Co-Occurrence of Moniliformin and Regulated Fusarium Toxins in Maize and Wheat Grown in Italy.

The co-occurrence of moniliformin (MON), fumonisins (FBs), and deoxynivalenol (DON) was evaluated in maize, durum, and common wheat grown in different experimental fields located in several Italian regions. MON was quantified using a LC-MS/MS method adding lanthanum ions in the mobile phase. In maize, MON contamination was widespread and considerable; the toxin was detected in almost all the samples (95.1%) and exceeded 500 and 1000 µg kg-1 in 42.0% and in 18.5% of samples, respectively. Significant positive correlation was found between MON and FB contamination levels. When there were not droughty climate conditions, a positive significant correlation was found between growing degree days (GDD) and MON values. In wheat, MON contamination was not widespread like in maize and it was lower in common wheat than in durum wheat. In durum wheat, MON was detected in 45.0% of the samples with only 6 samples (7.5%) exceeding 500 µg kg-1, while in common wheat the toxin was detected above the LOD in 18.7% of samples exceeding 100 µg kg-1 in only two samples (2.5%). No correlation was found with DON contamination. Climate conditions influenced both MON and DON occurrence.

A novel sandwich aptasensor for detecting T-2 toxin based on rGO-TEPA-Au@Pt nanorods with a dual signal amplification strategy.

T-2 toxin is a mycotoxin that can cause chronic illnesses, and the detection of T-2 toxin in food is critical for human health. Herein, a novel sandwich aptasensor with a dual signal amplification strategy was developed for the detection of T-2 toxin. Molybdenum disulfide-polyaniline-chitosan-gold nanoparticles (MoS2-PANI-Chi-Au) were processed to the modified glassy carbon electrode (GCE) and used as the aptasensor platform to expedite the electronics transport and immobilize the amino-terminated capture DNA probe by Au-N bonds. The reduced graphene oxide-tetraethylene pentamine-gold@platinum nanorods (rGO-TEPA-Au@Pt NRs) were first synthesized and immobilized with a signal DNA probe. Once T-2 toxin was added into the biosensing system, the aptamer would trap T-2 toxin to turn the signal off. Next, dissociative aptamer hybridized with the capture DNA probe in GCE and linked simultaneously to the signal DNA probe on rGO-TEPA-Au@Pt NRs with another end sequence of aptamer to turn the signal on. Owing to the efficient catalytic ability of bimetallic Au@Pt nanorods, the signal was perfectly amplified through the catalysis of hydrogen peroxide (H2O2) and recorded by chronoamperometry. With the outstanding augment response, the limit of detection reached 1.79 fg mL-1 (3SB/m) and a wide linear range from 10 fg mL-1 to 100 ng mL-1 was presented. The sensitivity of the aptasensor was 19.88 µA⋅µM-1⋅cm-2. Meanwhile, the DNA aptamer-bimetallic nanorod based sensing system presented excellent specificity. The developed aptasensor provides a new platform for T-2 toxin detection with low cost for real sample assays.

A universal multi-wavelength fluorescence polarization immunoassay for multiplexed detection of mycotoxins in maize.

Multi-analyte immunoassays have attracted increasing attention due to their short assay times, low sample consumption, and reduced detection costs per assay. In this work, we describe a homologous and high-throughput multi-wavelength fluorescence polarization immunoassay (MWFPIA) for the multiplexed detection of mycotoxins. Three typical Fusarium mycotoxins, deoxynivalenol (DON), T-2 toxin and fumonisin B1 (FB1), were labeled with different dyes. Tracers and specific monoclonal antibodies (mAbs) were employed in the MWFPIA to simultaneously detect the three mycotoxins. Under optimal conditions, the limits of detection using MWFPIA were 242.0 µg kg(-1) for DON, 17.8 µg kg(-1) for T-2 toxin and 331.5 µg kg(-1) for FB1, providing sufficient sensitivity to meet the action levels of these three contaminants in maize as set by the European Union. The use of a methanol/water (2:3, v/v) mixture for sample pretreatment allowed recoveries ranging from 76.5-106.3%, with coefficients of variation less than 21.7%. The total time of analysis, including sample preparation, was less than 30 min. Twenty naturally contaminated maize samples were tested using MWFPIA and HPLC-MS/MS, with correlation coefficients (R(2)) of 0.97 for DON and 0.99 for FB1. By changing the targets of interest, homologous MWFPIA, a method with high sensitivity, a simple procedure and a short analysis time, can easily be extended to other chemical contaminants. Thus, MWFPIA represents a versatile strategy for food safety analysis.

