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Banati H.,Agro Environmental Research Institute | Darvas B.,Agro Environmental Research Institute | Feher-Toth S.,Soft Flow Hungary R&D Ltd. | Czeh A.,Soft Flow Hungary R&D Ltd. | Szekacs A.,Agro Environmental Research Institute
Toxins | Year: 2017

Levels of mycotoxins produced by Fusarium species in genetically modified (GM) and near-isogenic maize, were determined using multi-analyte, microbead-based flow immunocytometry with fluorescence detection, for the parallel quantitative determination of fumonisin B1, deoxynivalenol, zearalenone, T-2, ochratoxin A, and aflatoxin B1. Maize varieties included the genetic events MON 810 and DAS-59122-7, and their isogenic counterparts. Cobs were artificially infested by F. verticillioides and F. proliferatum conidia, and contained F. graminearum and F. sporotrichoides natural infestation. The production of fumonisin B1 and deoxynivalenol was substantially affected in GM maize lines: F. verticillioides, with the addition of F. graminearum and F. sporotrichoides, produced significantly lower levels of fumonisin B1 (~300 mg·kg−1) in DAS-59122-7 than in its isogenic line (~580 mg·kg−1), while F. proliferatum, in addition to F. graminearum and F. sporotrichoides, produced significantly higher levels of deoxynivalenol (~18 mg·kg−1) in MON 810 than in its isogenic line (~5 mg·kg−1). Fusarium verticillioides, with F. graminearum and F. sporotrichoides, produced lower amounts of deoxynivalenol and zearalenone than F. proliferatum, with F. graminearum and F. sporotrichoides. T-2 toxin production remained unchanged when considering the maize variety. The results demonstrate the utility of the Fungi-Plex™ quantitative flow immunocytometry method, applied for the high throughput parallel determination of the target mycotoxins. © 2017 by the authors; Licensee MDPI, Basel, Switzerland.


Adanyi N.,aFood Science Research Institute | Majer-Baranyi K.,aFood Science Research Institute | Berki M.,aFood Science Research Institute | Darvas B.,Agro Environmental Research Institute | And 3 more authors.
Sensors and Actuators, B: Chemical | Year: 2017

For the quick and reliable quantification of special active substances derived from herbs, a new type of immunosensor based on optical waveguide lightmode spectroscopy (OWLS) detection was investigated. Artemisinin, an antimalarial drug derived from the sweet wormwood plant Artemisia annua is a sesquiterpene lactone endoperoxide, and it is distilled from the dried leaves or flower clusters of A. annua. Numerous derivatives of artemisinin, including artesunate and artemether, are also being used in the treatment of malaria, and these compounds have recently gained utility also as anticancer agents. To quantitatively determine the presence of these biologically effective substances in various herbs, a novel OWLS-based immunosensor was investigated. To create regenerable sensitized surfaces in the OWLS technique so that the sensor can be applied several times, the antigen or the antibody were immobilized by covalent attachment to the silanized surfaces of the OWLS chips. When measuring with the antibody capture mode antibodies raised against the appropriate artemisinin derivative (artemisinin, artesunate or artemether) were immobilized on the sensor surface and the linear measuring range was determined. During the antigen capture measurement the protein conjugate of the analyte was immobilized on the waveguide surface. Standard solutions containing different amounts of the appropriate standard were mixed with antibodies at optimized concentration, the mixture was incubated and injected into the flow-injection analysis system of OWLS. Binding of the antibodies in the sample to the coated surface is competed for with free antigen in the sample, and only antibodies that remained in free form in the mixture bound to immobilized antigen-conjugates. The amount of antibodies bound to the surface of the chip was inversely proportional to the active substance content in the samples. The dynamic measuring range and the relative substrate specificity of the serum on artemisinin and its derivatives were studied. Herbs of different origin and herbal supplement products were analyzed using the newly developed method and a correlation was studied with HPLC/DAD/MS reference method. © 2016 Elsevier B.V.


PubMed | Agro Environmental Research Institute, MicroVacuum Ltd. and Food Science Research Institute
Type: | Journal: Food chemistry | Year: 2016

Optical waveguide lightmode spectroscopy (OWLS) technique has been applied to label-free detection of aflatoxin B1 in a competitive immunoassay format, with the aim to compare the analytical goodness of the developed OWLS immunosenor with HPLC and enzyme-linked immunosorbent assay (ELISA) methods for the detection of aflatoxin in spice paprika matrix. We have also assessed applicability of the QuEChERS method prior to ELISA measurements, and the results were compared to those obtained by traditional solvent extraction followed by immunoaffinity clean-up. The AFB1 content of sixty commercial spice paprika samples from different countries were measured with the developed and optimized OWLS immunosensor. Comparing the results from the indirect immunosensor to that obtained by HPLC or ELISA provided excellent correlation (with regression coefficients above 0.94) indicating that the competitive OWLS immunosensor has a potential for quick determination of aflatoxin B1 in paprika samples.


