Proficiency of European GMO control laboratories to quantify MON89788 soybean in a meat pâté matrix.

GMO control laboratories in the EU routinely monitor the presence and content of genetically modified organisms (GMOs) in food and feed products collected from the EU market. As the vast majority of GMOs comprize genetically modified plants, most control samples have a plant-based origin. For the first time, a pilot proficiency test was organised requiring the analysis of GMOs in a meat matrix. Meat pâté, a product in which soybean is occasionally identified, was spiked with GM soybean event MON89788, homogenised by mixing, aliquoted in sachets and frozen. The assigned value was determined by two independent expert laboratories. Several DNA extraction methods were tested and proved to be insufficient for the removal of PCR inhibitors present in the DNA extracts, resulting in a GM content underestimated by at least 30%. This problem was solved either by using hot-start qPCR chemistry or by applying the same method in a digital PCR format. A total of 52 laboratories participated in the study. They were requested to verify the presence of any GM soybean in the test item and to quantify the GM event(s) identified by their method of choice. All but one laboratory identified the MON89788 soybean event present in the pâté matrix. The majority of the quantitative results reported were below the assigned value, but did not deviate more than 50% from it. This study demonstrated the proficiency of most GMO control laboratories for the analysis of GMOs in a meat-based product. It also shows that method optimisation for GMO analysis in meat products is nevertheless advisable.

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

Evaluation of plasmid and genomic DNA calibrants used for the quantification of genetically modified organisms.

The reliable quantification of genetically modified organisms (GMOs) by real-time PCR requires, besides thoroughly validated quantitative detection methods, sustainable calibration systems. The latter establishes the anchor points for the measured value and the measurement unit, respectively. In this paper, the suitability of two types of DNA calibrants, i.e. plasmid DNA and genomic DNA extracted from plant leaves, for the certification of the GMO content in reference materials as copy number ratio between two targeted DNA sequences was investigated. The PCR efficiencies and coefficients of determination of the calibration curves as well as the measured copy number ratios for three powder certified reference materials (CRMs), namely ERM-BF415e (NK603 maize), ERM-BF425c (356043 soya), and ERM-BF427c (98140 maize), originally certified for their mass fraction of GMO, were compared for both types of calibrants. In all three systems investigated, the PCR efficiencies of plasmid DNA were slightly closer to the PCR efficiencies observed for the genomic DNA extracted from seed powders rather than those of the genomic DNA extracted from leaves. Although the mean DNA copy number ratios for each CRM overlapped within their uncertainties, the DNA copy number ratios were significantly different using the two types of calibrants. Based on these observations, both plasmid and leaf genomic DNA calibrants would be technically suitable as anchor points for the calibration of the real-time PCR methods applied in this study. However, the most suitable approach to establish a sustainable traceability chain is to fix a reference system based on plasmid DNA.

Detection and traceability of genetically modified organisms in the food production chain.