Deoxynivalenol and other selected Fusarium toxins in Swedish wheat--occurrence and correlation to specific Fusarium species.

Wheat is often infected by Fusarium species producing mycotoxins, which may pose health risks to humans and animals. Deoxynivalenol (DON) is the most important Fusarium toxin in Swedish wheat and has previously been shown to be produced mainly by Fusarium graminearum. However, less is known about the co-occurrence of DON and F. graminearum with other toxins and Fusarium species in Sweden. This study examined the distribution of the most important toxigenic Fusarium species and their toxins in winter wheat (2009 and 2011) and spring wheat (2010 and 2011). DNA from seven species was quantified with qPCR and the toxin levels were quantified with a multitoxin analysis method based on liquid chromatography/electrospray ionisation-tandem mass spectrometry (HPLC/ESI-MS/MS). The method enabled detection of many fungal metabolites, including DON, zearalenone (ZEA), nivalenol (NIV), T-2 toxin, HT-2 toxins, moniliformin (MON), beauvericin (BEA), and enniatins (ENNs). It was found that Fusarium poae and Fusarium avenaceum were present in almost all samples. Other common Fusarium species were F. graminearum and F. culmorum, present in more than 70% of samples. Several species occurred at lower DNA levels in 2011 than in other years, but the reverse was true for F. graminearum and Fusarium langsethiae. The most prevalent toxins were ENNs, present in 100% of samples. DON was also common, especially in spring wheat, whereas ZEA and NIV were common in 2009 and in winter wheat, but less common in 2011 and in spring wheat. Only three samples of spring wheat contained T-2 or HT-2 above LOQ. Annual mean levels of several mycotoxins were significantly lower in 2011 than in other years, but the reverse applied for DON. The strongest correlations between mycotoxin and Fusarium DNA levels were found between F. avenaceum and ENNs (r(2) = 0.67) and MON (r(2) = 0.62), and F. graminearum and DON (r(2) = 0.74). These results show that several Fusarium species and toxins co-occur in wheat. The highest toxin levels were detected in spring wheat and DON and ENNs, the latter belonging to the group of so called "emerging toxins", which were the most prevalent toxins and those occurring at the highest levels.

Deoxynivalenol and other selected Fusarium toxins in Swedish oats--occurrence and correlation to specific Fusarium species.

