Report on the Verification of the Performance of a MON810 Event-specific Method on Maize Line MON810 Using Real-time PCR

The JRC as Community Reference Laboratory for GM Food and Feed (CRL-GMFF) (see Regulation EC No 1829/2003), has carried out an in-house verification study to assess the performance of the MON810 method to detect and quantify the MON810 transformation event in maize DNA (unique identifier MON-¿¿810-6). The method has previously undergone a full validation on samples represented by certified reference material. The present verification was conducted in order to verify the performance of the validated method on the control samples provided by the applicant as requested by Annex I.2.C.2 to Regulation (EC) No 641/2004 stating that ¿The method shall be applicable to samples of the food or feed, to the control samples and to the reference material, which is referred to in Articles 5(3)(j) and 17(3)(j) of Regulation (EC) No 1829/2003.¿ The study was conducted according to internationally accepted guidelines (1,2). In accordance with Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and to Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, the CRL-GMFF carried out a verification of the event-specific detection method previously validated by the Federal Institute for Risk Assessment (BfR) in collaboration with the American Association of Cereal Chemists (AACC), Joint Research Centre (JRC) of the European Commission (EC), Institute for Reference Material and Measurement (IRMM), the Institute for Health and Consumer Protection (IHCP) and GeneScan, Berlin; Monsanto Company provided the control samples (MON810 maize seeds and conventional maize seeds) used in the verification. The JRC prepared the in-house verification samples (calibration samples and blind samples at different GM percentages). The results of the in-house verification study were evaluated with reference to ENGL method performance requirements (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm) and to the results of the full validation (http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm). The results of CRL-GMFF in-house verification study are made publicly available at http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm).JRC.DDG.I.4-Molecular biology and genomic

Report on the Verification of the Performance of MON 89034, 1507 and NK603 Event-specific Methods on the Maize Event MON 89034 x 1507 x NK603 Using Real-Time PCR. Validation Report and Protocols

The European Union Reference Laboratory for GM Food and Feed EURL-GMFF), established by Regulation (EC) No 1829/2003, has carried out a verification study to assess the performance of three quantitative event-specific methods on the maize event MON 89034 x 1507 x NK603 (unique identifier MON-89Ø34-3 x DAS-Ø15Ø7-1 x MON-ØØ6Ø3-6) which combines the MON 89034, 1507 and NK603 transformation events. The three methods have been validated individually on single-trait events, to detect and quantify each event in maize samples. This study was conducted according to internationally accepted guidelines (1, 2). In accordance to Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and to Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, Monsanto and Dow AgroSciences provided the detection methods and the control samples: genomic DNA from homogenised seeds of MON 89034 x 1507 x NK603 maize (60-B) and from homogenised seeds of conventional maize (10001262-V). The EURL-GMFF prepared the verification samples (calibration samples and blind samples at different GM percentages [DNA/DNA]). The results of the verification study were evaluated with reference to ENGL method performance requirements (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm) and to the validation results on the individual parental events (http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm) The results of this EURL-GMFF verification study are publicly available at http://gmo-crl.jrc.ec.europa.eu/.JRC.DG.I.4 - Molecular biology and genomic

Event-specific Method for the Quantification of Soybean MON 87701 Using Real-time PCR

The European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF), established by Regulation (EC) No 1829/2003, has carried out a validation study to assess the performance of a quantitative event-specific method on the soybean event MON 87701 (unique identifier MON-877Ø1-2). The collaborative trial was conducted according to internationally accepted guidelines. In accordance to Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and to Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, Monsanto Company provided the detection method and the control samples. The EU-RL GMFF prepared the validation samples [calibration samples and blind samples at unknown GM percentages (DNA/DNA)]. The results of the international collaborative trial met the European Network of GMO Laboratories (ENGL) method performance requirements (http://gmocrl.jrc.ec.europa.eu/guidancedocs.htm). The method is therefore considered applicable to the control samples provided, in accordance with the requirements of Annex I – 2.C.2 to Regulation (EC) No 641/2004.JRC.I.3-Molecular Biology and Genomic

