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In the execution of its mission to promote a common European measurement system in support of EU policies, IRMM's Reference Materials Unit is currently involved in preparation of proficiency-testing samples and candidate reference materials. Recent work related to bovine spongiform encephalopathy in cows, genetically modified organisms, and a variety of environmental materials is described.
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There is a growing interest in producing food plants with increased amounts of flavonoids because of their potential health benefits. Tomatoes contain small amounts of flavonoids, most of which are located in the peel of the fruit. It has been shown that flavonoid accumulation in tomato flesh, and hence an overall increase in flavonoid levels in tomato fruit, can be achieved by means of simultaneous overexpression of the maize transcription factors LC and C1. Fruit from progeny of two modified lines (2027 and 2059) was selected for a detailed analysis and individual identification of flavonoids, at different stages of maturity. Nine major flavonoids were detected in the flesh of transgenic ripe tomatoes. LC/NMR, LC/MS, and LC/MS/MS enabled us to identify these as kaempferol-3,7-di-O-glucoside (1), kaempferol-3-O-rutinoside-7-O-glucoside (2), two dihydrokaempferol-O-hexosides (3 and 4), rutin (5), kaempferol-3-O-rutinoside (6), kaempferol-3-O-glucoside (7), naringenin-7-O-glucoside (8) and naringenin chalcone (9), which were quantified by HPLC/DAD. All but 5, 6, and 9 were detected in tomato for the first time. The total flavonoid glycoside content of ripe transgenic tomatoes of line 2059 was about 10-fold higher than that of the controls, and kaempferol glycosides accounted for 60% of this. Kaempferol glycosides comprised around 5% of the flavonoid glycoside content of ripe control tomatoes (the rest was rutin and naringenin chalcone). The rutin concentration in both transgenic and control fruits was similar.
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An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.
A DNA piezoelectric sensor has been developed for the detection of genetically modified organisms (GMOs). Single stranded DNA (ssDNA) probes were immobilised on the sensor surface of a quartz crystal microbalance (QCM) device and the hybridisation between the immobilised probe and the target complementary sequence in solution was monitored. The probe sequences were internal to the sequence of the 35S promoter (P) and Nos terminator (T), which are inserted sequences in the genome of GMOs regulating the transgene expression. Two different probe immobilisation procedures were applied: (a) a thiol-dextran procedure and (b) a thiol-derivatised probe and blocking thiol procedure. The system has been optimised using synthetic oligonucleotides, which were then applied to samples of plasmidic and genomic DNA isolated from the pBI121 plasmid, certified reference materials (CRM), and real samples amplified by the polymerase chain reaction (PCR). The analytical parameters of the sensor have been investigated (sensitivity, reproducibility, lifetime etc.). The results obtained showed that both immobilisation procedures enabled sensitive and specific detection of GMOs, providing a useful tool for screening analysis in food samples.
What is the relationship between biotechnology employees' beliefs about the moral outcomes of a controversial transgenic research project and their attitudes of acceptance towards the project? To answer this question, employees (n=466) of a New Zealand company, AgResearch Ltd., were surveyed regarding a project to create transgenic cattle containing a synthetic copy of the human myelin basic protein gene (hMBP). Although diversity existed amongst employees' attitudes of acceptance, they were generally: in favor of the project, believed that it should be allowed to proceed to completion, and that it is acceptable to use transgenic cattle to produce medicines for humans. These three items were aggregated to form a project acceptance score. Scales were developed to measure respondents' beliefs about the moral outcomes of the project for identified stakeholders in terms of the four principles of common morality (benefit, non-harm, justice, and autonomy). These data were statistically aggregated into an Ethical Valence Matrix fo the project. The respondents' project Ethical Valence Scores correlated significantly with their project acceptance scores (r=0.64, p<0.001), accounting for 41% of the variance in respondents' acceptance attitudes. Of the four principles, non-harm had the strongest correlation with attitude to the project (r=0.59), followed by benefit and justice (both r=0.54), then autonomy (r=0.44). These results indicate that beliefs about the moral outcomes of a research project, in terms of the four principles approach, are strongly related to, and may be significant determinants of, attitudes to the research project. This suggests that, for employees of a biotechnology organization, ethical reasoning could be a central mechanism for the evaluation of the acceptability of a project. We propose that the Ethical Valence Matrix may be used as a tool to measure ethical attitudes towards controversial issues, providing a metric for comparison of perceived ethical consequences for multiple stakeholder groups and for the evaluation and comparison of the ethical consequences of competing alternative issues or projects. The tool could be used to measure both public and special interest groups' ethical attitudes and results used for the development of socially responsible policy or by science organizations as a democratizing decision aid to selection amongst projects competing for scarce research funds.