Fusarium moulds frequently contaminate oats and other cereals world-wide, including those grown in Northern Europe. To investigate the presence of toxigenic Fusarium species and their toxins in oats, samples were taken during 2010 and 2011 in three geographical regions of Sweden (east, west, south). The samples were analysed by real-time PCR for the specific infection level of seven Fusarium species associated with oats and other cereals (Fusarium poae, Fusarium graminearum, Fusarium langsethiae, Fusarium culmorum, Fusarium tricinctum, Fusarium sporotrichioides and Fusarium avenaceum) and with a multi-mycotoxin method based on liquid chromatography/electrospray ionisation-tandem mass spectrometry (HPLC/ESI-MS/MS) for the detection of many fungal metabolites, including deoxynivalenol (DON), zearalenone (ZEA), nivalenol (NIV), T-2 toxin, HT-2 toxins, moniliformin (MON), beauvericin (BEA) and enniatins (ENNs). Most samples contained at least four of the seven Fusarium species analysed and F. poae, F. langsethiae and F. avenaceum were present in approximately 90-100% of all samples. The most common toxins detected were DON, NIV, BEA and ENNs, which were present in more than 90% of samples. Most Fusarium species and their toxins occurred in higher concentrations in 2010 than in 2011, with the exception of DON and its main producer F. graminearum. Significant regional differences were detected for some moulds and mycotoxins, with higher levels of F. graminearum, DON and ZEA in western Sweden than in the east (P<0.05) and higher levels of F. tricinctum and MON in the south (P<0.05). Correlation analysis showed significant correlations between many Fusarium species and toxin levels. For example, F. tricinctum was significantly correlated to F. avenaceum (r = 0.72, P<0.001), DON to ZEA (r = 0.52, P<0.001), DON to F. graminearum (r = 0.77, P<0.001) and the sum of T-2 and HT-2 to F. langsethiae (r = 0.77, P<0.001). The multi-toxin approach employed allowed simultaneous detection of many Fusarium mycotoxins in each sample. In combination with real-time PCR analysis of seven toxigenic Fusarium spp., the results gave an overall picture of the presence of Fusarium and their toxins in Swedish oats and revealed significant annual and regional differences. This is the first study of the so-called emerging mycotoxins (e.g., ENNs, MON and BEA) in oats grown in Sweden.

Detection of type A trichothecene di-glucosides produced in corn by high-resolution liquid chromatography-Orbitrap mass spectrometry.

The existence of di-glucosylated derivative of T-2 toxin in plant (corn powder) was confirmed for the first time in addition to that of HT-2 toxin. These masked mycotoxins (mycotoxin glucosides) were identified as T-2 toxin-di-glucoside (T2GlcGlc) and HT-2 toxin-di-glucoside (HT2GlcGlc) based on accurate mass measurements of characteristic ions and fragmentation patterns using high-resolution liquid chromatography-Orbitrap mass spectrometric (LC-Orbitrap MS) analysis. Although the absolute structure of T2GlcGlc was not clarified, two glucose molecules were suggested to be conjugated at 3-OH position in tandem when considering the structure of T-2 toxin. On the other hand, the specification of the structure seems to be more complicated in the case of HT2GlcGlc, since HT-2 toxin has two possible positions (at 3-OH and 4-OH) to be glusocylated. In addition, 15-monoacetoxyscirpenol-glucoside (MASGlc) was also detected in the identical sample.

Identification and characterization of new Fusarium masked mycotoxins, T2 and HT2 glycosyl derivatives, in naturally contaminated wheat and oats by liquid chromatography-high-resolution mass spectrometry.

The presence of glucoside derivatives of T-2 and HT-2 toxins (type A trichothecene mycotoxins) in naturally contaminated wheat and oats is reported for the first time. The use of advanced high-resolution mass spectrometry based on Orbitrap technology allowed to obtain molecular structure details by measuring exact masses of main characteristic fragments, with mass accuracy lower than 2.8 ppm (absolute value). A monoglucoside derivative of T-2 toxin and two monoglucoside derivatives of HT-2 toxin were identified and characterized. The analysis of their fragmentation patterns provided evidence for glucosylation at C-3 position for T-2 toxin and at C-3 or C-4 position for HT-2 toxin. A screening for the presence of these new masked forms of mycotoxins was carried out on a set of naturally contaminated wheat and oats samples. On the basis of peak area ratio between glucoside derivatives and free T-2 and HT-2 toxins, the presence of glucoside derivatives was more likely in wheat than in oats samples. The present work confirms the widespread occurrence of trichothecene glucosides in cereal grains naturally contaminated with the relevant unconjugated toxins, thus suggesting the importance of developing suitable analytical methods for their detection. Besides toxicity studies, tracking down these new masked forms of trichothecenes along the food/feed chain would enable to collect information on their relevance in human/animal exposure to mycotoxin risk.