In-house validation of an Event-specific Method for the Quantification of Oliseed Rape RF2 using Real-time PCR

The European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF), established by Regulation (EC) No 1829/2003, has carried out an in-house validation study to assess the performance of a quantitative event-specific method on the oilseed rape event RF2 (unique identifier ACS-BN002-5). In accordance to Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and to Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, Bayer CropScience provided the detection method and the control samples. The EU-RL GMFF prepared the validation samples [calibration samples and blind samples at different GM percentages (DNA/DNA)]. The results of the in-house validation were evaluated with respect to method acceptance criteria and method performance requirements recommended by the European Network of GMO Laboratories (ENGL) (http://gmocrl.jrc.ec.europa.eu/guidancedocs.htm) and to its applicability in different real-time PCR instruments. The results obtained indicate that the method complies with the ENGL criteria. The method is therefore considered applicable to the control samples provided, in accordance with the requirements of Annex I – 2.C.2 to Regulation (EC) No 641/2004.JRC.I.3-Molecular Biology and Genomic

Report on the Verification of the Performance of MON89034 and NK603 Event-specific Methods on the Maize Event MON89034 x NK603 Using Real-Time PCR

The JRC as Community Reference Laboratory for GM Food and Feed (CRL-GMFF), established by Regulation (EC) No 1829/2003, has carried out a verification study to assess the performance of two quantitative event-specific methods on the maize event MON89034 x NK603 (unique identifier MON 89Ø34-3 x MON-ØØ6Ø3-6) which combines the MON89034 and NK603 transformation events. The two methods have been validated individually on single-trait events, to detect and quantify each event in maize samples. This study was conducted according to internationally accepted guidelines (1, 2). In accordance to Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and to Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, Monsanto Company provided the detection methods and the control samples: genomic DNA extracted from seeds of MON89034 x NK603 maize (lot GLP-0701-17954-S), genomic DNA extracted from seeds of non-GM maize (lot GLP-0612-17871-S). The JRC prepared the verification samples (calibration samples and blind samples at different GM percentages). The results of the verification study were evaluated with reference to ENGL method performance requirements (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm) and to the validation results on the individual parental events (http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm). The results of this study are made publicly available at http://gmo-crl.jrc.ec.europa.eu/.JRC.DDG.I.4-Molecular biology and genomic

Report on the Verification of the Performance of MON87701 and MON89788 Event-specific Methods on the Soybean Event MON87701 x MON89788 Using Real-time PCR

The European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF), established by Regulation (EC) No 1829/2003, has carried out an in-house verification study to assess the performance of two quantitative event-specific methods on the soybean event MON87701 x MON89788 (unique identifier MON-877Ø1-2 x MON-89788-1) which combines the MON87701 and the MON89788 transformation events. The two methods have been validated individually on single-trait events, to detect and quantify each event in soybean samples. This study was conducted according to internationally accepted guidelines (1, 2). In accordance to Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and to Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, Monsanto Company provided the detection methods and the control samples: genomic DNA from homogenised seeds of MON87701 x MON89788 soybean and from homogenised seeds of conventional soybean. The EU-RL GMFF prepared the verification samples (calibration samples and blind samples at different unknown GM percentages [DNA/DNA]). The results of the verification study were evaluated with reference to ENGL method performance requirements (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm) and to the validation results on the individual parental events (http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm) The results of this EU-RL GMFF in-house verification studies are made publicly available at http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htmJRC.I.3-Molecular Biology and Genomic

Report on the Verification of the Performance of MON89034 and MON88017 Event-specific Methods on the Maize Event MON89034 x MON88017 Using Real-Time PCR

