Event-specific Method for the Quantification of Soybean DAS-68416-4 Using Real-time PCR: Validation report

In line with its mandate the European Union Reference Laboratory for GM Food and Feed (EU RL GMFF), in collaboration with the European Network of GMO Laboratories (ENGL), has validated an event-specific polymerase chain reaction (PCR) method for detecting and quantifying soybean event DAS-68416-4 (unique identifier DAS-68416-4). The validation study was conducted according to the EU-RL GMFF validation procedure (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm) and internationally accepted guidelines. In accordance with current EU legislation , Dow AgroSciences LLC provided the detection method and the positive and negative control samples (genomic DNA extracted from soybean kernels harbouring the DAS-68416-4 event as positive control DNA, genomic DNA extracted from conventional soybean kernels as negative control DNA). The EU-RL GMFF prepared the validation samples (calibration samples and blind samples at different GM percentage [DNA/DNA]), organised an international collaborative study and analysed the results. The study confirms that the method meets the method performance requirements as established by the EU-RL GMFF and the ENGL and according to Annex I-2.C.2 to Regulation (EC) No 641/2004 and it fulfils the analytical requirements of Regulation (EU) No 619/2011JRC.I.3-Molecular Biology and Genomic

Report on the Verification of the Performance of MON 87705 and MON 89788 Event-specific PCR-based Methods applied to DNA Extracted from GM Stack Soybean MON 87705 x MON 89788

The EU-RL GMFF has previously validated individually, and declared fit for purpose, the detection methods for the single line soybean events MON 87705 and MON 89788 and has published the corresponding reports (see http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.aspx). In line with the approach defined by the ENGL (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF therefore has carried out only an in-house verification of the performance of each validated method when applied to DNA extracted from the GM stack MON 87705 x MON 89788 soybean. The hereby reported in-house verification study led to the conclusion that the individual methods meet the ENGL requirements also when applied to DNA extracted from the GM stack MON 87705 x MON 89788 soybean.JRC.I.3-Molecular Biology and Genomic

MAD-4-MITO, a Multi Array of Detectors for ground-based mm/submm SZ observations

The last few years have seen a large development of mm technology and ultra-sensitive detectors devoted to microwave astronomy and astrophysics. The possibility to deal with large numbers of these detectors assembled into multi--pixel imaging systems has greatly improved the performance of microwave observations, even from ground--based stations, especially combining the power of multi--band detectors with their new imaging capabilities. Hereafter, we will present the development of a multi--pixel solution devoted to Sunyaev--Zel'dovich observations from ground--based telescopes, that is going to be operated from the Millimetre and Infrared Testagrigia Observatory.Comment: 5 pages, 3 figures, to be published in th eProceedings of the 2K1BC Workshop, July 9-13, 2001 - Breuil-Cervini

Viviparity and habitat restrictions may influence the evolution of male reproductive genes in tsetse fly (Glossina) species

11openInternationalInternational coauthor/editorBackground Glossina species (tsetse flies), the sole vectors of African trypanosomes, maintained along their long evolutionary history a unique reproductive strategy, adenotrophic viviparity. Viviparity reduces their reproductive rate and, as such, imposes strong selective pressures on males for reproductive success. These species live in sub-Saharan Africa, where the distributions of the main sub-genera Fusca, Morsitans, and Palpalis are restricted to forest, savannah, and riverine habitats, respectively. Here we aim at identifying the evolutionary patterns of the male reproductive genes of six species belonging to these three main sub-genera. We then interpreted the different patterns we found across the species in the light of viviparity and the specific habitat restrictions, which are known to shape reproductive behavior. Results We used a comparative genomic approach to build consensus evolutionary trees that portray the selective pressure acting on the male reproductive genes in these lineages. Such trees reflect the long and divergent demographic history that led to an allopatric distribution of the Fusca, Morsitans, and Palpalis species groups. A dataset of over 1700 male reproductive genes remained conserved over the long evolutionary time scale (estimated at 26.7 million years) across the genomes of the six species. We suggest that this conservation may result from strong functional selective pressure on the male imposed by viviparity. It is noteworthy that more than half of these conserved genes are novel sequences that are unique to the Glossina genus and are candidates for selection in the different lineages. Conclusions Tsetse flies represent a model to interpret the evolution and differentiation of male reproductive biology under different, but complementary, perspectives. In the light of viviparity, we must take into account that these genes are constrained by a post-fertilization arena for genomic conflicts created by viviparity and absent in ovipositing species. This constraint implies a continuous antagonistic co-evolution between the parental genomes, thus accelerating inter-population post-zygotic isolation and, ultimately, favoring speciation. Ecological restrictions that affect reproductive behavior may further shape such antagonistic co-evolution.openSavini, Grazia; Scolari, Francesca; Ometto, Lino; Rota-Stabelli, Omar; Carraretto, Davide; Gomulski, Ludvik M.; Gasperi, Giuliano; Abd-Alla, Adly M. M.; Aksoy, Serap; Attardo, Geoffrey M.; Malacrida, Anna R.Savini, G.; Scolari, F.; Ometto, L.; Rota-Stabelli, O.; Carraretto, D.; Gomulski, L.M.; Gasperi, G.; Abd-Alla, A.M.M.; Aksoy, S.; Attardo, G.M.; Malacrida, A.R