Molecular studies to identify the Fusarium species responsible for HT-2 and T-2 mycotoxins in UK oats.

High levels of Fusarium mycotoxins HT-2 and T-2 have been detected in UK oats since surveys started in 2002. Fusarium langsethiae and the closely related species F. sporotrichioides have previously been associated with the contamination of cereals with type A trichothecenes HT-2 and T-2 in Nordic countries. Preliminary microbiological analysis of UK oat samples with high concentrations of HT-2 and T-2 detected and isolated F. langsethiae and F. poae but not the other type A trichothecene producing species F. sporotrichioides, F. sibiricum and F. armeniacum. Two hundred and forty oat flour samples with a known mycotoxin profile were selected from a previous four year study (2002-2005) to cover the full concentration range from below the limit of quantification (<20 µg/kg) to 9,990 µg/kg HT-2+T-2 combined. All samples were analysed for the DNA of F. langsethiae, F. poae and F. sporotrichioides based on previously published PCR assays. F. langsethiae was detectable in nearly all samples; F. poae was detected in 90% of samples whereas F. sporotrichioides was not detected in any sample. A real-time PCR assay was developed to quantify F. langsethiae DNA in plant material. The assay could quantify as low as 10(-4)ngF. langsethiae DNA/µl. Based on this assay and a previously published assay for F. poae, both species were quantified in the oat flour samples with known HT-2+T-2 content. Results showed a good regression (P<0.001, r(2)=0.60) between F. langsethiae DNA and HT-2+T-2 concentration. F. poae DNA concentration was not correlated to HT2+T2 concentration (P=0.448) but was weakly correlated to nivalenol concentration (P<0.001, r(2)=0.09). Multiple regression with F. langsethiae and F. poae DNA as explanatory variates identified that both F. langsethiae and F. poae DNA were highly significant (P<0.001) but F. poae DNA only accounted for an additional 4% of the variance and the estimate was negative, indicating that higher concentrations of F. poae DNA were correlated with slightly lower concentrations of HT2+T2 detected. A stronger regression (P<0.001, r(2)=0.77) between F. langsethiae DNA and HT-2+T-2 was obtained after extraction and quantification of DNA and mycotoxins from individual oat grains. The results from this study provide strong evidence that F. langsethiae is the primary, if not sole, fungus responsible for high HT-2 and T-2 in UK oats.

Simultaneous and rapid detection of six different mycotoxins using an immunochip.

Mycotoxins are highly toxic contaminants in food, animal feed, and commodities. The study has developed an immunochip for quantifying the concentrations of six mycotoxins: aflatoxin B1, aflatoxin M1, deoxynivalenol, ochratoxin A, T-2 toxin, and zearalenone, which were added to drinking water. The complete antigens (Ags) of the mycotoxins were contact printed and immobilized onto agarose-modified glass slides with 12 physically isolated subarrays, based on the reaction of both diffusion and covalent bond. The optimal concentration of each antigen and antibody (Ab) was obtained using an Ag-Ab immunoassay. Based on the indirect competitive immunoassay for the simultaneous detection of six mycotoxins in one single chip, six standard curves with good logistic correlation (R(2)>0.97) were respectively plotted. The working ranges (0.04-1.69, 0.45-3.90, 20.20-69.23, 35.68-363.18, 0.11-1.81, and 0.08-7.47 ng/mL, respectively) were calculated, as well as the median inhibitory concentrations (0.31±0.04, 1.49±0.21, 34.54±1.30, 134.06±11.75, 0.49±0.05, and 1.54±0.22 ng/mL, respectively), when six mycotoxins were detected simultaneously. Finally, the recovery rates in drinking water generally ranged from 80% to 120% on the same chip, with an intra-assay coefficient of variation lower than 15%. We successfully established an immunochip for simultaneous detection of six mycotoxins within 4h, with advantages of using minimal samples and being visually semiquantitative with our naked eyes. In summary, the method could be developed on one single chip for detecting multiple contaminants in actual samples.

