A roadmap for the generation of benchmarking resources for antimicrobial resistance detection using next generation sequencing.

Next Generation Sequencing technologies significantly impact the field of Antimicrobial Resistance (AMR) detection and monitoring, with immediate uses in diagnosis and risk assessment. For this application and in general, considerable challenges remain in demonstrating sufficient trust to act upon the meaningful information produced from raw data, partly because of the reliance on bioinformatics pipelines, which can produce different results and therefore lead to different interpretations. With the constant evolution of the field, it is difficult to identify, harmonise and recommend specific methods for large-scale implementations over time.

Nuclear DNA barcodes for cod identification in mildly-treated and processed food products.

Gadoids are a group of fish with historical importance in the fishing industry. The high demand for cod is one of the reasons why cod products are often mislabelled, and numerous observations have been made on the replacement of Atlantic cod (Gadus morhua) by cheaper species or its illegal capture in contravention of fish quotas. Fish species identification is traditionally based on morphological features, but this may be difficult in case of heat-treated or processed products, or where the species look similar, as in the Gadoid group.

The challenges of designing a benchmark strategy for bioinformatics pipelines in the identification of antimicrobial resistance determinants using next generation sequencing technologies.

Next-Generation Sequencing (NGS) technologies are expected to play a crucial role in the surveillance of infectious diseases, with their unprecedented capabilities for the characterisation of genetic information underlying the virulence and antimicrobial resistance (AMR) properties of microorganisms.  In the implementation of any novel technology for regulatory purposes, important considerations such as harmonisation, validation and quality assurance need to be addressed.  NGS technologies pose unique challenges in these regards, in part due to their reliance on bioinformatics for the processing and proper interpretation of the data produced.

Identification of single target taxon-specific reference assays for the most commonly genetically transformed crops using digital droplet PCR.

Knowledge of the number of DNA sequences targeted by the taxon-specific reference assays is essential for correct GM quantification and is key to the harmonisation of measurement results. In the present study droplet digital PCR (ddPCR) was used to determine the number of DNA target copies of taxon-specific assays validated for real-time PCR for the four main genetically modified (GM) crops. The transferability of experimental conditions from real-time PCR to ddPCR was also explored, as well as the effect of DNA digestion.

Novel nuclear barcode regions for the identification of flatfish species.

The development of an efficient seafood traceability framework is crucial for the management of sustainable fisheries and the monitoring of potential substitution fraud across the food chain. Recent studies have shown the potential of DNA barcoding methods in this framework, with most of the efforts focusing on using mitochondrial targets such as the and genes. In this article, we show the identification of novel targets in the nuclear genome, and their associated primers, to be used for the efficient identification of flatfishes of the family.

Molecular characterization of an unauthorized genetically modified Bacillus subtilis production strain identified in a vitamin B feed additive.

Many food and feed additives result from fermentation of genetically modified (GM) microorganisms. For vitamin B2 (riboflavin), GM Bacillus subtilis production strains have been developed and are often used. The presence of neither the GM strain nor its recombinant DNA is allowed for fermentation products placed on the EU market as food or feed additive.

Baseline Practices for the Application of Genomic Data Supporting Regulatory Food Safety.

The application of new data streams generated from next-generation sequencing (NGS) has been demonstrated for food microbiology, pathogen identification, and illness outbreak detection. The establishment of best practices for data integrity, reproducibility, and traceability will ensure reliable, auditable, and transparent processes underlying food microbiology risk management decisions. We outline general principles to guide the use of NGS data in support of microbiological food safety.

Development and applicability of a ready-to-use PCR system for GMO screening.

With the growing number of GMOs introduced to the market, testing laboratories have seen their workload increase significantly. Ready-to-use multi-target PCR-based detection systems, such as pre-spotted plates (PSP), reduce analysis time while increasing capacity. This paper describes the development and applicability to GMO testing of a screening strategy involving a PSP and its associated web-based Decision Support System.

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.

JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms.

The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event.

Development, optimization, and single laboratory validation of an event-specific real-time PCR method for the detection and quantification of Golden Rice 2 using a novel taxon-specific assay.

In this study, we developed, optimized, and in-house validated a real-time PCR method for the event-specific detection and quantification of Golden Rice 2, a genetically modified rice with provitamin A in the grain. We optimized and evaluated the performance of the taxon (targeting rice Phospholipase D α2 gene)- and event (targeting the 3' insert-to-plant DNA junction)-specific assays that compose the method as independent modules, using haploid genome equivalents as unit of measurement. We verified the specificity of the two real-time PCR assays and determined their dynamic range, limit of quantification, limit of detection, and robustness.