Szekacs A.,Agro Environmental Research Institute | Mortl M.,Agro Environmental Research Institute | Darvas B.,Agro Environmental Research Institute
Journal of Chemistry | Year: 2015

Over 2000 surface, ground and raw drinking water samples have been analyzed in the frame of different monitoring projects in Hungary and watercourses in neighboring countries between 1990 and 2015. Effects of pesticide contamination on ecological farming and drinking water supply have been assessed. Main water pollutant ingredients of agricultural origin in Hungary are herbicides related to maize production. After EU pesticide re-registration, diazinon, atrazine, and trifluralin gradually disappeared as contaminants. High levels of water soluble pollutants (e.g., acetochlor) in surface water result in temporarily enhanced levels in raw drinking water as well. Extreme levels observed for herbicide residues were of agrochemical industrial origin. © 2015 András Székács et al.


Paszti-Gere E.,Szent Istvan University | Jerzsele A.,Szent Istvan University | Balla P.,Semmelweis University | Ujhelyi G.,Semmelweis University | Szekacs A.,Agro Environmental Research Institute
Oxidative Medicine and Cellular Longevity | Year: 2016

Objectives. The relationship among matriptase function, cellular redox status, and maintenance of intestinal barrier integrity has not been established yet. The aim of this study is to reveal if the crosstalk between matriptase activators and intestinal epithelial monolayers can lead to perturbations in physiological redox regulation in vitro. Methods. The effects of suramin and sphingosine-1-phosphate (S1P) were tested on viability of intestinal porcine epithelial IPEC-J2 cells using MTS assay. Measurements of transepithelial electrical resistance (TER) were performed to determine changes in barrier integrity of cell monolayers. Amplex Red assay was used to monitor extracellular hydrogen peroxide production. Occludin distribution pattern was detected prior to and after matriptase activation using immunofluorescent staining technique. Results. TER reduction was observed in suramin-treated IPEC-J2 cell monolayers, which could be attributed to cell cytotoxic properties of 48 hr 50 M suramin administration. In contrast, S1P treatment increased TER significantly and elevated occludin accumulation in tight junctions. It was also found that extracellular hydrogen peroxide levels were maintained in IPEC-J2 cells exposed to matriptase activators. Discussion. S1P administration not accompanied by redox imbalance might be one of the key strategies in the improvement of barrier function and consequently in the therapy of intestinal inflammations. © 2016 E. Pászti-Gere et al.


Seres A.,Szent Istvan University | Kiss I.,Szent Istvan University | Nagy P.,Szent Istvan University | Saly P.,Hungarian Academy of Sciences | And 2 more authors.
Plant, Soil and Environment | Year: 2014

Despite the fact that, on average, approximately 5–6 metric tons/ha of Bt maize stubble enter the soil on more than 170 million of hectares worldwide, the environmental impact of genetically modified maize plants on the arbuscular mycorrhizal fungi (AMF) is poorly known. In this study, the mycorrhizal colonisation on the roots of Bt maize (DAS-59122-7) and its near isogenic line was examined during the whole vegetation period. Cry3 toxin-producing Bt maize and its near isogenic line were grown in an experimental field in Julianna-major, Nagykovácsi, Hungary. DAS-59122-7 maize produces Cry34Ab1, Cry35Ab1 toxins and pat proteins for herbicide tolerance. The study assessed whether similar arbuscular mycorrhizal colonisation can be observed on the root of the Bt and near isogenic maize line and whether there are any differences in the temporal dynamics of AMF development. The arbuscular, hyphal and the arbuscular mycorrhizal fungi colonisation were higher in the near isogenic line as compared to its Bt counterpart, but no significant effect of the maize line was found as regards vesicle colonisation. The intensity of the arbuscular infection increased over time during plant maturation. DAS-59122-7 Bt maize had a negative effect on the initial development of AMF under field conditions, but no difference was seen in the case of the last two sampling dates (day 82 and 135). The reason of the latter is still not known. © 2014, Institute of Agricultural and Food Information. All rights reserved.


PubMed | Agro Environmental Research Institute, University of Caen Lower Normandy and CRIIGEN
Type: Journal Article | Journal: International journal of environmental research and public health | Year: 2016

Pesticide formulations contain declared active ingredients and co-formulants presented as inert and confidential compounds. We tested the endocrine disruption of co-formulants in six glyphosate-based herbicides (GBH), the most used pesticides worldwide. All co-formulants and formulations were comparably cytotoxic well below the agricultural dilution of 1% (18-2000 times for co-formulants, 8-141 times for formulations), and not the declared active ingredient glyphosate (G) alone. The endocrine-disrupting effects of all these compounds were measured on aromatase activity, a key enzyme in the balance of sex hormones, below the toxicity threshold. Aromatase activity was decreased both by the co-formulants alone (polyethoxylated tallow amine-POEA and alkyl polyglucoside-APG) and by the formulations, from concentrations 800 times lower than the agricultural dilutions; while G exerted an effect only at 1/3 of the agricultural dilution. It was demonstrated for the first time that endocrine disruption by GBH could not only be due to the declared active ingredient but also to co-formulants. These results could explain numerous in vivo results with GBHs not seen with G alone; moreover, they challenge the relevance of the acceptable daily intake (ADI) value for GBHs exposures, currently calculated from toxicity tests of the declared active ingredient alone.