Report on the Verification of the Performance of Bt11, DAS-59122-7, MIR604, TC 1507 and GA21 Event-specific PCR-based Methods Applied to DNA Extracted from GM Stack Bt11 x DAS-59122-7 x MIR604 x TC 1507 x GA21 Maize

An application was submitted by Syngenta Crop Protection AG to request the authorisation of genetically modified stack (GM stack) Bt11 x 59122 x MIR604 x TC1507 x GA21 maize (tolerant to herbicide products containing glufosinate ammonium/glyphosate and resistant to certain lepidopteran/coleopteran pests) and all sub-combinations of the individual events as present in the segregating progeny (except for 1507 x 59122), for food and feed uses, and import and processing, in accordance with articles 5 and 17 of Regulation (EC) No 1829/2003 on GM Food and Feed. The unique identifier assigned to GM stack Bt11 x 59122 x MIR604 x TC1507 x GA21 maize is SYN-BTØ11-1 × DAS-59122-7 × SYN-IR6Ø4-5 × DAS-Ø15Ø7-1 × MON-ØØØ21-9. The GM stack Bt11 x 59122 x MIR604 x TC1507 x GA21 maize has been obtained by conventional crossing of genetically modified maize events: Bt11, 59122, MIR604, TC1507 and GA21, without any new genetic modification. The European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) has previously validated individually, and declared fit for purpose, the detection methods for the single events Bt11, 59122, MIR604, TC1507 and GA21 (see http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.aspx). In line with the approach defined by the European Network of GMO Laboratories (ENGL) (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF has carried out only an in-house verification of the performance of each validated method when applied to genomic DNA extracted from GM stack Bt11 x 59122 x MIR604 x TC1507 x GA21 maize. The results of the in-house verification led to the conclusion that the individual methods meet the ENGL performance criteria also when applied to genomic DNA extracted from the GM stack Bt11 x 59122 x MIR604 x TC1507 x GA21 maize.JRC.I.3-Molecular Biology and Genomic

Extraction of DNA from Choline Chloride Feed Additive (CC) and from derived Pre-Mixes (PMCC) and Screening of CC and PMCC for (a) presence of rice and (b) presence of Bt63

Choline Chloride 60% (CC) is a feed additive that is imported in significant quantities from China. In a number of cases GM rice, harbouring the event Bt63, has been found in imported CC batches but not in derived pre-mixes. The Member States were asked to provide positive CC samples and derived pre-mixes to the EU-RL GMFF in order to allow the EU-RL GMFF establishing practical approaches to DNA extraction and the subsequent testing for presence of (a) rice and (b) the Bt63 rice event. This technical note has been derived based on data provided from National Control Laboratories and experience made by the EU-RL GMFF when re-testing nine feed additive samples and ten pre-mixes samples. The EU-RL GMFF found that the extraction of DNA from the feed additive Choline Chloride (CC) does not normally pose specific problems. It can be carried out following the available protocols or using available standard DNA extraction kits. The extraction of DNA from derived pre-mixes (PMCC) is, however, posing problems because of observed strong inhibition and the presence of DNA from additional sources than those present in the CC. The EU-RL GMFF has tried a number of protocols and extraction kits on PMCC samples. On this basis it is concluded that it is advisable to carefully purify the DNA, to verify the possible presence of inhibition effects and eventually to try to reduce any inhibition observed. Concerning testing the extracted DNA for rice and Bt63 rice event presence, this does not pose a problem in case of the CC while for PMCC rice was not anymore detectable or was detected in trace amounts. In any case testing for Bt63 presence was impossible. A possible explanation could be that much of the extracted DNA is from other plants than rice and hence the concentration of rice DNA is below the LOD of the rice-taxon specific method. In order to verify this, the EU-RL GMFF has analysed the available rice (taxon-) specific methods by means of bio-informatics and (partly) in the laboratory and concludes that they all should be suitable for specifically and reliably detecting rice from the Oryza sativa species. The re-testing by the EU-RL GMFF of CC and PMCC samples has confirmed the results of the initial tests.JRC.I.3-Molecular Biology and Genomic

