results suggest that a higher HO-1 by macrophages in our patient population might be an adaptive response to tissue injury and inflammation, reflecting damage due to the transplantation procedure that finally results in TCAD

Application of passive sampling to characterise the fish exometabolome

The endogenous metabolites excreted by organisms into their surrounding environment, termed the exometabolome, are important for many processes including chemical communication. In fish biology, such metabolites are also known to be informative markers of physiological status. While metabolomics is increasingly used to investigate the endogenous biochemistry of organisms, no non-targeted studies of the metabolic complexity of fish exometabolomes have been reported to date. In environmental chemistry, Chemcatcher® (Portsmouth, UK) passive samplers have been developed to sample for micro-pollutants in water. Given the importance of the fish exometabolome, we sought to evaluate the capability of Chemcatcher® samplers to capture a broad spectrum of endogenous metabolites excreted by fish and to measure these using non-targeted direct infusion mass spectrometry metabolomics. The capabilities of C18 and styrene divinylbenzene reversed-phase sulfonated (SDB-RPS) Empore™ disks for capturing non-polar and polar metabolites, respectively, were compared. Furthermore, we investigated real, complex metabolite mixtures excreted from two model fish species, rainbow trout (Oncorhynchus mykiss) and three-spined stickleback (Gasterosteus aculeatus). In total, 344 biological samples and 28 QC samples were analysed, revealing 646 and 215 m/z peaks from trout and stickleback, respectively. The measured exometabolomes were principally affected by the type of Empore™ (Hemel Hempstead, UK) disk and also by the sampling time. Many peaks were putatively annotated, including several bile acids (e.g., chenodeoxycholate, taurocholate, glycocholate, glycolithocholate, glycochenodeoxycholate, glycodeoxycholate). Collectively these observations show the ability of Chemcatcher® passive samplers to capture endogenous metabolites excreted from fish

Non-targeted UHPLC-MS metabolomic data processing methods: A comparative investigation of normalisation, missing value imputation, transformation and scaling

INTRODUCTION: The generic metabolomics data processing workflow is constructed with a serial set of processes including peak picking, quality assurance, normalisation, missing value imputation, transformation and scaling. The combination of these processes should present the experimental data in an appropriate structure so to identify the biological changes in a valid and robust manner. OBJECTIVES: Currently, different researchers apply different data processing methods and no assessment of the permutations applied to UHPLC-MS datasets has been published. Here we wish to define the most appropriate data processing workflow. METHODS: We assess the influence of normalisation, missing value imputation, transformation and scaling methods on univariate and multivariate analysis of UHPLC-MS datasets acquired for different mammalian samples. RESULTS: Our studies have shown that once data are filtered, missing values are not correlated with m/z, retention time or response. Following an exhaustive evaluation, we recommend PQN normalisation with no missing value imputation and no transformation or scaling for univariate analysis. For PCA we recommend applying PQN normalisation with Random Forest missing value imputation, glog transformation and no scaling method. For PLS-DA we recommend PQN normalisation, KNN as the missing value imputation method, generalised logarithm transformation and no scaling. These recommendations are based on searching for the biologically important metabolite features independent of their measured abundance. CONCLUSION: The appropriate choice of normalisation, missing value imputation, transformation and scaling methods differs depending on the data analysis method and the choice of method is essential to maximise the biological derivations from UHPLC-MS datasets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-016-1030-9) contains supplementary material, which is available to authorized users

Using AMF inoculum to improve the nutritional status of Prunella vulgaris plants in green roof substrate during establishment

Arbuscular mycorrhizal fungi (AMF) have been shown to improve the growth, health, nutrient uptake, flowering and drought tolerance of many terrestrial plant species. Green roofs are generally deficient in nutrients, organic matter and water, and therefore AMF could be extremely beneficial in improving green roof plant performance. Despite this there is a lack of empirical research into artificially introducing AMF into green roof substrates. In this study, a commercial AMF inoculum was applied to Prunella vulgaris green roof plugs grown in small modules on a flat roof in Sheffield, UK. The modules were filled with commercial green roof substrate (80% small particle sized crushed brick, 20% green waste compost) to a depth of 100 mm. AMF inoculum was applied as four treatments: (i) directly with plug, (ii) mixed evenly into surrounding substrate, (iii) split between plug and substrate, (iv) control treatment with no inoculum added. Significantly greater levels of AMF colonisation of P. vulgaris roots was detected in all AMF treatments compared to the control. Low levels of AMF colonisation of P. vulgaris roots were also observed in the control treatment, confirming that low levels of AMF inoculum were present in this commercial substrate. Shoot phosphorous (P) concentration was improved in all AMF treatments, however there was no significant effect of any AMF treatment on P. vulgaris growth rate or biomass production. The highest AMF colonisation of P. vulgaris roots was observed when AMF inoculum was directly added to just the plug. Promisingly, P. vulgaris flowering time at the end of the first growing season was also extended in the plug AMF treatment only. This study has confirmed that commercial AMF inoculum can be used to successfully colonise plants and introduce AMF networks into green roof substrate. Although AMF inoculum was naturally present in the substrate used in this study, levels were extremely low, and unlikely to have any significant effect on plants. This study indicates that care should be taken in the use of AMF inoculum on green roofs, as the growth and health benefits of AMF are not always immediately apparent for green roof plants. In addition much more research is required in order to fully assess the extent of the benefits of AMF on green roof plants and to determine if their use can be financially viable

Genotyping a second growth coast redwood forest : a high throughput methodology

The idea that excitonic (electronic) coherences are of fundamental importance to natural photosynthesis gained popularity when slowly dephasing quantum beats (QBs) were observed in the two-dimensional electronic spectra of the Fenna–Matthews–Olson (FMO) complex at 77 K. These were assigned to superpositions of excitonic states, a controversial interpretation, as the strong chromophore–environment interactions in the complex suggest fast dephasing. Although it has been pointed out that vibrational motion produces similar spectral signatures, a concrete assignment of these oscillatory signals to distinct physical processes is still lacking. Here we revisit the coherence dynamics of the FMO complex using polarization-controlled two-dimensional electronic spectroscopy, supported by theoretical modelling. We show that the long-lived QBs are exclusively vibrational in origin, whereas the dephasing of the electronic coherences is completed within 240 fs even at 77 K. We further find that specific vibrational coherences are produced via vibronically coupled excited states. The presence of such states suggests that vibronic coupling is relevant for photosynthetic energy transfer