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The ability to produce exopolysaccharides (EPS) is widespread among lactic acid bacteria (LAB), although the physiological role of these molecules has not been clearly established yet. Some EPS confer on LAB a "ropy" character that can be detected in cultures that form long strands when extended with an inoculation loop. When EPS are produced in situ during milk fermentation they can act as natural biothickeners, giving the product a suitable consistency, improving viscosity, and reducing syneresis. In addition, some of these EPS may have beneficial effects on human health. The increasing demand by consumers of novel dairy products requires a better understanding of the effect of EPS on existing products and, at the same time, the search for new EPS-producing strains with desirable properties. The use of genetically modified organisms capable of producing high levels of EPS or newly designed biopolymers is still very limited. Therefore, exploration of the biodiversity of wild LAB strains from natural ecological environments is currently the most suitable approach to search for the desired EPS-phenotype. The screening of ropy strains and the isolation and characterization of EPS responsible for this characteristic have led to the application over the past years of a wide variety of techniques. This review summarizes the available information on methods and procedures used for research on this topic. The information provided deals with methods for screening of EPS-producing LAB, detection of the ropy phenotype, and the physicochemical and structural characterization of these molecules, including parameters related to their viscosifying properties. To our knowledge, this is the first compilation of methods available for the study of EPS produced by LAB.
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The role of parental behaviour in the development of food preferences is considered. Food preferences develop from genetically determined predispositions to like sweet and salty flavours and to dislike bitter and sour tastes. Particularly towards the second year of life, there is a tendency to avoid novel foods (neophobia). Food aversions can be learnt in one trial if consumption is followed by discomfort. There is a predisposition to learn to like foods with high-energy density. However, from birth genetic predispositions are modified by experience and in this context during the early years parents play a particularly important role. Parental style is a critical factor in the development of food preferences. Children are more likely to eat in emotionally positive atmospheres. Siblings, peers and parents can act as role models to encourage the tasting of novel foods. Repeated exposure to initially disliked foods can breakdown resistance. The offering of low-energy-dense foods allows the child to balance energy intake. Restricting access to particular foods increases rather than decreases preference. Forcing a child to eat a food will decrease the liking for that food. Traditionally, educational strategies have typically involved attempts to impart basic nutritional information. Given the limited ability of information to induce changes in behaviour, an alternative strategy would be to teach parents about child development in the hope that an understanding of the characteristic innate tendencies and developmental stages can be used to teach healthy food preferences.
As rich sources of vitamins and minerals, fruits are an important part of our diet. However, they are highly perishable. As the biochemical basis for fruit quality is being unraveled, so genetic approaches are being developed to modify the way in which fruit ripen, for the benefit of the consumer.
At present, the development of plants with improved traits like superior quality, high yield, or stress resistance, are highly desirable in agriculture. Accelerated crop improvement, however, must capitalize on revolutionary new plant breeding technologies, like genetically modified and gene-edited crops, to heighten food crop traits. Genome editing still faces ineffective methods for the transformation and regeneration of different plant species and must surpass the genotype dependency of the transformation process. Tomato is considered an alternative plant model system to rice and Arabidopsis, and a model organism for fleshy-fruited plants. Furthermore, tomato cultivars like Micro-Tom are excellent models for tomato research due to its short life cycle, small size, and capacity to grow at high density. Therefore, we developed an indirect somatic embryo protocol from cotyledonary tomato explants and used this to generate epigenetically edited tomato plants for the SlWRKY29 gene via CRISPR-activation (CRISPRa). We found that epigenetic reprogramming for SlWRKY29 establishes a transcriptionally permissive chromatin state, as determined by an enrichment of the H3K4me3 mark. A whole transcriptome analysis of CRISPRa-edited pro-embryogenic masses and mature somatic embryos allowed us to characterize the mechanism driving somatic embryo induction in the edited tomato cv. Micro-Tom. Furthermore, we show that enhanced embryo induction and maturation are influenced by the transcriptional effector employed during CRISPRa, as well as by the medium composition and in vitro environmental conditions such as osmotic components, plant growth regulators, and light intensity.
Genetic manipulation of durum wheats by tobacco rab-1 genes influence the trafficking of gluten proteins through the secretory system by up- or down-regulating the transport step from the ER to the Golgi apparatus which may in turn modify functional performance of the grain. Gluten proteins were extracted from two genetically manipulated lines - Svevo B730 1-1 and Ofanto B688 1-2 - and their control lines and were analyzed by two dimensional gel electrophoresis. When the two-dimensional maps were compared by image analysis no significant differences between the GM line with an up-regulated trafficking containing the wild type tobacco rab1 (Svevo B730 1-1) and its control (Svevo control). By contrast, significant differences were found between the GM line with a down-regulated trafficking due to the tobacco rab1 mutant form (Ofanto B688 1-2) and its control (Ofanto control). Of the new protein spots detected in the down-regulated Ofanto B688 1-2 map, only a beta-amylase was identified. The remaining spots were susceptible to chymotripsin action but not to trypsin one, as in the case of the gluten protein. Rheological measurements showed that gluten quality was enhanced in the down-regulated Ofanto B688 1-2 without an increase in the amount of gluten. Proteomics is a useful and powerful tool for investigating protein changes in GMOs and in understanding events in food science and technology.