Clustering and classification of reference documents from large-scale literature searches: Support to the SAM explanatory note "New Techniques in Agricultural Biotechnology"

Searches of the existing scientific literature are the cornerstone of scientific research and reporting. With the constant growth of the rate at which scientific studies and reviews are being published, the information produced by these searches can be delicate to manage. Narrowing the search increases the risk of overlooking important documents, while broadening it can produce too many documents to be reasonably processed. This report describes a set of strategies designed to process large sets of scientific references (such as those obtained by broad literature searches) and assist in the identification of documents relevant for specific purposes. These strategies take advantage of metadata associated to each document in SCOPUS, the database of peer-reviewed literature maintained by Elsevier and accessible through an Application Programming Interface (API). These strategies were developed and applied in support to the European Commission's Scientific Advice Mechanism (SAM) in managing the results of literature searches in the context of the exploratory note "New Techniques in Agricultural Biotechnology".JRC.F.7-Knowledge for Health and Consumer Safet

PlasmaDNA: a free, cross-platform plasmid manipulation program for molecular biology laboratories

<p>Abstract</p> <p>Background</p> <p>Most molecular biology experiments, and the techniques associated with this field of study, involve a great deal of engineering in the form of molecular cloning. Like all forms of engineering, perfect information about the starting material is crucial for successful completion of design and strategies.</p> <p>Results</p> <p>We have generated a program that allows complete <it>in silico </it>simulation of the cloning experiment. Starting with a primary DNA sequence, PlasmaDNA looks for restriction sites, open reading frames, primer annealing sequences, and various common domains. The databases are easily expandable by the user to fit his most common cloning needs. PlasmaDNA can manage and graphically represent multiple sequences at the same time, and keeps in memory the overhangs at the end of the sequences if any. This means that it is possible to virtually digest fragments, to add the digestion products to the project, and to ligate together fragments with compatible ends to generate the new sequences. Polymerase Chain Reaction (PCR) fragments can also be virtually generated using the primer database, automatically adding to the fragments any 5' extra sequences present in the primers.</p> <p>Conclusion</p> <p>PlasmaDNA is a program available both on Windows and Apple operating systems, designed to facilitate molecular cloning experiments by building a visual map of the DNA. It then allows the complete planning and simulation of the cloning experiment. It also automatically updates the new sequences generated in the process, which is an important help in practice. The capacity to maintain multiple sequences in the same file can also be used to archive the various steps and strategies involved in the cloning of each construct. The program is freely available for download without charge or restriction.</p

Literature and Bioinformatics Analyses of Wheat-specific Detection Methods

In view of the recent necessity to perform testing for the detection of genetically modified common wheat (Triticum aestivum), the need arises for a taxon-specific method for this organism. However, no such method has yet been officially validated. Multiple species of wheat exist on the market, such as common wheat, durum wheat, emmer wheat, etc. These plants have complex genomes, composed of different combinations (from diploid to hexaploid) of common sets of chromosomes. The specificity of a method then depends on which set of chromosome the targeted region is located, which increases the complexity of identifying methods specific to Triticum aestivum. Often, such methods were developed for the specific regulatory need of differentiating durum and common wheat (for example, in alimentary pasta labeling), with minimal concerns for non-specific detection of other plants. This document summarises the review performed by the EU-RL GMFF, complemented with in-house bioinformatics analyses, in order to identify and characterise Triticum aestivum-specific detection methods that have been described in the scientific literature. Methods with apparent specificity (based on results shown and bioinformatics analyses) and promising performance (based on results shown) are highlighted and their primers and probe sequences reported. Those methods are the 'SS II-D' and ' SS II ex7' methods described in Matsuoka et al. (2012) and the 'wx012' method described in Iida et al. (2005), and they represent good candidates to uniquely identify common wheat in complex food samples.JRC.I.3-Molecular Biology and Genomic

Report on the Verification of the Performance of a Method for the Detection of Event MON71800 in Wheat Using Real-Time PCR