Both labelling and traceability of genetically modified organisms are current issues that are considered in trade and regulation. Currently, labelling of genetically modified foods containing detectable transgenic material is required by EU legislation. A proposed package of legislation would extend this labelling to foods without any traces of transgenics. These new legislations would also impose labelling and a traceability system based on documentation throughout the food and feed manufacture system. The regulatory issues of risk analysis and labelling are currently harmonised by Codex Alimentarius. The implementation and maintenance of the regulations necessitates sampling protocols and analytical methodologies that allow for accurate determination of the content of genetically modified organisms within a food and feed sample. Current methodologies for the analysis of genetically modified organisms are focused on either one of two targets, the transgenic DNA inserted- or the novel protein(s) expressed- in a genetically modified product. For most DNA-based detection methods, the polymerase chain reaction is employed. Items that need consideration in the use of DNA-based detection methods include the specificity, sensitivity, matrix effects, internal reference DNA, availability of external reference materials, hemizygosity versus homozygosity, extrachromosomal DNA, and international harmonisation. For most protein-based methods, enzyme-linked immunosorbent assays with antibodies binding the novel protein are employed. Consideration should be given to the selection of the antigen bound by the antibody, accuracy, validation, and matrix effects. Currently, validation of detection methods for analysis of genetically modified organisms is taking place. In addition, new methodologies are developed, including the use of microarrays, mass spectrometry, and surface plasmon resonance. Challenges for GMO detection include the detection of transgenic material in materials with varying chromosome numbers. The existing and proposed regulatory EU requirements for traceability of genetically modified products fit within a broader tendency towards traceability of foods in general and, commercially, towards products that can be distinguished from each other. Traceability systems document the history of a product and may serve the purpose of both marketing and health protection. In this framework, segregation and identity preservation systems allow for the separation of genetically modified and non-modified products from "farm to fork". Implementation of these systems comes with specific technical requirements for each particular step of the food processing chain. In addition, the feasibility of traceability systems depends on a number of factors, including unique identifiers for each genetically modified product, detection methods, permissible levels of contamination, and financial costs. In conclusion, progress has been achieved in the field of sampling, detection, and traceability of genetically modified products, while some issues remain to be solved. For success, much will depend on the threshold level for adventitious contamination set by legislation.

A microbial biosensor to predict bioavailable nickel in soil and its transfer to plants.

Ralstonia eutropha strain AE2515 was constructed and optimised to serve as a whole-cell biosensor for the detection of bioavailable concentrations of Ni2+ and Co2+ in soil samples. Strain AE2515 is a Ralstonia eutropha CH34 derivative containing pMOL1550, in which the cnrYXH regulatory genes are transcriptionally fused to the bioluminescent luxCDABE reporter system. Strain AE2515 was standardised for its specific responses to Co2+ and Ni2+. The detection limits for AE2515 were 0.1 microM Ni2+ and 9 microM Co2+, respectively. The signal to noise (S/N) bioluminescence response and the metal cation concentration could be linearly correlated: for Ni2+ this was applicable within the range 0.1-60 microM, and between 9 and 400 microM for Co2+. The AE2515 biosensor strain was found to be highly selective for nickel and cobalt: no induction was observed with Zn(II), Cd(II), Mn(II), Cu(III) and Cr(VI). In mixed metal solutions, the bioluminescent response always corresponded to the nickel concentrations. Only in the presence of high concentrations of Co2+ (2 mM), the sensitivity to nickel was reduced due to metal toxicity. AE2515 was used to quantify the metal bioavailability in various nickel-enriched soils, which had been treated with additives for in situ metal immobilisation. The data obtained with strain AE2515 confirmed that the bioavailability of nickel was greatly reduced following the treatment of the soils with the additives beringite and steel shots. Furthermore, the data were found to correlate linearly with those on the biological accumulation of Ni2+ in specific parts of important agricultural crops, such as maize and potato. Therefore, the test can be used to assess the potential transfer of nickel to organisms of higher trophic levels, in this case maize and potato plants grown on nickel-enriched soils, and the potential risk of transfer of these elements to the food chain.

Heavy metals bioremediation of soil.

Historical emissions of old nonferrous factories lead to large geographical areas of metals-contaminated sites. At least 50 sites in Europe are contaminated with metals like Zn, Cd, Cu, and Pb. Several methods, based on granular differentiation, were developed to reduce the metals content. However, the obtained cleaned soil is just sand. Methods based on chemical leaching or extraction or on electrochemistry do release a soil without any salts and with an increased bioavailability of the remaining metals content. In this review a method is presented for the treatment of sandy soil contaminated with heavy metals. The system is based on the metal solubilization on biocyrstallization capacity of Alcaligenes eutrophus CH34. The bacterium can solubilize the metals (or increase their bioavailability) via the production of siderophores and adsorb the metals in their biomass on metal-induced outer membrane proteins and by bioprecipitation. After the addition of CH34 to a soil slurry, the metals move toward the biomass. As the bacterium tends to float quite easily, the biomass is separated from the water via a flocculation process. The Cd concentration in sandy soils could be reduced from 21 mg Cd/kg to 3.3 mg Cd/kg. At the same time, Zn was reduced from 1070 mg Zn/kg to 172 mg Zn/kg. The lead concentration went down from 459 mg Pb/kg to 74 mg Pb/kg. With the aid of biosensors, a complete decrease in bioavailability of the metals was measured.

