Impact of Nanoparticle-Based TiO2 Surfaces on Norovirus Capsids and Genome Integrity.

Human noroviruses (HuNoVs) are among the main causes of acute gastroenteritis worldwide. HuNoVs can survive for several days up to weeks at room temperature in the environment, on food, and on food handling and processing surfaces. As a result, this could lead to viral spread through the ingestion of food in contact with contaminated surfaces. The development of stable surface materials with antiviral activity might be useful to reduce viral outbreaks. Metal-based compounds, including photoactivated titanium nanoparticles (TiO2 NPs), are known for their antiviral activity. In this study, we tested the impact of 2000 µg/mL TiO2 NPs, with or without UV activation, on HuNoV GII and murine norovirus. Their recovery rates were reduced by 99.6%. We also evaluated a new TiO2 NP-coating process on a polystyrene surface. This process provided a homogenous coated surface with TiO2 NPs ranging between 5 nm and 15 nm. Without photoactivation, this TiO2 NP-coated polystyrene surface reduced the recovery rates of intact HuNoV GII by more than 94%. When a capsid integrity treatment with PtCl4 or a longer reverse transcription polymerase chain detection approach was used to evaluate virus integrity following contact with the TiO2 NP-coated polystyrene, the HuNoV GII recovery yield reduction varied between 97 and 100%. These results support the hypothesis that TiO2 NP-coated surfaces have the potential to prevent viral transmission associated with contaminated food surfaces.

Impact of Capsid and Genomic Integrity Tests on Norovirus Extraction Recovery Rates.

Human norovirus (HuNoV) is the leading pathogen responsible for food-borne illnesses. However, both infectious and non-infectious HuNoV can be detected by RT-qPCR. This study evaluated the efficiency of different capsid integrity treatments coupled with RT-qPCR or a long-range viral RNA (long RT-qPCR) detection to reduce the recovery rates of heat inactivated noroviruses and fragmented RNA. The three capsid treatments evaluated (RNase, the intercalating agent PMAxx and PtCl4) reduced the recovery of heat inactivated HuNoV and murine norovirus (MNV) spiked on lettuce, when combined with the ISO 15216-1:2017 extraction protocols. However, PtCl4 also reduced non-heat-treated noroviruses recovery as estimated by RT-qPCR. The PMAxx and RNase treatments had a similar effect on MNV only. The most efficient approaches, the RNase and PMAxx treatments, reduced the heat-inactivated HuNoV recovery rates estimated using RT-qPCR by 2 and >3 log, respectively. The long RT-qPCR detection approach also reduced the recovery rates of heat inactivated HuNoV and MNV by 1.0 and 0.5 log, respectively. Since the long-range viral RNA amplification could be applied to verify or confirm RT-qPCR results, it also provides some advantages by reducing the risk of false positive HuNoV results.

Study of the Antibacterial Potency of Electroactivated Solutions of Calcium Lactate and Calcium Ascorbate on Bacillus cereus ATCC 14579 Vegetative Cells.

Bacillus cereus is a pathogenic bacterium, Gram-positive, aerobic, and facultative anaerobic that can produce spores and different toxins. It is involved in serious foodborne illnesses such as the diarrheal and emetic syndromes, depending on the ingested toxin. This work is aimed to study the potency of electroactivated solutions (EAS) of calcium lactate, calcium ascorbate, and their mixture as antibacterial agents against B. cereus ATCC 14579 vegetative cells. The solutions used were electroactivated under electric current intensities of 250, 500, and 750 mA for 30 min. The obtained EAS were tested in direct contact with B. cereus (107 CFU/mL) for different durations ranging from 5 s to 2 min. Moreover, standard lactic and ascorbic acids were tested as controls at equivalent titratable acidity as that of the corresponding electroactivated solutions. The obtained results showed that EAS exhibit high antibacterial efficacy against B. cereus vegetative cells. The EAS obtained after electroactivation of calcium lactate and calcium ascorbate were more efficient than those of their corresponding standard acids (lactic and ascorbic). The observed antibacterial effect of the EAS resulted in a reduction of 7 log CFU/mL after 5 s of direct contact in some specific cases. Scanning (SEM) and transmission (TEM) electron microscopic observations provided conclusive evidence of the antibacterial activity of the used EAS. These results outlined the highly antimicrobial potency of EAS against B. cereus vegetative cells and that they can be considered in an eventual strategy to ensure food safety, surface cleaning, as well as replacement of hazardous disinfecting chemicals.