Novel MIPs-Parabens based SPE Stationary Phases Characterization and Application

In this work, the synthesis, characterization, and application of novel parabens imprinted polymers as highly selective solid-phase extraction (SPE) sorbents have been reported. The imprinted polymers were created using sol–gel molecular imprinting process. All the seven parabens were considered herein in order to check the phase selectivity. By means of a validated HPLC-photodiode array detector (PDA) method all seven parabens were resolved in a single chromatographic run of 25 min. These SPE sorbents, in-house packed in SPE empty cartridges, were first characterized in terms of extraction capability, breakthrough volume, retention volume, hold-up volume, number of theoretical plates, and retention factor. Finally, the device was applied to a real urine sample to check the method feasibility on a very complex matrix. The new paraben imprinted SPE sorbents, not yet present in the literature, potentially encourage the development of novel molecularly imprinted polymers (MIPs) to enhance the extraction efficiency, and consequently the overall analytical performances, when the trace quantification is required

Multi-centre and multi-vendor reproducibility of a standardized protocol for quantitative susceptibility Mapping of the human brain at 3T

: Quantitative Susceptibility Mapping (QSM) is an MRI-based technique allowing the non-invasive quantification of iron content and myelination in the brain. The RIN - Neuroimaging Network established an optimized and harmonized protocol for QSM across ten sites with 3T MRI systems from three different vendors to enable multicentric studies. The assessment of the reproducibility of this protocol is crucial to establish susceptibility as a quantitative biomarker. In this work, we evaluated cross-vendor reproducibility in a group of six traveling brains. Then, we recruited fifty-one volunteers and measured the variability of QSM values in a cohort of healthy subjects scanned at different sites, simulating a multicentric study. Both voxelwise and Region of Interest (ROI)-based analysis on cortical and subcortical gray matter were performed. The traveling brain study yielded high structural similarity (∼0.8) and excellent reproducibility comparing maps acquired on scanners from two different vendors. Depending on the ROI, we reported a quantification error ranging from 0.001 to 0.017 ppm for the traveling brains. In the cohort of fifty-one healthy subjects scanned at nine different sites, the ROI-dependent variability of susceptibility values, of the order of 0.005-0.025 ppm, was comparable to the result of the traveling brain experiment. The harmonized QSM protocol of the RIN - Neuroimaging Network provides a reliable quantification of susceptibility in both cortical and subcortical gray matter regions and it is ready for multicentric and longitudinal clinical studies in neurological and pychiatric diseases

Normative values of the topological metrics of the structural connectome: A multi-site reproducibility study across the Italian Neuroscience network

Purpose: The use of topological metrics to derive quantitative descriptors from structural connectomes is receiving increasing attention but deserves specific studies to investigate their reproducibility and variability in the clinical context. This work exploits the harmonization of diffusion-weighted acquisition for neuroimaging data performed by the Italian Neuroscience and Neurorehabilitation Network initiative to obtain normative values of topological metrics and to investigate their reproducibility and variability across centers. / Methods: Different topological metrics, at global and local level, were calculated on multishell diffusion-weighted data acquired at high-field (e.g. 3 T) Magnetic Resonance Imaging scanners in 13 different centers, following the harmonization of the acquisition protocol, on young and healthy adults. A “traveling brains” dataset acquired on a subgroup of subjects at 3 different centers was also analyzed as reference data. All data were processed following a common processing pipeline that includes data pre-processing, tractography, generation of structural connectomes and calculation of graph-based metrics. The results were evaluated both with statistical analysis of variability and consistency among sites with the traveling brains range. In addition, inter-site reproducibility was assessed in terms of intra-class correlation variability. / Results: The results show an inter-center and inter-subject variability of <10%, except for “clustering coefficient” (variability of 30%). Statistical analysis identifies significant differences among sites, as expected given the wide range of scanners’ hardware. / Conclusions: The results show low variability of connectivity topological metrics across sites running a harmonised protocol