Comparative sensitivity of 20 bioassays for soil quality.

Increasing evidence suggests that the use of a single bioassay will never provide a full picture of the quality of the environment. Only a test battery, composed of bioassays of different animal and plant species from different trophic levels will reduce uncertainty, allowing an accurate assessment of the quality of the environment. In the present study, a test battery composed of 20 bioassays of varying biological endpoints has been compared. Apart from lethality and reproductive failure in earthworms, springtails, nematoda, algae and vascular plants, these endpoints also included bioavailibility of metals (bacteria), heat-shock induction (nematodes, algae), DNA damage (bacteria, earthworm, vascular plants), beta-galactosidase (Daphnia) and esterase activity (algae) and a range of immunological parameters (earthworm). Four chemicals (cadmium, phenol, pentachlorophenol and trifluralin)--each representing a different toxic mode of action--were applied in a dilution series (from 1 mg/kg up to 1000 mg/kg) onto OECD standard soil. The tests have been performed both on these artificially contaminated soil samples and on aqueous extracts subsequently obtained from these soils. The results show that the immunological parameters and the loss of weight in the earthworms were among the most sensitive solid-phase assays. Esterase inhibition and heat-shock induction in algae were shown to be extremely sensitive when applied to soil extracts. As previously shown at the species level, no single biological endpoint was shown to be the most sensitive for all four modes of toxic action.

Effect of the siderophore alcaligin E on the bioavailability of Cd to Alcaligenes eutrophus CH34.

Alcaligin E, the siderophore of the heavy metal-resistant A. eutrophus strain CH34, was shown to interact with Cd and consequently affect its bioavailability and toxicity. The addition of alcaligin E markedly stimulated the growth in the presence of Cd of an alcaligin E-deficient CH34 derivative. Using bioluminescence assays, this effect could be assigned to a decrease in bioavailability of Cd in the presence of alcaligin E. However, Cd-uptake studies showed no influence of alcaligin E on the cellular concentration of Cd. Furthermore, by scanning electron microscopy, the morphology of precipitated Cd crystals was shown to be altered by alcaligin E. These data suggest that alcaligin E, besides its function in iron supply to the cell, provides a protection against heavy metal toxicity. A link between the A. eutrophus CH34 siderophore system and the czc-mediated Cd-efflux system is hypothesized.

Plasmids for heavy metal resistance in Alcaligenes eutrophus CH34: mechanisms and applications.

Alcaligenes eutrophus CH34 is the main representative of a group of strongly related strains (mostly facultative chemolithotrophs) that are well adapted to environments containing high levels of heavy metals. It harbors the megaplasmids pMOL28 and pMOL30 which carry resistance determinants to Co2+, Ni2+, CrO(4)2-, Hg2+, Tl+, Cd2+, Cu2+ and Zn2+. Among the best characterized determinants are the cnr operon (resistance to Co, Ni) on pMOL28 and the czc operon on pMOL30 (resistance to Co, Cd and Zn). Although the two systems reveal a significant degree of amino acid similarity in the structural genes, the regulation of the operons is different. The resistance mechanism in both cases is based on efflux. The efflux mechanism leads to a pH increase outside of the cytoplasmic membrane. Metals are sequestered from the external medium through the bioprecipitation of metal carbonates formed in the saturated zone around the cell. This latter phenomenon can be exploited in bioreactors designed to remove metals from effluents. The bacteria are immobilized on composite membranes in a continuous tubular membrane reactor (CTMR). The effluent continuously circulates through the intertubular space, while the external surface of the tubes is in contact with the growth medium. Metal crystals are eventually removed by the effluent stream and collected on a glass bead column. The system has been applied to effluents containing Cd, Zn, Co, Ni and Cu. By introducing catabolic plasmids involved in the aerobic degradation of PCBs and 2,4-D into metal-resistant A. eutrophus strains, the application range was widened to include effluents polluted with both organic and inorganic substances. Biosensors have been developed which are based on the fusion of genes induced by metals to a reporter system, the lux operon of Vibrio fischeri. Bacterial luciferases produce light through the oxidation of fatty aldehydes. The gene fusions are useful both for the study of regulatory genes and for the determination of heavy metal concentrations in the environment.