Detection and Sequencing of Multiple Human Norovirus Genotypes from Imported Frozen Raspberries Linked to Outbreaks in the Province of Quebec, Canada, in 2017.

Human noroviruses are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several norovirus food-related outbreaks. However, the extraction of norovirus RNA from frozen raspberries remains challenging. Recovery yields are low and PCR inhibitors limit the sensitivity of the detection methodologies. In 2017, 724 people from various regions of the Province of Quebec, Canada, were infected by noroviruses and the outbreak investigation pointed to frozen raspberries as a putative source. A new magnetic silica bead approach was used for the extraction of viruses from different outbreak samples. The RNA extracts were tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and five samples were confirmed positive for norovirus by RT-qPCR amplicon sequencing. A multiplex long-range two-step RT-PCR approach was developed to amplify norovirus ORF2 and ORF3 capsid genes from the positive frozen raspberry RNA extracts and other sequencing strategies were also explored. These capsid genes were sequenced by Next-Generation Sequencing. Phylogenetic analyses confirmed the presence of multiple genotypes (GI.3, GI.6, and GII.17) and intra-genotype variants in some of the frozen raspberry samples. Variants of genotype GI.3 and GI.6 had 100% homology with sequences from patient samples. Similar strains were also reported in previous outbreaks. Confirmation approaches based on sequencing the norovirus capsid genes using Next-Generation Sequencing can be applied at trace level contaminations and could be useful to assess risk and assist in source tracking.

Extraction of human noroviruses from leafy greens and fresh herbs using magnetic silica beads.

Consumption of leafy greens and to a lesser extent fresh herbs has been associated with several foodborne outbreaks including human norovirus (HuNoV). However, the extraction and detection of viruses from these matrices present multiple challenges such as low recovery yields and relatively high PCR inhibition. A new magnetic silica bead based (MSB) extraction protocol was developed and used to recover norovirus from leafy greens and fresh herbs. The performance results were compared to the ISO 15216-1:2017 standard. The HuNoV GII.4 and GI.5 recovery yields from spiked lettuce using the MSB extraction protocol range from 33 to 82%. There was a good correlation between murine norovirus (MNV) and HuNoV recovery yields from fresh herbs and leafy greens. No reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) inhibition was detected from leafy green extracts using the MSB methodology. The selected commercial RT-qPCR detection kit had a major impact on RT-qPCR inhibition levels detected in the ISO 15216-1:2017 RNA extracts. RNase treatment was used to estimate genome recovery from HuNoV with intact capsids. This treatment resulted in similar HuNoV and MNV recovery yields. Between 2019 and 2020, the MSB protocol was used to conduct a survey of HuNoV in domestic and imported leafy greens and fresh herbs sold at retail in Canada. All of the 280 samples tested were negative. Overall, the use of MSB was shown to be an efficient approach to recover HuNoV from leafy greens and certain types of fresh herbs and to conduct surveys.

Study of the impacts of electro-activated solutions of calcium lactate, calcium ascorbate and their equimolar mixture combined with moderate heat treatments on the spores of Bacillus cereus ATCC 14579 under model conditions and in fresh salmon.