The JRC as Community Reference Laboratory for GM Food and Feed (CRL-GMFF), established by Regulation (EC) No 1829/2003, has carried out an in-house verification study to assess the performance of two quantitative event-specific methods on the maize event MON89034 x MON88017 (unique identifier MON-89Ø34-3 x MON-88Ø17-3) which combines the MON89034 and MON88017 transformation events. The two methods have been validated individually on single-trait events, to detect and quantify each event in maize samples. This study was conducted according to internationally accepted guidelines (1, 2). In accordance to Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and to Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, Monsanto Company provided the detection methods and the control samples: genomic DNA extracted from seeds of MON89034 x MON88017 maize, genomic DNA extracted from seeds of non-GM maize, seeds of MON89034 x MON88017 and seed of conventional maize. The JRC prepared the in-house verification samples (calibration samples and blind samples at different GM percentages). The results of the in-house verification study were evaluated with reference to ENGL method performance requirements (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm) and to the validation results on the parental events (http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm). The results of this CRL-GMFF in-house verification studies are made publicly available at http://gmo-crl.jrc.ec.europa.eu/JRC.DDG.I.4-Molecular biology and genomic

Fast running modelling techniques for an IC engine by means of the Gasdyn code

LAUREA MAGISTRALEL’obiettivo di questo lavoro è quello di velocizzare la simulazione di un modello monodimensionale di un motore a combustione interna aspirato, ad accensione comandata, mantenendo l’accuratezza dei risultati al livello più alto possibile. Tale obiettivo viene raggiunto attraverso una semplificazione della geometria del modello dettagliato, in parallelo con un incremento della mesh di calcolo adottata nelle simulazioni. Queste due strategie combinate fra loro, comportano a una riduzione del numero di nodi di calcolo del modello, riducendo così i tempi di calcolo. Inizialmente, il modello base è stato analizzato attraverso un incremento del passo di discretizzazione, evidenziandone i limiti di accuratezza. Dopodiché è stata modificata la configurazione relativa alla sezione di scarico; il modello finale è stato costruito semplificando anche la sezione di aspirazione, e prende il nome di Fast Running Model. Per svolgere questa tesi, è stato utilizzato il software di modellazione numerica monodimensionale Gasdyn, sviluppato dal gruppo di Motori a Combustione Interna del Politecnico di Milano. Tutti i risultati sono stati ottenuti adottando due differenti tecniche numeriche: una a differenze finite, Gascon-Corberan, e una a volumi finiti, il 3D Cell. Sono state effettuate simulazioni, sia del modello semplificato al solo sistema di scarico che del Fast Running Model, con entrambi i metodi numerici e applicando diverse lunghezze di discretizzazione. Le performance sono state valutate in termini di parametri operativi, di conservazione della portata, di onde di pressione; i risultati ottenuti poi sono stati comparati con quelli relativi al modello base, cosicché possa essere valutata l’accuratezza delle semplificazioni. Infine, i dati relativi alla precisione dei risultati e delle velocità di calcolo sono stati raccolti e confrontati. Attraverso questa procedura si è potuto trovare un compromesso tra l’accuratezza dei risultati e la durata computazionale delle simulazioni. Entrambi i metodi hanno dato buoni risultati, tuttavia si è dimostrato che il 3D Cell è più appropriato per questo scopo, anche soprattutto grazie alla sua peculiare abilità di conservare perfettamente la massa attraverso tutte le sezioni del modello del motore.The objective of this work is to speed up the simulation of a 1D model of a naturally aspirated, spark ignition internal combustion engine, maintaining as much as possible the accuracy in the results. The aim is achieved through a geometry simplification of the detailed model, in parallel with an increase of the mesh dimension adopted for the calculations. These two strategies combined together, lead to a reduction in the total number of the calculation nodes in the model, and so, to a reduction in computation time. At first, the base model is analysed and tested by means of an enlargement of the discretization length, highlighting the accuracy limits that this model has. Afterwards, the configuration at the exhaust system is modified; the final model is built simplifying also the intake system and takes the name of Fast Running Model. To accomplish this work, the numerical one-dimensional simulation tool Gasdyn, developed by the Internal Combustion Engines Group of Politecnico di Milano, is used. All the numerical results are achieved applying two different numerical techniques: a finite-difference method, the Gascon-Corberan, and a finite-volume method, the 3D Cell. The simulations of both the exhaust only simplified model, and of the Fast Running Model have been performed for the two numerical methods and applying several discretization lengths. The performances are then evaluated in terms of operating parameters, mass conservation, pressure waves motion; the obtained results are compared with the ones of the base model. So, the accuracy of the simplifications can be assessed. In the end, the data relative to the results accuracy and of the computational times are collected and evaluated. By means of this procedure a trade-off between accuracy and computational time has been found. Good results are achieved by both methods but has been demonstrated that the 3D Cell method is more appropriate to reach the purpose, mainly thanks to his peculiar ability to conserve much better the mass flow rate through all the engine model