Following the United States Department of Agriculture's (USDA) Animal and Plant Health Inspection Service (APHIS) announcement that test results confirmed the finding of unauthorised GM glyphosate-resistant wheat "volunteer" plants harbouring the event MON71800 on a farm in Oregon, the European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) was requested to provide as soon as possible a method to test wheat consignments for the presence of this genetically modified organism (GMO) to the National Reference Laboratories (NRLs) for GMOs of the EU Member States. In response, the EU-RL put together a testing strategy, based on readily available screening tests which was published here (http://gmo-crl.jrc.ec.europa.eu/GM_wheat.htm). Upon request, Monsanto provided in May 2013 the EU-RL with the procedure “Roundup Ready® Wheat MON71800 Event Specific Endpoint TaqMan® PCR with acc Internal Control for Seed Pools of 1:15” that had previously been made available to, and was used by USDA. The EU-RL GMFF tested this protocol on positive control samples consisting of MON71800 crude lysate, also provided by Monsanto. Our results can be summarised as follow: The method is apparently event-specific. Our specificity-tests did not show cross-reactivity on genomic DNA from a wide selection of similar GMO. The sensitivity of the method was found to be in agreement with previous findings of USDA, i.e. the relative limit of detection lies at 0.5% in a background of 301 ng of total wheat genomic DNA. The absolute limit of detection (LODabs) was determined between 5 and 10 copies of MON71800 target. The latter was not indicated by the USDA. For seed/grains the application of a sub-sampling strategy could allow detection below 0.5% but would require significant additional efforts, including the analysis of numerous sub-samples. Our tests also indicated that the duplex PCR system at the tested stage of optimisation is characterised by poor efficiency at increasing background DNA concentration in reaction. Based on the scientific evidence described in the present report, the EU-RL suggest that its testing strategy (http://gmo-crl.jrc.ec.europa.eu/GM_wheat.htm), making use of validated element and construct-specific methods and found to be more sensitive, is used to test for presence of MON71800 GM-wheat. The verified event specific method of Monsanto could be used to confirm positive findings at GM-target concentration equal or above 0.5% or it could be used for detection of GM-event MON71800 below 0.5% but it would require a costly sub-sampling strategy, which, in addition, is only possible in seeds/grains.JRC.I.3-Molecular Biology and Genomic

Report on the Verification of the Performance of a Testing Strategy for the Detection of Wheat MON71800 Event Using Real-Time PCR

In response to a request of DG SANCO to provide National Reference Laboratories (NRLs) as soon as possible with a method to test soft white wheat consignments for the presence of unauthorised GM glyphosate-resistant wheat harbouring the event MON71800, the European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) developed, in collaboration with the European Network of GMO Laboratories (ENGL), a testing strategy intended to be immediately implementable by EU NRLs. The testing strategy is based on a combination of three validated screening methods that allow excluding (detectable) presence of Monsanto’s GM glyphosate-resistant wheat (MON71800) in wheat grain or food/feed products and confirming its presence whenever other GMOs can be excluded. The present report describes the results of the tests carried out by the EU-RL GMFF to verify the testing strategy proposed; the tests were conducted using the positive control sample represented by a crude DNA lysate of MON71800 provided by Monsanto and genomic DNA samples of genetically modified organisms harbouring the CTP2-CP4epsps element for which a validated event-specific method is available. The sensitivity of the three methods was assessed by verifying the relative limit of detection (LODrel) on MON71800 wheat DNA. The LODrel is approximately 0.03% for the P-35S and for T-nos methods and 0.06% for the CTP2-CP4epsps method in 300 nanograms of wheat genomic DNA. Further experimental evidence confirmed that the three methods react against genomic DNA extracted from GM events containing the CTP2-CP4epsps element for which a validated event-specific method is available. The experimental verification hereby reported confirmed the validity of the EU-RL GMFF guidance on testing for GM glyphosate-resistant wheat (MON71800) in wheat grain or in food/feed products containing wheat flour originating or consigned from the US, provided that DNA of acceptable quality can be obtained.JRC.I.3-Molecular Biology and Genomic

Towards plant species identification in complex samples: a bioinformatics pipeline for the identification of novel nuclear barcode candidates

Monitoring of the food chain to fight fraud and protect consumer health relies on the availability of methods to correctly identify the species present in samples, for which DNA barcoding is a promising candidate. The nuclear genome is a rich potential source of barcode targets, but has been relatively unexploited until now. Here, we show the development and use of a bioinformatics pipeline that processes available genome sequences to automatically screen large numbers of input candidates, identifies novel nuclear barcode targets and designs associated primer pairs, according to a specific set of requirements. We applied this pipeline to identify novel barcodes for plant species, a kingdom for which the currently available solutions are known to be insufficient. We tested one of the identified primer pairs and show its capability to correctly identify the plant species in simple and complex samples, validating the output of our approach.JRC.I.3-Molecular Biology and Genomic