Widespread in very diverse environments, the spores of Bacillus cereus are highly resistant to hostile conditions and can contaminate a huge variety of food products, posing a potential health hazard to consumers. Given this significant risk, the objective of this research work was to study the impacts of electro-activated solutions (EAS) made with calcium ascorbate, calcium lactate, and their equimolar mixture on Bacillus cereus ATCC 14579 spores in model conditions and food matrix, the fresh Atlantic salmon. The model conditions consisted of a direct application of the EAS to the spores, which avoided any interference with factors external to those of the solutions. Salmon was chosen as a food model because it is a product sensitive to bacterial spoilage and can be eaten raw. To achieve this, the solutions were prepared by electro-activation using an electric current with an intensity of 750 mA for 30 min, resulting in mean pH values of 1.94 ± 0.15-2.16 ± 0.01 and titratable acidity of 0.102 ± 0.001-0.109 ± 0.001 mol/L, depending on the type of solution. These conditions were chosen because of their excellent antibacterial efficacy previously demonstrated against vegetative cells of B. cereus. The results showed high sporicidal activities of the EAS against B. cereus with a 7 to 9 log reduction, using an initial spore population of 109 CFU/mL, depending on the conditions evaluated, namely: in direct contact (2-30 min), in salmon used as a food matrix (2-7 min), and in combination with moderate heat treatments from 60 to 90 °C (0.5-2 min). In addition, it was observed that the sporicidal capacity of the EAS increased with temperature and the contact time. Otherwise, analysis of the color and lipids of the salmon have not shown any major impacts of the use of EAS as a rinsing solution for this highly perishable food. Furthermore, micrographs taken by scanning and transmission electron microscopy revealed the destructive effects of the EAS used in the vital structures/components of the spores. In general, this study has demonstrated that the electro-activation technology is effective in producing EAS capable of destroying/inactivating B. cereus spores and that they can be used for the improvement of food safety and preservation.

Study of the Electro-Activation Process of Calcium Lactate, Calcium Ascorbate Solutions, and Their Equimolar Mixture: Assessment of Their Physicochemical Properties.

The aim of this study was to prepare electro-activated solutions (EAS) from calcium lactate, calcium ascorbate, and an equimolar mixture of these two salts to obtain their corresponding acids and to study their physicochemical characteristics, in particular, pH, titratable acidity, pK a, and antioxidant activity. Indeed, the solutions were electro-activated in a reactor comprising three compartments (anodic, central, and cathodic) separated by anionic and cationic exchange membranes, respectively. The electric current intensities used were set at 250, 500, and 750 mA for a maximum period of 30 min. In general, the EAS obtained at 750 mA for 30 min showed the lowest pH (2.16, 2.08, 1.94) and pK a (3.13, 3.07, 2.90) values and the highest titratable acidity (0.107, 0.102, 0.109 mol/L) for calcium lactate, the mixture, and calcium ascorbate, respectively. In addition, the obtained results have demonstrated that the pH, titratable acidity, and pK a of the EAS varied proportionally and significantly (p < 0.001) with the duration of the experiment and the intensity of the electric current applied. To evaluate the migration of calcium (Ca2+) between the central and the cathodic compartments of the reactor, the concentration of Ca2+ was determined especially in the cathodic section by inductively coupled plasma optical emission spectroscopy (ICP-OES). The results showed that the migration of Ca2+ varied proportionally with the electric current intensity. In this context, analysis by Fourier transform infrared (FTIR) spectroscopy, high-performance liquid chromatography (HPLC), and differential scanning calorimetry (DSC) have confirmed the production of lactic acid and ascorbic acid compared to standards. In addition, analysis by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging technique confirmed high antioxidant activities of >90 and >83% for calcium ascorbate and the mixture, respectively, in comparison to the standard ascorbic acid (85%). Overall, this research has clearly demonstrated the eventual potential of electro-activation to produce highly reactive organic acids from their conjugated salts. These EAS can become excellent antimicrobial and sporicidal agents in the food processing industry.

Norovirus Extraction from Frozen Raspberries Using Magnetic Silica Beads.

Human noroviruses (HuNoV) are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several HuNoV food-related outbreaks. However, the extraction of HuNoV RNA from frozen raspberries remains challenging. Recovery yields are low, and real-time quantitative reverse transcriptase PCR (RT-qPCR) inhibitors limit the sensitivity of the detection methodologies. A new approach using fine magnetic silica beads was developed for the extraction of HuNoV spiked on frozen raspberries. Relatively low recovery yields were observed with both the magnetic silica bead and the reference ISO 15216-1:2017 methods. High RT-qPCR inhibition was observed with the ISO 15216-1:2017 recommended amplification kit but could be reduced by using an alternative kit. Reducing RT-qPCR inhibition is important to limit the number of inconclusive HuNoV assays thus increasing the capacity to assess the HuNoV prevalence in frozen raspberries.