Event-specific Method for the Quantification of Soybean Line 40-3-2 Using Real-time PCR - Validation Report and Protocol - Report on the Validation of a DNA Extraction Method for Soybean Seeds

The JRC as Community Reference Laboratory for GM Food and Feed (CRL-GMFF), established by Regulation (EC) No 1829/2003, in collaboration with the European Network of GMO Laboratories (ENGL), has carried out a collaborative study to assess the performance of a quantitative event-specific method to detect and quantify the 40-3-2 transformation event in soybean DNA (unique identifier MON-¿4¿32-6). The collaborative trial was conducted according to internationally accepted guidelines (1, 2). In accordance with Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and with Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, Monsanto provided the detection method and the samples (soybean seeds containing the transformation event and conventional soybean seeds). The JRC prepared the validation samples (calibration samples and blind samples at unknown GM percentage [DNA/DNA]). The collaborative trial involved fourteen laboratories from nine European countries. The results of the international collaborative trial met the ENGL performance requirements and the scientific understanding about satisfactory method performance. Therefore, the CRL-GMFF considers the method validated as fit for the purpose of regulatory compliance. The results of the collaborative study are made publicly available at http://gmo-crl.jrc.it/.JRC.I.6-Biotechnology and GMO

Effect of external PEEP in patients under controlled mechanical ventilation with an auto-PEEP of 5 cmH2O or higher.

In some patients with auto-positive end-expiratory pressure (auto-PEEP), application of PEEP lower than auto-PEEP maintains a constant total PEEP, therefore reducing the inspiratory threshold load without detrimental cardiovascular or respiratory effects. We refer to these patients as complete PEEP-absorbers. Conversely, adverse effects of PEEP application could occur in patients with auto-PEEP when the total PEEP rises as a consequence. From a pathophysiological perspective, all subjects with flow limitation are expected to be complete PEEP-absorbers, whereas PEEP should increase total PEEP in all other patients. This study aimed to empirically assess the extent to which flow limitation alone explains a complete PEEP-absorber behavior (i.e., absence of further hyperinflation with PEEP), and to identify other factors associated with it.One hundred patients with auto-PEEP of at least 5 cmH2O at zero end-expiratory pressure (ZEEP) during controlled mechanical ventilation were enrolled. Total PEEP (i.e., end-expiratory plateau pressure) was measured both at ZEEP and after applied PEEP equal to 80 % of auto-PEEP measured at ZEEP. All measurements were repeated three times, and the average value was used for analysis.Forty-seven percent of the patients suffered from chronic pulmonary disease and 52 % from acute pulmonary disease; 61 % showed flow limitation at ZEEP, assessed by manual compression of the abdomen. The mean total PEEP was 7 ± 2 cmH2O at ZEEP and 9 ± 2 cmH2O after the application of PEEP (p < 0.001). Thirty-three percent of the patients were complete PEEP-absorbers. Multiple logistic regression was used to predict the behavior of complete PEEP-absorber. The best model included a respiratory rate lower than 20 breaths/min and the presence of flow limitation. The predictive ability of the model was excellent, with an overoptimism-corrected area under the receiver operating characteristics curve of 0.89 (95 % CI 0.80-0.97).Expiratory flow limitation was associated with both high and complete PEEP-absorber behavior, but setting a relatively high respiratory rate on the ventilator can prevent from observing complete PEEP-absorption. Therefore, the effect of PEEP application in patients with auto-PEEP can be accurately predicted at the bedside by measuring the respiratory rate and observing the flow-volume loop during manual compression of the abdomen