Report on the In-house Validation of a DNA Extraction Method from Oilseed rape Grains and Validated Method

In accordance with relevant EU legislation , Pioneer Overseas Corporation provided to the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) a DNA extraction method for oilseed rape grains and the relevant samples (ground oilseed rape grains). In line with its mandate , the EU RL GMFF has conducted an in-house validation of this DNA extraction method. To this end it tested the DNA extraction method on the samples provided and evaluated its performance in terms of DNA yield, integrity and quality. The in-house validation study confirmed that the method meets the method performance requirements as established by the EU-RL GMFF and the ENGL , and that it satisfies the provisions of Annex I-2.C.2 to Regulation (EC) No 641/2004. The method is therefore fit for the purpose of producing rapeseed DNA of suitable quantity and quality for subsequent PCR-based analysis. This report is published at http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.htm.JRC.I.3-Molecular Biology and Genomic

Event-specific Method for the Quantification of Maize DAS-40278-9 by Real-time PCR - Validation Report and Validated Method

In line with its mandate1 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 maize event DAS-40278-9 (unique identifier DAS-4Ø278-9). The validation study was conducted according to the EU-RL GMFF validation procedure [http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm] and the internationally accepted guidelines2. In accordance with current EU legislation1, Dow AgroSciences LLC has provided the detection method and the positive and negative control samples (genomic DNA from maize seeds harbouring the DAS-40278-9 event as positive control DNA, genomic DNA from conventional maize seeds 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 in Annex I-2.C.2 to Regulation (EC) No 641/20041 and it fulfils the analytical requirements of Regulation (EU) No 619/2011. This report is published at http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm.JRC.I.3-Molecular Biology and Genomic

Report on the single-laboratory validation of a PCR-based Detection Method for Identification of Florigene™ IFD-25958-3 GM Carnation -Validation Report and Validated Method

In the context of the application for marketing submitted by Florigene Pty Ltd for a genetically modified carnation line (C/NL/09/01) IFD-25958-3, the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) has carried out a single-laboratory validation to assess the performance of a polymerase chain reaction (PCR)-based detection method for detecting and identifying the carnation GM line IFD-25958-3. This report describes the results of tests carried out by the EU-RL GMFF on control samples provided by the method developer and according to the detection method described by the applicant. The taxon-specific method correctly detects the endogenous gene target in genomic DNA of a conventional carnation line (negative control) and in the genomic DNA of the GM carnation line; the same method can also detect the GM target DNA in IFD-25958-3 GM line (positive control) in the experimental conditions described in this report. The Limit of Detection (LOD) of the method has been estimated to be at least 50 copies for the taxon-specific gene and at least 100 copies for the GM insert, based on haploid genome copy number.JRC.I.3-Molecular Biology and Genomic

Event-specific Method for the Quantification of Soybean Line A5547-127 Using Real-time PCR

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 A5547-127 transformation event in soybean DNA (unique identifier ACS-GMØØ6-4). 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¿, Bayer CropScience provided the detection method and the samples (genomic DNA from leaves of plants harbouring the A5547-127 event and from leaves of conventional A5547 soybean plants). The JRC prepared the validation samples (calibration samples and blind samples at unknown GM percentage [DNA/DNA]). The collaborative trial involved twelve laboratories from eight European countries. The results of the international collaborative trial met the ENGL performance requirements. The method is, therefore, considered applicable to the control samples provided, in accordance with the requirements of Annex I-2.C.2 to Commission Regulation (EC) No 641/2004. The results of the collaborative study are made publicly available at http://gmo-crl.jrc.it/.JRC.DDG.I.4-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

3D Bioprinting of Human Adipose-Derived Stem Cells and Their Tenogenic Differentiation in Clinical-Grade Medium

Defining the best combination of cells and biomaterials is a key challenge for the development of tendon tissue engineering (TE) strategies. Adipose-derived stem cells (ASCs) are ideal candidates for this purpose. In addition, controlled cell-based products adherent to good manufacturing practice (GMP) are required for their clinical scale-up. With this aim, in this study, ASC 3D bioprinting and GMP-compliant tenogenic differentiation were investigated. In detail, primary human ASCs were embedded within a nanofibrillar-cellulose/alginate bioink and 3D-bioprinted into multi-layered square-grid matrices. Bioink viscoelastic properties and scaffold ultrastructural morphology were analyzed by rheology and scanning electron microscopy (SEM). The optimal cell concentration for printing among 3, 6 and 9 × 106 ASC/mL was evaluated in terms of cell viability. ASC morphology was characterized by SEM and F-actin immunostaining. Tenogenic differentiation ability was then evaluated in terms of cell viability, morphology and expression of scleraxis and collagen type III by biochemical induction using BMP-12, TGF-β3, CTGF and ascorbic acid supplementation (TENO). Pro-inflammatory cytokine release was also assessed. Bioprinted ASCs showed high viability and survival and exhibited a tenocyte-like phenotype after biochemical induction, with no inflammatory response to the bioink. In conclusion, we report a first proof of concept for the clinical scale-up of ASC 3D bioprinting for tendon TE

Event-specific Method for the Quantification of Cotton MON 88913 Using Real-time PCR