Control of T-2 toxin in Fusarium langsethiae and Geotrichum candidum co-culture.

Due to contamination of barley grains by Fusarium langsethiae, T-2 toxin can be present in the brewing process. It has been observed that the presence of the yeast Geotrichum candidum during malting can reduce the final concentration of this mycotoxin in beer. In this work, a co-culture method was carried out for both microorganisms in order to evaluate the effect on T-2 mycotoxin concentration in comparison with the pure culture of F. langsethiae in the same conditions. The microbial growth of both microorganisms was assessed using three different methods: dry weight, DOPE-FISH, and DNA quantification. In coculture, both microorganisms globally developed less than in pure cultures but G. candidum showed a better growth than F. langsethiae. The concentration of T-2 was reduced by 93 % compared to the pure culture. Hence, the interaction between G. candidum and F. langsethiae led to a drastic mycotoxin reduction despite the only partial inhibition of fungal growth.

A rapid fluorescence polarization immunoassay for the determination of T-2 and HT-2 toxins in wheat.

A rapid fluorescence polarization (FP) immunoassay has been developed for the simultaneous determination of T-2 and HT-2 toxins in naturally contaminated wheat samples. Syntheses of four fluorescein-labelled T-2 or HT-2 toxin tracers were carried out and their binding response with seven monoclonal antibodies was evaluated. The most sensitive antibody-tracer combination was obtained by using an HT-2-specific antibody and a fluorescein-HT-2 tracer. The developed competitive FP immunoassay in solution showed high cross-reactivity for T-2 toxin (CR% = 100%) while a very low CR% for neosolaniol (0.12%) and no cross-reactivity with other mycotoxins frequently occurring in wheat. A rapid extraction procedure using 90% methanol was applied to wheat samples prior to FP immunoassay. The average recovery from spiked wheat samples (50 to 200 µg kg(-1)) was 96% with relative standard deviation generally lower than 8%. A limit of detection of 8 µg kg(-1) for the combined toxins was determined. Comparative analyses of 45 naturally contaminated and spiked wheat samples by both the FP immunoassay and high-performance liquid chromatography/immunoaffinity clean-up showed a good correlation (r = 0.964). These results, combined with the rapidity (10 min) and simplicity of the assay, show that this method is suitable for high throughput screening as well as for quantitative determination of T-2 and HT-2 toxins in wheat.

Determination of ochratoxin A and T-2 toxin in alcoholic beverages by hollow fiber liquid phase microextraction and ultra high-pressure liquid chromatography coupled to tandem mass spectrometry.

A new method for the determination of ochratoxin A and T-2 toxin in alcoholic beverages (wine and beer) by hollow fiber liquid microextraction was optimized. The extraction step was followed by ultra high-pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The extraction procedure was based on the extraction of mycotoxins from the sample to the organic solvent (1-octanol) immobilized in the fiber, and afterwards, they were desorbed in a mixture of acetonitrile/water (80:20, v/v) at pH 7 prior to chromatographic determination. Different variables affecting the extraction process such as organic solvent, salt content, extraction time and desorption solution were studied. The developed method was validated in wine and beer, using white wine and alcoholic beer as representative matrices for both types of samples. Relative recoveries higher than 70% were obtained for the selected mycotoxins. Good linearity (R(2)>0.993) was obtained and quantification limits (0.02-0.09 microg L(-1)) below European regulatory levels were achieved. Repeatability, expressed as relative standard deviation, was always lower than 12%, whereas interday precision was lower than 21%. The proposed method was applied to the analysis of several types of wines and beers and ochratoxin A was detected in a rosé wine at 1.1 microg L(-1).

