A theoretical introduction to “Combinatory SYBR®Green qPCR Screening”, a matrix-based approach for the detection of materials derived from genetically modified plants

The detection of genetically modified (GM) materials in food and feed products is a complex multi-step analytical process invoking screening, identification, and often quantification of the genetically modified organisms (GMO) present in a sample. “Combinatory qPCR SYBR®Green screening” (CoSYPS) is a matrix-based approach for determining the presence of GM plant materials in products. The CoSYPS decision-support system (DSS) interprets the analytical results of SYBR®GREEN qPCR analysis based on four values: the Ct- and Tm values and the LOD and LOQ for each method. A theoretical explanation of the different concepts applied in CoSYPS analysis is given (GMO Universe, “Prime number tracing”, matrix/combinatory approach) and documented using the RoundUp Ready soy GTS40-3-2 as an example. By applying a limited set of SYBR®GREEN qPCR methods and through application of a newly developed “prime number”-based algorithm, the nature of subsets of corresponding GMO in a sample can be determined. Together, these analyses provide guidance for semi-quantitative estimation of GMO presence in a food and feed product

Sequencing strategies for the traceability of GMOs - methods and related quality aspects

DNA sequencing in GMO analysis has undergone a rapid repositioning from a technology that may be used as an additional confirmatory step to a technique that deserves consideration as a routine methodology. The methodology may be applied in different ways, from initial screening for authorised as well as unauthorised GMOs in complex samples to, for specific cases, a strategy to establish the identity of possible new GMOs, as well as a range of options in between these two alternative applications. As the overall costs of DNA sequencing are reducing, broader applications are becoming feasible for routine testing in GMO laboratories. The actual application, however, requires further harmonisation and standardisation to maintain current quality standards when applying innovative GMO analytical strategies that include DNA sequencing steps. In the present report, a summary is provided of the results of the dedicated Working Group of the European Network of GMO Laboratories (ENGL) on good practice/quality of DNA sequencing data. The Working Group has addressed both Sanger sequencing as well as massive parallel DNA sequencing within the framework of GMO analysis, with emphasis on the latter. General information is provided in relation to quality aspects of massive parallel DNA sequencing, from sample DNA extraction and preparation, through template amplification and DNA sequencing, to quality assessment of the sequencing results and quality criteria for the use of sequencing data analysis pipelines. In addition, specific GMO-related aspects of DNA sequencing methodologies are discussed considering current GMO detection and identification strategies in different sample types (i.e. simple versus complex food/feed matrices, known versus unknown GMOs), and the intended purpose (i.e. characterisation of the full genome of a genetically modified microorganism (GMM), the full identification of an inserted genetic element and its flanking regions, or the screening for multiple genetically modified (GM) elements in a single genome or in a mixture). The Working Group identified four scenarios covering current real-life situations in GMO analysis strategies that include massive parallel DNA sequencing steps: two targeted sequencing approaches focusing on multiple known or partially known sequences, respectively, and two non-targeted sequencing approaches focusing on either whole genome sequencing of a single organism or, alternatively, applying metagenomics in complex samples. Quality considerations and criteria common to all scenarios have been tentatively established, as well as specific ones relevant for individual scenarios. On a global level, one harmonised definition of a GMO is no longer applicable, especially for genome-edited organisms that contain minor modifications, such as single nucleotide mutations. For these organisms, there is no international consensus on whether or not they should fall within the scope of the GMO regional legislations. In this context, global discussions on the safety aspects and the traceability of these organisms are affected and the exchange of information on the (potential) presence of GMO in food/feed samples and related raw materials has become hampered.JRC.F.5 - Food and Feed Complianc

The HEV Ventilator

HEV is a low-cost, versatile, high-quality ventilator, which has been designed in response to the COVID-19 pandemic. The ventilator is intended to be used both in and out of hospital intensive care units, and for both invasive and non-invasive ventilation. The hardware can be complemented with an external turbine for use in regions where compressed air supplies are not reliably available. The standard modes provided include PC-A/C(Pressure Assist Control),PC-A/C-PRVC(Pressure Regulated Volume Control), PC-PSV (Pressure Support Ventilation) and CPAP (Continuous Positive airway pressure). HEV is designed to support remote training and post market surveillance via a web interface and data logging to complement the standard touch screen operation, making it suitable for a wide range of geographical deployment. The HEV design places emphasis on the quality of the pressure curves and the reactivity of the trigger, delivering a global performance which will be applicable to ventilator needs beyond theCOVID-19 pandemic. This article describes the conceptual design and presents the prototype units together with their performance evaluation.Comment: 34 pages, 18 figures, Extended version of the article submitted to PNA

TcOPT3, a Member of Oligopeptide Transporters from the Hyperaccumulator Thlaspi caerulescens, Is a Novel Fe/Zn/Cd/Cu Transporter

BACKGROUND: Thlaspi caerulescens is a natural selected heavy metal hyperaccumulator that can not only tolerate but also accumulate extremely high levels of heavy metals in the shoots. Thus, to identify the transportors involved in metal long-distance transportation is very important for understanding the mechanism of heavy metal accumulation in this hyperaccumulator. METHODOLOGY/PRINCIPAL FINDINGS: We cloned and characterized a novel gene TcOPT3 of OPT family from T. caerulescens. TcOPT3 was pronouncedly expressed in aerial parts, including stem and leaf. Moreover, in situ hybridization analyses showed that TcOPT3 expressed in the plant vascular systems, especially in the pericycle cells that may be involved in the long-distance transportation. The expression of TcOPT3 was highly induced by iron (Fe) and zinc (Zn) deficiency, especially in the stem and leaf. Sub-cellular localization showed that TcOPT3 was a plasma membrane-localized protein. Furthermore, heterogonous expression of TcOPT3 by mutant yeast (Saccharomyces cerevisiae) complementation experiments demonstrated that TcOPT3 could transport Fe(2+) and Zn(2+). Moreover, expression of TcOPT3 in yeast increased metal (Fe, Zn, Cu and Cd) accumulation and resulted in an increased sensitivity to cadmium (Cd) and copper (Cu). CONCLUSIONS: Our data demonstrated that TcOPT3 might encode an Fe/Zn/Cd/Cu influx transporter with broad-substrate. This is the first report showing that TcOPT3 may be involved in metal long-distance transportation and contribute to the heavy metal hyperaccumulation

The influence of cadmium stress on the content of mineral nutrients and metal-binding proteins in arabidopsis halleri

We investigated the influence of cadmium stress on zinc hyperaccumulation, mineral nutrient uptake, and the content of metal-binding proteins in Arabidopsis halleri. The experiments were carried out using plants subjected to long-term cadmium exposure (40 days) in the concentrations of 45 and 225 μM Cd2+. Inductively coupled plasma-mass spectrometry, size exclusion chromatography coupled with plasma-mass spectrometry, and laser ablation inductively coupled plasma-mass spectrometry used for ablation of polyacylamide gels were employed to assess the content of investigated elements in plants as well as to identify metal-binding proteins. We found that A. halleri is able to translocate cadmium to the aerial parts in high amounts (translocation index >1). We showed that Zn content in plants decreased significantly with the increase of cadmium content in the growth medium. Different positive and negative correlations between Cd content and mineral nutrients were evidenced by our study. We identified more than ten low-molecular-weight (<100 kDa) Cd-binding proteins in Cd-treated plants. These proteins are unlikely to be phytochelatins or metallothioneins. We hypothesize that low-molecular-weight Cd-binding proteins can be involved in cadmium resistance in A. halleri