JRC GMO-Matrix: a web application to support Genetically Modified Organisms detection strategies.

Background: The polymerase chain reaction (PCR) is the current state of the art technique for DNA-based detection of Genetically Modified Organisms (GMOs). A typical control strategy starts by analyzing a sample for the presence of target sequences (GM-elements) known to be present in many GMOs. Positive findings from this "screening" are then confirmed with GM (event) specific test methods.

Somatic progenitor cell vulnerability to mitochondrial DNA mutagenesis underlies progeroid phenotypes in Polg mutator mice.

Somatic stem cell (SSC) dysfunction is typical for different progeroid phenotypes in mice with genomic DNA repair defects. MtDNA mutagenesis in mice with defective Polg exonuclease activity also leads to progeroid symptoms, by an unknown mechanism. We found that Polg-Mutator mice had neural (NSC) and hematopoietic progenitor (HPC) dysfunction already from embryogenesis.

The regulatory use of the Local Lymph Node Assay for the notification of new chemicals in Europe.

The regulatory use of the Local Lymph Node Assay (LLNA) for new chemicals registration was monitored by screening the New Chemicals Database (NCD), which was managed by the former European Chemicals Bureau (ECB) at the European Commission Joint Research Centre (JRC). The NCD centralised information for chemicals notified after 1981, where toxicological information has been generated predominantly according to approved test methods. The database was searched to extract notifications for which the information for skin sensitisation labelling was based on results derived with the LLNA.

Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010.

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commission's Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e.

Timing of the cell cycle exit of differentiating hippocampal neural stem cells.

Neural stem cells contribute to mammalian brain tissue turnover in specific locations throughout life. Differentiation of stem cells is associated with terminal mitosis and cell cycle exit, but it is unclear how the timing and signaling of these are interlinked. Here, we have investigated the cell cycle exit characteristics in comparison with morphological changes during hippocampal stem cell differentiation in an adult mammalian cell line.

Myc increases self-renewal in neural progenitor cells through Miz-1.

The mechanisms underlying the decision of a stem or progenitor cell to either self-renew or differentiate are incompletely understood. To address the role of Myc in this process, we expressed different forms of the proto-oncogene Myc in multipotent neural progenitor cells (NPCs) using retroviral transduction. Expression of Myc in neurospheres increased the proportion of self-renewing cells fivefold, and 1% of the Myc-overexpressing cells, but none of the control cells, retained self-renewal capacity even under differentiation-inducing conditions.

KIT overexpression induces proliferation in astrocytes in an imatinib-responsive manner and associates with proliferation index in gliomas.

Activating gene mutations, gene amplifications and overexpressed proteins may be useful as targets for novel therapies. Alterations at chromosome locus 4q12 are associated with gliomas and the region harbors the receptor tyrosine kinase gene KIT, which is frequently amplified in gliomas, and also overexpressed in a subset of gliomas. KIT and its ligand stem cell factor are widely expressed in embryonic and adult mouse brain, and they play a role in many signal transduction pathways involved in cellular proliferation, differentiation and cancer cell metastasis.

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

Background: 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.

Results: We have generated a program that allows complete in silico simulation of the cloning experiment.

SRC regulates actin dynamics and invasion of malignant glial cells in three dimensions.

Malignant glioma is the major brain tumor in adults and has a poor prognosis. The failure to control invasive cell subpopulations may be the key reason for local glioma recurrence after radical tumor resection and may contribute substantially to the failure of the other treatment modalities such as radiation therapy and chemotherapy. As a model for this invasion, we have implanted spheroids from a human glioma cell line (U251) in three-dimensional collagen type I matrices, which these cells readily invade.

GDNF promotes tubulogenesis of GFRalpha1-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 alpha1 (GFRalpha1) and Ret receptor tyrosine kinase.

K252a and CEP1347 are neuroprotective compounds that inhibit mixed-lineage kinase-3 and induce activation of Akt and ERK.

K252a is best known as a Trk inhibitor, but is also a neuroprotective compound. CEP1347, a K252a derivative, retains neuroprotective properties, but does not inhibit TrkA. CEP1347 has recently been shown to directly inhibit MAPKKKs, including MLK3, but the effect of K252a on MAPKKKs remains unknown.