Fusarial toxin-induced toxicity in cultured cells and in isolated mitochondria involves PTPC-dependent activation of the mitochondrial pathway of apoptosis.

Mycotoxins produced by the Fusarium molds can cause a variety of human diseases and economic losses in livestock. Fusaria produce predominantly two types of mycotoxins: the nonestrogenic trichothecenes including T-2 toxin and the mycoestrogens such as zearalenone (ZEN). In a previous report, we demonstrated that the hepatotoxicity of these mycotoxins involves the mitochondrial pathway of apoptosis. Here, we observed that both fusarotoxins induced cell death by a mitochondria-dependent apoptotic process which includes opening of the mitochondrial permeability transition pore complex (PTPC), loss of mitochondrial transmembrane potential, increase in O(2)(.-) production, mitochondrial relocalization of Bax, cytochrome c release, and caspase activation. Studies performed on isolated mouse liver mitochondria showed that both ZEN and T-2 toxin might act directly on mitochondria to induce a PTPC-dependent permeabilization of mitochondrial membranes. Moreover, they may target different members of PTPC. Indeed, although the inner membrane protein adenine nucleotide translocase could be the target of T-2 toxin, ZEN seems to target the outer membrane protein voltage-dependent anion channel. Cells pretreatment with the p53 inhibitor pifithrin-alpha suggested that ZEN but not T-2 toxin triggered a p53-dependent mitochondrial apoptotic pathway. Finally, mitochondrial alterations induced by ZEN and T-2 toxin are mediated by Bcl-2 family proteins, such as Bax, and prevented by Bcl-x(L) and to a lesser extent by Bcl-2. Taken together, these data indicate that mitochondria play a pivotal role in both ZEN- and T-2 toxin-induced apoptosis and that PTPC members and proteins of Bcl-2 family should be interesting targets to overcome fusarotoxin toxicity.

Determination of T-2 and HT-2 toxins in cereals including oats after immunoaffinity cleanup by liquid chromatography and fluorescence detection.

A reliable method for the determination of T-2 toxin and HT-2 toxin in different cereals, including oats, as well as in cereal products was developed. After extraction with methanol/water (90/10, v/v) and dilution with a 4% NaCl solution, the toxins were purified with immunoaffinity columns, derivatized with 1-anthroylnitrile, separated by HPLC, and determined using fluorescence detection. Due to the unspecific derivatization reagents, validation parameters were matrix dependent: in the range 10-200 microg/kg, recovery rates of 74-120% with relative standard deviations between 0.5 and 20.3% were obtained. On average, the limit of quantitation was shown to be 8 microg/kg for each toxin. For naturally contaminated samples, comparable results were obtained when analysis was performed according to this method without derivatization as well as according to a method based on a SPE cleanup utilizing tandem mass spectrometric detection in both cases. Using aqueous acetonitrile as extractant resulted in incorrectly high toxin concentrations due to water absorption of dry samples and toxin accumulation in the organic phase in the subsequent phase separation of the extractant. Furthermore, when comparing the commercially available immunoaffinity columns for T-2 and HT-2 toxins, significant differences regarding capacity and cleanup performance were observed.

Phytotoxic effects of trichothecenes on the growth and morphology of Arabidopsis thaliana.