Influence of the energetic pattern of mitochondria in cell ageing.

Injections of mitochondria isolated from liver of young or old rats have been performed in young or old WI-38 human fibroblasts and the survival of the injected cells was followed with time. Cells having received young mitochondria behave as the control non-injected cells while cells having received old mitochondria showed signs of degeneration after a few days. Such a behaviour could however be obtained with young mitochondria when partially uncoupled. The negative effect of the presence of uncoupled mitochondria could be overcome by addition of a ketone body: D(-) beta-hydroxybutyrate. When comparisons were performed between injected young and old fibroblasts, old cells were found less efficient in counteracting the presence of uncoupled mitochondria. These results clearly indicate that old cells contain partly altered mitochondria which are less able to fulfil their energy requirement so that a general lowering of homeostasis but also an increased susceptibility, toward unfavorable situations is obtained.

luxAB gene fusions with the arsenic and cadmium resistance operons of Staphylococcus aureus plasmid pI258.

pC101, a novel shuttle vector between Escherichia coli and Staphylococcus aureus carrying the lux genes encoding luciferase from Vibrio harveyi, selectable ampicillin and chloramphenicol markers and origins of replication for Gram-negative and Gram-positive bacteria has been constructed. The inducibility of the arsenic and cadmium operon from S. aureus plasmid pI258 to different ions has been tested in E. coli and in S. aureus with two fusions in pC101: an arsB-luxAB and a cadA-luxAB transcriptional gene fusion. Patterns of induction are influenced by the host strain and are slightly different from previous reports using the blaZ gene as reporter gene.

Involvement of mitochondria in cell degeneration.

Human WI-38 fibroblasts were microinjected with isolated mitochondria, and survival of the injected cells was followed. More than 95% of the cells were alive and able to divide when they were injected with fresh mitochondrial preparations having a high respiratory control ratio (RCR). The presence of lysosomes was found to be toxic to the cells, and hence mitochondria had to be isolated without being contaminated by lysosomes. The microinjection of isolated mitochondria from old rats induced a 20% degeneration of the injected cells. The proportion of dead cells was also found to be dependent on the metabolic control of the injected mitochondria. Moreover, an easily metabolized energy substrate such as D(-)-beta-hydroxybutyrate sodium salt was able to inhibit, in a dose-dependent manner, cell degeneration induced by microinjection of uncoupled mitochondria. These results suggest that modifications of mitochondria leading to their uncoupling are harmful to the cells, and this can explain some of the degenerative processes observed in natural or externally induced cell death.

Respiratory activity of isolated rat liver mitochondria following in vitro exposure to oxygen species: a threshold study.

Respiratory activity of isolated rat liver mitochondria was assayed following in vitro exposure to oxygen radicals. Our results show that mitochondrial respiration is more sensitive to O2.(-) than to H2O2. However, ferrous ions drastically enhance the toxicity of the enzymatic system generating H2O2 because of the production of the hydroxyl radicals. A protection against those oxygen species could be given by SOD in the xanthine/xanthine oxidase system and by catalase with the glucose/glucose oxidase system. The most damaging system was the combination of Fe2+ with H2O2. In this case, OH. is formed in a Fenton-like reaction. The fact that the OH. is the most damaging molecule accounts for the finding that catalase and desferrioxamine were efficient protectors in this system. Threshold levels of O2.(-) and H2O2 able to inhibit the mitochondrial respiration have been estimated. It is concluded that under normal respiration such thresholds are not reached in vivo and that the impairment of the mitochondrial respiratory activity does not seem to originate only from the natural free radical production in those organelles. However, if the production of free radicals is such to exceed the defense capability, like under oxidative stress, then the critical threshold can be surpassed and the respiration impaired leading to irreversible damages.

Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals.

Glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase are the most important enzymes of the cell antioxidant defense system. However, these molecules are themselves susceptible to oxidation. The aim of this work was to estimate to what extent this system could be inactivated by its own substrates. We tested the effect of hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and hydroxyl and superoxide radicals on GPX, SOD and catalase. For GPX, a 50% inactivation was observed at 10(-1) M (30 min, 37 degrees C) for hydrogen peroxide, 3 x 10(-4) M (15 min, 37 degrees C) for cumene hydroperoxide and 5 x 10(-5) M (11 min, 37 degrees C) for t-butyl hydroperoxide. Unlike the hydroxyl radicals, superoxide anions did not inactivate this enzyme. Catalase was inactivated by hydroxyl radicals and by superoxide anions but organic peroxides had no effect. SOD was inactivated by 50% by hydrogen peroxide at 4 x 10(-4) M (20 min, 37 degrees C), but organic peroxides and hydroxyl radicals were ineffective on this enzyme. Since the three enzymes of the antioxidant system are susceptible to at least one of the oxidative reactive molecules, in the case of high oxidative stresses such an inhibition could take place, leading to an irreversible autocatalytical process in which the production rate of the oxidants will continuously increase, leading to cell death.

Importance of a threshold for error accumulation in cell degenerative processes. I. Modulation of the threshold in a model of free radical-induced cell degeneration.

Antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) have been injected into human fibroblasts exposed to 2 atm O2 in order to test if the threshold of oxidative damage versus antioxidant defenses could be modulated and if the damage remains reversible beyond the threshold. Cell damage was estimated by thymidine incorporation and cell survival curves. The proportion of dividing cells, measured by thymidine incorporation, rapidly decreased after O2 incubation: no cells could divide after 15 h of hyperoxia. However, cells incubated for a short time and injected with a high concentration of any of the three enzymes divided like non-oxygen-incubated cells: the enzymes could protect the cells against their loss of division potential. However, when cells were incubated for a longer period and/or when the injected enzyme concentration was lower, cells were either less or not protected and could no longer divide. These results suggest the presence of a threshold for the oxidative damage which cannot be totally repaired and which impairs the cell division; this threshold can, however, be modulated by supplementation of antioxidant enzymes, glutathione peroxidase being the most efficient.

A new technique for highly sensitive detection of superoxide dismutase activity by chemiluminescence.

A stable enzymatic free radical generation system has been developed which allows a precise production of 02-. and its detection by chemiluminescence between 2 pmol and 8 nmol. This test has been used for assaying superoxide dismutase (SOD) by inhibition of the chemiluminescence (CL) signal. No inhibition was observed with catalase, which excludes the participation of H2O2 in lucigenin CL. N,N-Diethyldithiocarbamate gives 100% inhibition of SOD activity either from a purified enzymatic preparation or from biological samples, which confirms the specificity of the CL assay. SOD assay can be performed either on a purified enzymatic preparation or on biological materials such as cultured cells.

[Properties of histones on hydrophobic chromatography]. / Le comportement des histones en chromatographie hydrophobe

The hydrophobic chromatography on alkylated Sepharose allows to separate the histones into three groups which exhibit an increasing affinity for the support HI less than H2A-H2B less than H3-H4. In this fractionation procedure, the behaviour of the histones taken separately or pair-associated, is discussed in relation with the ability of these proteins to complex each other in vitro and in vivo.