Porcine reproductive and respiratory syndrome virus (PRRSV) in pig meat.

Porcine reproductive and respiratory syndrome, caused by the porcine reproductive and respiratory syndrome virus (PRRSV), is an economically important disease in the swine industry. Previous studies demonstrated the presence of the virus in pig meat and its transmissibility by oral consumption. This study further analyzed the infectivity of PRRSV in commercial pig meat. Fresh bottom meat pieces (n = 1500) randomly selected over a period of 2 y from a pork ham boning plant located in Quebec, Canada, were tested by reverse transcriptase polymerase chain reaction (RT-PCR). Each trimmed meat was stored in the plant freezer, subsampled weekly for up to 15 wk, and tested with quantitative RT-PCR to determine the viral load. Meat infectivity was evaluated using specific pathogen-free piglets, each fed with approximately 500 g of meat at the end of the storage time. Genotype-specific RT-PCR confirmed the presence of PRRSV mainly during cold weather in 0.73% of the fresh meat pieces. Wild and vaccine strains of genotype 2 were detected. Porcine reproductive and respiratory syndrome virus nucleic acid was stable in meat stored at around -20°C during the 15 wk. Serological and molecular analysis showed the transmission of infection by a majority of PRRSV positive meat pieces (5/9) fed orally to naïve recipients. The results confirmed a low prevalence of PRRSV in market's pig meat, and virus transmissibility by oral consumption to naïve recipients even after several weeks of storage in a commercial freezer. It occurred mainly with meat harboring the highest PRRSV RNA copies, in the range of 109 copies per 500 g of meat, with both wild type and vaccine-related strains.

Le syndrome reproducteur et respiratoire porcin, causé par le virus du syndrome respiratoire et reproducteur porcin (vSRRP), est une maladie ayant un impact économique important pour l'industrie porcine. Des études antérieures ont démontré la présence du virus dans la viande de porc ainsi que sa transmissibilité par ingestion. La présente étude poursuit l'analyse de l'infectiosité du vSRRP dans la viande commerciale de porc. Des coupes de fesses de porc fraîches (n = 1500) sélectionnées aléatoirement sur une période de deux ans dans une usine de désossage située au Québec (Canada), ont été testées en utilisant une transcription réverse suivie d'une amplification en chaîne par polymérase (RT-PCR). Chaque pièce de viande parée a été entreposée dans les congélateurs à l'usine, échantillonnée hebdomadairement pendant 15 semaines, et testée par RT-PCR quantitatif afin de calculer la charge virale. Le potentiel infectieux a été évalué sur des porcelets exempts d'agent pathogène spécifique qui ont été nourris avec approximativement 500 g de viande à la fin de la période d'entreposage. Une RT-PCR spécifique au génotype a confirmé la présence du vSRRP principalement durant les temps froids, dans 0,73 % des pièces de viandes fraîches. Des souches sauvages et vaccinales du génotype 2 ont été détectées. L'acide nucléique du virus du syndrome respiratoire et reproducteur porcin est demeuré stable dans la viande durant la période d'entreposage de 15 semaines à −20 °C. L'analyse sérologique et moléculaire a démontré la transmission de l'infection par une majorité des pièces de viande positives au vSRRP (5/9) chez les porcelets naïfs ayant consommé la viande. Les résultats confirment la faible prévalence du vSRRP dans la viande distribuée sur le marché ainsi que la transmissibilité du virus par consommation orale chez des hôtes naïfs même après plusieurs semaines d'entreposage dans un congélateur commercial. La transmission s'est produite surtout avec les viandes ayant un nombre de copies d'ARN de vSRRP plus élevés, environ 109 copies par 500 g de viande, associées à des souches de type tant sauvage que vaccinal.(Traduit par les auteurs).

Identification of Novel Zoonotic Activity of Bartonella spp., France.