GDNF promotes tubulogenesis of GFRα1-expressing MDCK cells by Src-mediated phosphorylation of Met receptor tyrosine kinase

Glial cell line–derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are multifunctional signaling molecules in embryogenesis. HGF binds to and activates Met receptor tyrosine kinase. The signaling receptor complex for GDNF typically includes both GDNF family receptor α1 (GFRα1) and Ret receptor tyrosine kinase. GDNF can also signal independently of Ret via GFRα1, although the mechanism has remained unclear. We now show that GDNF partially restores ureteric branching morphogenesis in ret-deficient mice with severe renal hypodysplasia. The mechanism of Ret-independent effect of GDNF was therefore studied by the MDCK cell model. In MDCK cells expressing GFRα1 but no Ret, GDNF stimulates branching but not chemotactic migration, whereas both branching and chemotaxis are promoted by GDNF in the cells coexpressing Ret and GFRα1, mimicking HGF/Met responses in wild-type MDCK cells. Indeed, GDNF induces Met phosphorylation in several ret-deficient/GFRα1-positive and GFRα1/Ret-coexpressing cell lines. However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable. Met activation is mediated by Src family kinases. The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not. Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling

Enhancing fish species identification using novel markers and emerging technologies

Establishing an efficient traceability framework for fish products is crucial for consumer protection and fisheries management and conservation. This is well reflected in the EU legislation. The EU general food law emphasizes strongly that European citizens must have access to safe and wholesome food of the highest standard. Consumer protection is supported by a stringent traceability concept as stipulated in Regulation (EC) 178/2002. This notion is also expressed in the Common Fisheries Policy (CFP) basic regulation (EU) 1380/2013, according to which fishing and aquaculture must be environmentally, economically and socially sustainable while providing a source of healthy food for all EU citizens. Under the CFP the need for traceability is not exclusively raised in the context of consumer protection, but also as a necessary component for fisheries control and enforcement in Regulation (EU) 1224/2009 and in the context of the EU’s ambitious strategy to fight Illegal, Unreported and Unregulated (IUU) fishing under the remit of Regulation (EC) 1005/2008. Recent scientific advances, particularly in the fields of genetics and genomics, have led to the development of novel and improved technologies, and efforts are under way to harness their potential for the species identification of unknown fish samples or products. This report reviews these efforts, describing the technologies and the early results obtained for fish product traceability. Each of these technologies have the potential to fill some specific existing gaps, although they come with their own individual set of disadvantages. Understanding those and monitoring progress is thus crucial for their proper integration in existing traceability frameworks.JRC.F.7-Knowledge for Health and Consumer Safet

The Human Gut Microbiota: Overview and analysis of the current scientific knowledge and possible impact on healthcare and well-being

Recent years have seen a fast increase in the analytical capacity to read genetic information and in the ability to understand the link between the genetic information and the functioning of organisms. This has increased the scientific knowledge in previously underexploited fields. One example is the human microbiota and the understanding of the vital role that the microbiota plays in the physiological and psychological human health status and well-being. Brain degenerative diseases like Alzheimer and Parkinson are, for example, now considered to be linked to abnormalities in the functioning of the human gut microbiota. This understanding may have revolutionary impact on (personal) healthcare but this promise has not yet been fully recognized by the general public or the policy community and for example today, microbiota-related policy interventions are mostly restricted to the marketing and health claims of possible probiotic foods and food supplements. As the JRC is holding the responsibility for the knowledge management of health-related scientific information for policy, we present and discuss here the most recent information available on the vital role of the human gut microbiota and the associated opportunities for human health and well-being. This report provides the state-of-the-art of scientific progress and details how we are only starting to learn its importance for human health, food and chemicals safety, as well as for our protection against environmental stressors. We also indicate why and how the human gut microbiota is going to have an impact on healthcare, nutrition and well-being and how this may change the way we assess the risks of the food, drugs and chemicals we are in contact with.JRC.F.7-Knowledge for Health and Consumer Safet