Non-volatile sesquiterpenoids, a trichothecene family of phytotoxins such as deoxynivalenol (DON) and T-2 toxin, contain numerous molecular species and are synthesized by phytopathogenic Fusarium species. Although trichothecene chemotypes might play a role in the virulence of individual Fusarium strains, the phytotoxic action of individual trichothecenes has not been systematically studied. To perform a comparative analysis of the phytotoxic action of representative trichothecenes, the growth and morphology of Arabidopsis thaliana growing on media containing these compounds was investigated. Both DON and diacetoxyscirpenol (DAS) preferentially inhibited root elongation. DON-treated roots were less organized compared with control roots. Moreover, preferential inhibition of root growth by DON was also observed in wheat plants. In addition, T-2 toxin-treated seedlings exhibited dwarfism with aberrant morphological changes (e.g. petiole shortening, curled dark-green leaves, and reduced cell size). These results imply that the phytotoxic action of trichothecenes differed among their molecular species. Cycloheximide (CHX)-treated seedlings displayed neither feature, although it is known that trichothecenes inhibit translation in eukaryotic ribosomes. Microarray analyses suggested that T-2 toxin caused a defence response, the inactivation of brassinosteroid (BR), and the generation of reactive oxygen species in Arabidopsis. This observation is in agreement with our previous reports in which trichothecenes such as T-2 toxin have an elicitor-like activity when infiltrated into the leaves of Arabidopsis. Since it has been reported that BR plays an important role in a broad range of disease resistance in tobacco and rice, inactivation of BR might affect pathogenicity during the infection of host plants by trichothecene-producing fungi.

Inhibition of sodium glucose cotransporter-I expressed in Xenopus laevis oocytes by 4-acetoxyscirpendiol from Cordyceps takaomantana (anamorph = Paecilomyces tenuipes).

Cordyceps contains many health-promoting constituents. Recent studies revealed that the fruiting body of cordyceps significantly alleviates hyperglycemia which usually accompanies diabetes mellitus. The mechanism of the anti-hyperglycemic effect by cordyceps, however, is not fully understood. In this study, methanolic extracts were prepared from fruiting bodies of Paecilomyces tenuipes, and 4-beta acetoxyscirpendiol (ASD) was eventually purified from the extracts. The Na+/ glucose transporter-1 (SGLT-1) was expressed in Xenopus oocytes, and the effect of ASD on it was analyzed using voltage clamp and 2-deoxy-D-glucose (2-DOG) uptake studies. Fluorescence microscopy was performed to monitor the effect of ASD on glucose uptake using HEK293 cells expressing recombinant SGLT-1. ASD inhibited SGLT-1 activity, and its two derivatives (2-acetoxyscirpenol and 15-acetoxyscirpendiol), were also effective; 15-acetoxyscirepenol was as inhibitory as ASD while diacetoxyscirpenol had less effect. Thus, the ASD in P. tenuipes may play an important role in lowering blood sugar in the circulatory system along with its derivatives as specific inhibitors of SGLT-1.

Feasibility of immunodiagnostic devices for the detection of ricin, amanitin, and T-2 toxin in food.

Qualitative and quantitative comparisons were conducted of commercially available immunodiagnostic devices for the detection of three select agents with oral LD50 values > or = 0.1 mg/kg of body weight. Ricin (oral LD50 > 1 mg/kg), amanitin (oral LD50 approximately 0.1 mg/kg), and T-2 toxin (oral LD50 > 1 mg/kg) were spiked into beverages, produce, dairy, and baked goods and assayed using commercially available enzyme-linked immunosorbent assays (ELISAs) and lateral flow devices. In all cases, the commercial diagnostic kits successfully detected all three select agents at concentrations below what might be a health concern. The considerable difference between the limit of detection of the immunodiagnostic devices employed (typically < or = 0.020 microg/g) and the amount of the select agent necessary to pose a health threat in a single serving of food facilitated the design of protocols for the high throughput screening of food samples. These protocols entailed simple extraction methods followed by sample dilution. Lateral flow devices and sandwich ELISAs for the detection of ricin had no significant background problems due to the food matrices. Competitive ELISAs, which typically have unacceptably high background reactions with food samples, successfully detected amanitin and T-2 toxin.

4-acetoxyscirpendiol of Paecilomyces tenuipes inhibits Na(+)/D-glucose cotransporter expressed in Xenopus laevis oocytes.