Certain Bartonella species are known to cause afebrile bacteremia in humans and other mammals, including B. quintana, the agent of trench fever, and B. henselae, the agent of cat scratch disease. Reports have indicated that animal-associated Bartonella species may cause paucisymptomatic bacteremia and endocarditis in humans. We identified potentially zoonotic strains from 6 Bartonella species in samples from patients who had chronic, subjective symptoms and who reported tick bites. Three strains were B. henselae and 3 were from other animal-associated Bartonella spp. (B. doshiae, B. schoenbuchensis, and B. tribocorum). Genomic analysis of the isolated strains revealed differences from previously sequenced Bartonella strains. Our investigation identifed 3 novel Bartonella spp. strains with human pathogenic potential and showed that Bartonella spp. may be the cause of undifferentiated chronic illness in humans who have been bitten by ticks.

Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules.

Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. In the model legume Medicago truncatula, NO production has been detected in the nitrogen fixation zone of the nodule, but the systems responsible for its synthesis are yet unknown and its role in symbiosis is far from being elucidated. In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions. When transferred from normoxia to hypoxia, nodule NO production was rapidly increased, indicating that NO production capacity is present in functioning nodules and may be promptly up-regulated in response to decreased oxygen availability. Contrary to roots and leaves, nodule NO production was stimulated by nitrate and nitrite and inhibited by tungstate, a nitrate reductase inhibitor. Nodules obtained with either plant nitrate reductase RNA interference double knockdown (MtNR1/2) or bacterial nitrate reductase-deficient (napA) and nitrite reductase-deficient (nirK) mutants, or both, exhibited reduced nitrate or nitrite reductase activities and NO production levels. Moreover, NO production in nodules was found to be inhibited by electron transfer chain inhibitors, and nodule energy state (ATP-ADP ratio) was significantly reduced when nodules were incubated in the presence of tungstate. Our data indicate that both plant and bacterial nitrate reductase and electron transfer chains are involved in NO synthesis. We propose the existence of a nitrate-NO respiration process in nodules that could play a role in the maintenance of the energy status required for nitrogen fixation under oxygen-limiting conditions.

Detection and identification of multiple genetically modified events using DNA insert fingerprinting.

Current screening and event-specific polymerase chain reaction (PCR) assays for the detection and identification of genetically modified organisms (GMOs) in samples of unknown composition or for the detection of non-regulated GMOs have limitations, and alternative approaches are required. A transgenic DNA fingerprinting methodology using restriction enzyme digestion, adaptor ligation, and nested PCR was developed where individual GMOs are distinguished by the characteristic fingerprint pattern of the fragments generated. The inter-laboratory reproducibility of the amplified fragment sizes using different capillary electrophoresis platforms was compared, and reproducible patterns were obtained with an average difference in fragment size of 2.4 bp. DNA insert fingerprints for 12 different maize events, including two maize hybrids and one soy event, were generated that reflected the composition of the transgenic DNA constructs. Once produced, the fingerprint profiles were added to a database which can be readily exchanged and shared between laboratories. This approach should facilitate the process of GMO identification and characterization.

Prolonged root hypoxia induces ammonium accumulation and decreases the nutritional quality of tomato fruits.

Here we examined the effects of root hypoxia (1-2% oxygen) on the physiology of the plant and on the biochemical composition of fruits in tomato (Solanum lycopersicum cv. Micro-Tom) plants submitted to gradual root hypoxia at first flower anthesis. Root hypoxia enhanced nitrate absorption with a concomitant release of nitrite and ammonium into the medium, a reduction of leaf photosynthetic activity and chlorophyll content, and an acceleration of fruit maturation, but did not affect final fruit size. Quantitative metabolic profiling of mature pericarp extracts by (1)H NMR showed that levels of major metabolites including sugars, organic acids and amino acids were not modified. However, ammonium concentration increased dramatically in fruit flesh, and ascorbate and lycopene concentrations decreased. Our data indicate that the unfavorable effects of root hypoxia on fruit quality cannot be explained by two of the well-known effects of root hypoxia on the plant, namely a decrease in photosynthesis or an excess in ethylene production, but may instead result from disturbances in the supply of either growth regulators or ammonium, by the roots.