PubMed | Balaton Limnological Research Institute, Agro Environmental Research Institute and The National Pedagogical University
Type: | Journal: Aquatic toxicology (Amsterdam, Netherlands) | Year: 2015

Neonicotinoids are highly potent and selective systemic insecticides, but their widespread use also has a growing impact on non-target animals and contaminates the environment, including surface waters. We tested the neonicotinoid insecticides commercially available in Hungary (acetamiprid, Mospilan; imidacloprid, Kohinor; thiamethoxam, Actara; clothianidin, Apacs; thiacloprid, Calypso) on cholinergic synapses that exist between the VD4 and RPeD1 neurons in the central nervous system of the pond snail Lymnaea stagnalis. In the concentration range used (0.01-1 mg/ml), neither chemical acted as an acetylcholine (ACh) agonist; instead, both displayed antagonist activity, inhibiting the cholinergic excitatory components of the VD4-RPeD1 connection. Thiacloprid (0.01 mg/ml) blocked almost 90% of excitatory postsynaptic potentials (EPSPs), while the less effective thiamethoxam (0.1 mg/ml) reduced the synaptic responses by about 15%. The ACh-evoked membrane responses of the RPeD1 neuron were similarly inhibited by the neonicotinoids, confirming that the same ACh receptor (AChR) target was involved. We conclude that neonicotinoids act on nicotinergic acetylcholine receptors (nAChRs) in the snail CNS. This has been established previously in the insect CNS; however, our data indicate differences in the background mechanism or the nAChR binding site in the snail. Here, we provide the first results concerning neonicotinoid-related toxic effects on the neuronal connections in the molluscan nervous system. Aquatic animals, including molluscs, are at direct risk while facing contaminated surface waters, and snails may provide a suitable model for further studies of the behavioral/neuronal consequences of intoxication by neonicotinoids.


PubMed | Agro Environmental Research Institute, Szent Istvan University and Max Planck Institute of Biochemistry
Type: | Journal: Journal of microbiological methods | Year: 2015

A variety of protein expression tags with different biochemical properties has been used to enhance the yield and solubility of recombinant proteins. Ubiquitin, SUMO (small ubiquitin-like modifier) and prokaryotic ubiquitin like MoaD (molybdopterin synthase, small subunit) fusion tags are getting more popular because of their small size. In this paper we report on the use of ubiquitin-like small archaeal modifier proteins (SAMPs) as fusion tags since they proved to increase expression yield, stability and solubility in our experiments. Equally important, they did not co-purify with proteins of the expression host and there was information that their specific JAB1/MPN/Mov34 metalloenzyme (JAMM) protease can recognize the C-terminal VSGG sequence when SAMPs fused, either branched or linearly to target proteins, and cleave it specifically. SAMPs and JAMM proteases from Haloferax volcanii, Thermoplasma acidophilum, Methanococcoides burtonii and Nitrosopumilus maritimus were selected, cloned, expressed heterologously in Escherichia coli and tested as fusion tags and cleaving proteases, respectively. Investigated SAMPs enhanced protein expression and solubility on a wide scale. T. acidophilum SAMPs Ta0895 and Ta01019 were the best performing tags and their effect was comparable to the widely used maltose binding protein (MBP) and N utilization substance protein A (NusA) tags. Moreover, H. volcanii SAMP Hvo_2619 contribution was mediocre, whereas M. burtonii Mbur_1415 could not be expressed. Out of four investigated JAMM proteases, only Hvo_2505 could cleave fusion tags. Interestingly, it was found active not only on its own partner substrate Hvo_2619, but it also cleaved off Ta0895.


PubMed | Agro Environmental Research Institute, Szent Istvan University and Semmelweis University
Type: | Journal: Oxidative medicine and cellular longevity | Year: 2016

Objectives. The relationship among matriptase function, cellular redox status, and maintenance of intestinal barrier integrity has not been established yet. The aim of this study is to reveal if the crosstalk between matriptase activators and intestinal epithelial monolayers can lead to perturbations in physiological redox regulation in vitro. Methods. The effects of suramin and sphingosine-1-phosphate (S1P) were tested on viability of intestinal porcine epithelial IPEC-J2 cells using MTS assay. Measurements of transepithelial electrical resistance (TER) were performed to determine changes in barrier integrity of cell monolayers. Amplex Red assay was used to monitor extracellular hydrogen peroxide production. Occludin distribution pattern was detected prior to and after matriptase activation using immunofluorescent staining technique. Results. TER reduction was observed in suramin-treated IPEC-J2 cell monolayers, which could be attributed to cell cytotoxic properties of 48hr 50M suramin administration. In contrast, S1P treatment increased TER significantly and elevated occludin accumulation in tight junctions. It was also found that extracellular hydrogen peroxide levels were maintained in IPEC-J2 cells exposed to matriptase activators. Discussion. S1P administration not accompanied by redox imbalance might be one of the key strategies in the improvement of barrier function and consequently in the therapy of intestinal inflammations.

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