Cordyceps, an entomopathogenic fungus, contains many health-promoting ingredients. Recent reports indicate that the consumption of cordyceps helps reduce blood-sugar content in diabetics. However, the mechanism underlying this reduction in circulatory sugar content is not fully understood. Methanolic extracts were prepared from the fruiting bodies of Paecilomyces tenuipes, and 4-beta acetoxyscirpendiol (4-ASD) was eventually isolated and purified. Na(+)/Glucose transporter-1 (SGLT-1) was expressed in Xenopus oocytes, and the effect of 4-ASD on SGLT-1 was analyzed utilizing a voltage clamp and by performing 2-deoxy-D-glucose (2-DOG) uptake studies. 4-ASD was shown to significantly inhibit SGLT-1 activity compared to the non-treated control in a dose-dependent manner. In the presence of the derivatives of 4-ASD (diacetoxyscirpenol or 15-acetoxyscirpendiol), SGLT-1 activity was greatly inhibited in an 4-ASD-like manner. Of these derivatives, 15-acetoxyscirepenol inhibited SGLT-1 as well as 4-ASD, whereas diacetoxyscirpenol was slightly less effective. Taken together, these results strongly indicate that 4-ASD in P. tenuipes may lower blood sugar levels in the circulatory system. We conclude that 4-ASD and its derivatives are effective SGLT-1 inhibitors.

Apoptosis induction by 4beta-acetoxyscirpendiol from Paecilomyces tenuipes in human leukaemia cell lines.

The carpophores of Paecilomyces tenuipes are known in the Orient for their strong antitumor activity. In continuation of our study on acetoxyscirpendiol (ASD, 4beta-acetoxyscirpene-3alpha,15-diol) as a cytotoxic component from this fungus, we report particularly on the mode of action of ASD in inducing apoptosis in human MOLT-4, THP-1 and Jurkat T cell leukaemia in vitro. The antiproliferative effects of ASD seem attributable to its induction of apoptosis in the cells, as it blocked the cell cycle, induced hypodiploidity and bound annexin V and also cleaved poly-(ADP-ribose) polymerase (PARP) in these cell lines. The 50% inhibitory concentrations (IC50) of ASD on MOLT-4, THP-1 and Jurkat T cells were found to be 60, 85 and 60 ng/ml, respectively. ASD arrested the cell cycle at the G1/S transition and showed hypodiploidity due to the accumulation of sub-G0 population. Annexin V binding was increased in the presence of ASD in the MOLT-4 cell line in a time-dependent manner. ASD and three of its derivatives also induced cleavage of PARP in both MOLT-4 and Jurkat T cell lines. From these data, it is suggested that ASD exerts its cytotoxic activity by inducing apoptosis in leukaemia cell lines in vitro.

A collaborative study to validate novel field immunoassay kits for rapid mycotoxin detection.

Kits designed to detect ochratoxin A (OA) and T-2 toxin by a membrane-based flow-through enzyme immunoassay were studied collaboratively by screening cereals (wheat, rye, maize and barley) for the presence of these mycotoxins. Sample preparation and test procedure were clearly described in the instruction leaflets included in the kits. A simple methanol-based extraction followed by filtration and dilution steps was prescribed. Reagents were successively pipetted to the membrane of the device, then colour development was evaluated visually. Limits of detection for the ochratoxin A and T-2 toxin tests were 4 and 50 microg kg(-1), respectively. Five laboratories took part in the first stage of this study, and five more joined the second stage. Cereal samples (blank, spiked or inoculated) were shipped with the kits to the participating laboratories, while results obtained were confirmed by high-performance liquid chromatography with fluorescence detection and by gas chromatography-mass spectrometry for ochratoxin A and T-2 toxin, respectively. Some initial difficulties were encountered. In the second stage, four ochratoxin A and four T-2 toxin kits were used by 10 collaborators to analyse 21 cereal samples. For the ochratoxin A kits, the percentage of false positive and false negative results were 2% and 4%, respectively. The results of one T-2 toxin kit were outliers and when excluded, the overall percentage false positive and false negative results were 6% and 3%, respectively.