Modifications in endopeptidase and 20S proteasome expression and activities in cadmium treated tomato (Solanum lycopersicum L.) plants.

The effects of cadmium (Cd) on cellular proteolytic responses were investigated in the roots and leaves of tomato (Solanum lycopersicum L., var Ibiza) plants. Three-week-old plants were grown for 3 and 10 days in the presence of 0.3-300 microM Cd and compared to control plants grown in the absence of Cd. Roots of Cd treated plants accumulated four to fivefold Cd as much as mature leaves. Although 10 days of culture at high Cd concentrations inhibited plant growth, tomato plants recovered and were still able to grow again after Cd removal. Tomato roots and leaves are not modified in their proteolytic response with low Cd concentrations (< or =3 microM) in the incubation medium. At higher Cd concentration, protein oxidation state and protease activities are modified in roots and leaves although in different ways. The soluble protein content of leaves decreased and protein carbonylation level increased indicative of an oxidative stress. Conversely, protein content of roots increased from 30 to 50%, but the amount of oxidized proteins decreased by two to threefold. Proteolysis responded earlier in leaves than in root to Cd stress. Additionally, whereas cysteine- and metallo-endopeptidase activities, as well as proteasome chymotrypsin activity and subunit expression level, increased in roots and leaves, serine-endopeptidase activities increased only in leaves. This contrasted response between roots and leaves may reflect differences in Cd compartmentation and/or complexation, antioxidant responses and metabolic sensitivity to Cd between plant tissues. The up-regulation of the 20S proteasome gene expression and proteolytic activity argues in favor of the involvement of the 20S proteasome in the degradation of oxidized proteins in plants.

A metabolic flux analysis to study the role of sucrose synthase in the regulation of the carbon partitioning in central metabolism in maize root tips.

In order to understand the role of sucrose synthase (SuSy) in carbon partitioning, metabolic fluxes were analyzed in maize root tips of a double mutant of SuSy genes, sh1 sus1 and the corresponding wild type, W22. [U-(14)C]-glucose pulse labeling experiments permitted the quantification of unidirectional fluxes into sucrose, starch and cell wall polysaccharides. Isotopic steady-state labeling with [1-(13)C]-, [2-(13)C]- or [U-(13)C]-glucose followed by the quantification by (1)H-NMR and (13)C-NMR of enrichments in carbohydrates and amino acids was also performed to determine 29 fluxes through central metabolism using computer-aided modeling. As a consequence of the suppression of SUS1 and SH1 isozymes, maize root tips diameter was significantly decreased and respiratory metabolism reduced by 30%. Our result clearly established that, in maize root tips, starch is produced from ADP-Glc synthesized in the plastid and not in the cytosol by sucrose synthase. Unexpectedly, the flux of cell wall synthesis was increased in the double mutant. This observation indicates that, in maize root tips, SH1 and SUS1 are not specific providers for cellulose biosynthesis.

Substrate cycles in the central metabolism of maize root tips under hypoxia.

Substrate cycles, also called "futile" cycles, are ubiquitous and lead to a net consumption of ATP which, in the normoxic maize root, have been estimated at about 50% of the total ATP produced [Alonso, A.P., Vigeolas, H., Raymond, P., Rolin, D., Dieuaide-Noubhani, M., 2005. A new substrate cycle in plants. Evidence for a high glucose-phosphate-to-glucose turnover from in vivo steady-state and pulse-labeling experiments with [(13)C] glucose and [(14)C] glucose. Plant Physiol. 138, 2220-2232]. To evaluate their role we studied the substrate cycles of maize root tips under an oxygen limitation of respiration (3% O(2)). Short-time labeling experiments with [U-(14)C]-Glc were performed to quantify the fluxes through sucrose and starch cycles of synthesis and degradation. Steady-state labeling with [1-(13)C]-Glc followed by (1)H NMR and (13)C NMR analysis of sugars and free alanine was used to quantify fluxes in the central metabolic pathways, including the Glc-P/Glc cycle and the fructose-P/triose-P cycle of glycolysis. Comparison with results previously obtained in normoxia [Alonso et al., as mentioned above] showed that 3% O(2) induced fermentation and reduced respiration, which led to a lesser amount of ATP produced. The rates of Glc consumption, glycolytic flux and all substrate cycles were lower, but the proportion of ATP consumed in the substrate cycles remained unchanged. These findings suggest that substrate cycles are not a luxury but an integral part of the organization of the plant central metabolism.