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 MON 88913 transformation event in cotton DNA (unique identifier MON-88913-8). 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: genomic DNA from cotton seeds harbouring the MON 88913 event (line ST 4664) and from conventional cotton seeds (line ST 474). The JRC prepared the validation samples (calibration samples and blind samples at unknown GM percentage [DNA/DNA]). The collaborative trial involved twelve laboratories from nine European countries. The results of the international collaborative trial met the ENGL performance requirements. 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. The results of the collaborative study are made publicly available at http://gmo-crl.jrc.ec.europa.eu/JRC.DDG.I.4-Molecular biology and genomic

Physicochemical characterisation of gold, silica and silver nanoparticles in water and in serum-containing cell culture media

This report presents the results from a study organised under the coordination of JRC as part of a project aiming at the adaptation of the in vitro micronucleus test (Test Guideline 487) for the assessment of manufactured NMs. The aim of the first step of the project was to evaluate the physicochemical characterisation of selected representative nanomaterials (5 nm gold, 30 nm gold, 22 nm silica, 30 nm citrate and 30 nm PVP stabilised silver nanoparticles) in pure water and in different complete culture media. The results of the study show that using a combination of different characterisation techniques is important to providing reliable information about the agglomeration behaviour of the tested nanoparticles in complete cell culture media (CCM). Most of the materials exhibited mild agglomeration in serum containing CCM. Only the PVP functionalised silver nanoparticles showed a size distribution change in all of the culture media that is so small that it could be attributed to solely protein adsorption without notable agglomeration. Silica nanoparticles were found to be the most sensitive to interaction with serum containing CCM, showing massive concentration and time dependent agglomeration strongly affected by the CCM composition. Extensive agglomeration might lead also to the accelerated sedimentation of the particles changing drastically the true, effective dose that the cells will receive under in vitro conditions1, 2. Thus, it has to be investigated in more detail and taken in account when designing in vitro experiments in the next phase of the project.JRC.F.2-Consumer Products Safet

Possible implications for SARS-CoV-2 impact on brain development

Funding Information: The authors would like to thank Dr. Marc Peschanski (I-Stem, Évry, France) for providing IMR90-hiPSCs, and Dr. Anna Navarro Cuenca for providing the license for the use of BioRender.com. Publisher Copyright: © 2022The possible neurodevelopmental consequences of SARS-CoV-2 infection are presently unknown. In utero exposure to SARS-CoV-2 has been hypothesized to affect the developing brain, possibly disrupting neurodevelopment of children. Spike protein interactors, such as ACE2, have been found expressed in the fetal brain, and could play a role in potential SARS-CoV-2 fetal brain pathogenesis. Apart from the possible direct involvement of SARS-CoV-2 or its specific viral components in the occurrence of neurological and neurodevelopmental manifestations, we recently reported the presence of toxin-like peptides in plasma, urine and fecal samples specifically from COVID-19 patients. In this study, we investigated the possible neurotoxic effects elicited upon 72-hour exposure to human relevant levels of recombinant spike protein, toxin-like peptides found in COVID-19 patients, as well as a combination of both in 3D human iPSC-derived neural stem cells differentiated for either 2 weeks (short-term) or 8 weeks (long-term, 2 weeks in suspension + 6 weeks on MEA) towards neurons/glia. Whole transcriptome and qPCR analysis revealed that spike protein and toxin-like peptides at non-cytotoxic concentrations differentially perturb the expression of SPHK1, ELN, GASK1B, HEY1, UTS2, ACE2 and some neuronal-, glia- and NSC-related genes critical during brain development. Additionally, exposure to spike protein caused a decrease of spontaneous electrical activity after two days in long-term differentiated cultures. The perturbations of these neurodevelopmental endpoints are discussed in the context of recent knowledge about the key events described in Adverse Outcome Pathways relevant to COVID-19, gathered in the context of the CIAO project (https://www.ciao-covid.net/).publishersversionpublishe

Interlaboratory comparison study of the Colony Forming Efficiency assay for assessing cytotoxicity of nanomaterials

Nanotechnology has gained importance in the past years as it provides opportunities for industrial growth and innovation. However, the increasing use of manufactured nanomaterials (NMs) in a number of commercial applications and consumer products raises also safety concerns and questions regarding potential unintended risks to humans and the environment. Since several years the European Commission’s Joint Research Centre (JRC) is putting effort in the development, optimisation and harmonisation of in vitro test methods suitable for screening and hazard assessment of NMs. Work is done in collaboration with international partners, in particular the Organisation for Economic Co-operation and Development (OECD). This report presents the results from an interlaboratory comparison study of the in vitro Colony Forming Efficiency (CFE) cytotoxicity assay performed in the frame of OECD's Working Party of Manufactured Nanomaterials (WPMN). Twelve laboratories from European Commission, France, Italy, Japan, Poland, Republic of Korea, South Africa and Switzerland participated in the study coordinated by JRC. The results show that the CFE assay is a suitable and robust in vitro method to assess cytotoxicity of NMs. The assay protocol is well defined and is easily and reliably transferable to other laboratories. The results obtained show good intra and interlaboratory reproducibility of the assay for both the positive control and the tested nanomaterials. In conclusion the CFE assay can be recommended as a building block of an in vitro testing battery for NMs toxicity assessment. It could be used as a first choice method to define dose-effect relationships for other in vitro assays.JRC.I.4-Nanobioscience