Metabolic flux analysis: recent advances in carbon metabolism in plants.

Isotopic tracers are used to both trace metabolic pathways and quantify fluxes through these pathways. The use of different labeling methods recently led to profound changes in our views of plant metabolism. Examples are taken from primary metabolism, with sugar interconversions, carbon partitioning between glycolysis and the pentose phosphate pathway, or metabolite inputs into the tricarboxylic acid (TCA) cycle, as well as from secondary metabolism with the relative contribution of the plastidial and cytosolic pathways to the biosynthesis of terpenoids. While labeling methods are often distinguished according to the instruments used for label detection, emphasis is put here on labeling duration. Short time labeling is adequate to study limited areas of the metabolic network. Long-term labeling, when designed to obtain metabolic and isotopic steady-state, allows to calculate various fluxes in large areas ofcentral metabolism. After longer labeling periods, large amounts of label accumulate in structural or storage compounds: their detailed study through the retrobiosynthetic method gives access to the biosynthetic pathways of otherwise undetectable precursors. This chapter presents the power and limits of the different methods, and illustrates how they can be associated with each other and with other methods of cell biology, to provide the information needed for a rational approach of metabolic engineering.

The occurrence of antibiotic resistance genes in Taq polymerases and a decontamination method applied to the detection of genetically modified crops.

Different antibiotic resistance (AR) genes, such as Bla, Tet and NPTII, contaminate commercially available Taq polymerases. The specificity of the AR gene PCR can be increased when using a restriction enzyme-based decontamination of polymerase. The elimination of Taq polymerase contamination allows the use of PCR tests to screen seeds (corn) and processed food for the presence of genetically modified organisms (GMO) based on the detection of AR genes. Without a decontamination procedure for AR genes, PCR screening tests should be interpreted with caution.

Development of a liquid chromatography-tandem mass spectrometry method using capillary liquid chromatography and nanoelectrospray ionization-quadrupole time-of-flight hybrid mass spectrometer for the detection of milk allergens.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the tryptic digest of a cleaned-up food matrix extract was used for the detection of milk allergens. The emphasis of this study was on casein, which is the most abundant milk protein and is also considered the most allergenic. A sample cleanup method was developed using an ion exchange column and centriprep device. Cookies spiked with milk powder from 0 to 1250 ppm were extracted, cleaned up, and either digested directly by trypsin or further cleaned up by gel electrophoresis before digestion. The peptide mixture was analyzed on a capillary LC-quadrupole time-of-flight system. Two marker peptides from alphaS1-casein were identified and used for prescreening. The MS/MS data from the mass spectrometry system were processed with Masslynx v4.0 and submitted for database search using either ProteinLynx Global Server or Mascot for protein identification. The LC-MS/MS method, using casein enzyme-linked immunosorbent assay as a reference, was tested on the cookie matrix and was extended to other sample matrices. There were good agreements between the two. This LC-MS/MS method provides a valuable confirmatory method for the presence of casein. It also allows the simultaneous detection of other milk allergens.

Identification of genetically modified potato (Solanum tuberosum) cultivars using event specific polymerase chain reaction.

Several genetically modified (GM) cultivars are registered in Canada although they are not currently in commercial production. The GM cultivars can be distinguished from the non-GM and other GM cultivars by analyzing the DNA nucleotide sequence at the insertion site of the transgene corresponding to a single transformation event in the plant genome. Techniques based on modified polymerase chain reaction (PCR) strategies were used to generate sequence information from the plant genome flanking the insertion site of transgenic DNA for specific GM potato events. The plant genome sequence adjacent to the transgenic insertion was used to design PCR primers, which could be used in combination with a primer annealing to one of the nearby inserted genetic elements to amplify an event specific DNA fragment. The event specific PCR fragments generated were sequenced to confirm the specificity of the method.