Activation of Notch Signaling Pathway is involved in Extracellular Matrix Degradation in human induced pluripotent stem cells chondrocytes induced by HT-2 toxin.

Notch signaling regulates cartilage formation and homeostasis. Kashin-Beck Disease (KBD), an endemic osteochondropathy, is characterized by severe cartilage degradation. The etiology of KBD is related to the exposure of HT-2 toxin, a mycotoxin and primary metabolite of T-2 toxin. This study aims to explore the role of HT-2 toxin in the Notch signaling regulation and extracellular matrix (ECM) metabolism of hiPSCs-Chondrocytes. Immunohistochemistry and qRT-PCR were employed to investigate the expression of Notch pathway molecules in KBD articular cartilage and primary chondrocytes. hiPSCs-Chondrocytes, derived from hiPSCs, were treated with 100 ng/mL HT-2 toxin and the γ-secretase inhibitor (DAPT) for 48h, respectively. The markers related to the Notch signaling pathway and ECM were assessed using qRT-PCR and Western blot. Notch pathway dysregulation was prominent in KBD cartilage. HT-2 toxin exposure caused cytotoxicity in hiPSCs-Chondrocytes, and activated Notch signaling by increasing the mRNA and protein levels of NOTCH1 and HES1. HT-2 toxin also upregulated ECM catabolic enzymes and downregulated ECM components (COL2A1 and ACAN), indicating ECM degradation. DAPT-mediated Notch signaling inhibition suppressed the mRNA and protein level of ADAMTS5 expression while enhancing ECM component expression in hiPSCs-Chondrocytes. This study suggests that HT-2 toxin may induce ECM degradation in hiPSCs-Chondrocytes through activating Notch signaling.

Impact of Environmental Conditions on the Concentrations of Trichothecenes, Their Glucosides, and Emerging Fusarium Toxins in Naturally Contaminated, Irradiated, and Fusarium langsethiae Inoculated Oats.

Trichothecenes produced by Fusarium species are commonly detected in oats. However, the ratios of the concentrations of free trichothecenes and their conjugates and how they are impacted by different interacting environmental conditions are not well documented. This study aims to examine the effect of water activity (0.95 and 0.98 aw) and temperature (20 and 25 °C) stress on the production of T-2 and HT-2 toxins, deoxynivalenol and their conjugates, as well as diacetoxyscirpenol (DAS). Multiple mycotoxins were detected using liquid chromatography-tandem mass spectrometry from 64 contaminated oat samples. The highest concentrations of HT-2-glucoside (HT-2-Glc) were observed at 0.98 aw and 20 °C, and were higher than other type A trichothecenes in the natural oats' treatments. However, no statistical differences were found between the mean concentrations of HT-2-Glc and HT-2 toxins in all storage conditions analysed. DAS concentrations were generally low and highest at 0.95 aw and 20 °C, while deoxynivalenol-3-glucoside levels were highest at 0.98 aw and 20 °C in the naturally contaminated oats. Emerging mycotoxins such as beauvericin, moniliformin, and enniatins mostly increased with a rise in water activity and temperature in the naturally contaminated oats treatment. This study reinforces the importance of storage aw and temperature conditions in the high risk of free and modified toxin contamination of small cereal grains.

Co-occurrence of T-2 and HT-2 Mycotoxins and α and ß Anomers of Their Glucosides in Wheat and Oat Grains.

The aim of this study was to simultaneously determine T-2 and HT-2 toxins and the α and ß anomers of their glucosides to assess their content in wheat and oat grains harvested in Poland (2020-2022). Of 298 wheat samples, only 14 (5%) contained the sum of the T-2 and HT-2 toxins (average 34.2 µg/kg; 10.6-67.7 µg/kg). In oat (n = 129), these compounds were detected much more frequently (70% of samples) at an average level of 107.5 µg/kg (6.9-949.1 µg/kg). The sum of T-2 and HT-2 glucosides was detectable in 3% of the wheat (average 16.3 µg/kg; 7.1-39.4 µg/kg) and 65% of the oat samples (average 35.1 µg/kg; 4.0-624.1 µg/kg). Following the study, T-2-3-α-glucoside was identified as the only naturally occurring anomer, while both anomers of HT-2-3-glucosides were detected with higher contents and occurrence rates of HT-2-3-ß-glucoside than the α anomer of this compound.

Results of multi-actor collaboration in risk analysis: a simplified risk assessment toolkit for rapid detection of emerging risks.

The dynamic field of food safety faces continuous challenges, prompting stakeholders to develop collaborative actions for improved food safety systems. As part of these actions, the EU-FORA fellowship programme was dedicated to a multi-actor collaboration addressing risks of the unregulated mycotoxins T-2 and HT-2 toxins in oats. Critical gaps in risk assessment procedures were identified, leading to a joint effort to develop a strategy for rapid data collection and risk assessment, including the development of a risk assessment toolkit comprising of a training manual and two intuitive Microsoft® Excel files. The toolkit enables efficient data collection and processing, facilitating risk assessment calculations and rapid risk detection. Applying the toolkit to assess T-2 and HT-2 toxin risks in Belgian oats revealed minimal concerns, except for children aged 3-9 years, likely due to an overestimation. The toolkit is available on the FoodSafety4EU Platform and will be refined based on user feedback, promoting better risk assessment practices. This approach empowers stakeholders, from professionals to policymakers, fostering collaboration and enhancing food safety practices.

Potential Toxicity and Mechanisms of T-2 and HT-2 Individually or in Combination on the Intestinal Barrier Function of Porcine Small Intestinal Epithelial Cells.

Under natural conditions, T-2 toxin can be easily metabolized to HT-2 toxin by deacetylation, and T-2 and HT-2 are usually co-contaminated in grain and feed at a high detected rate. Our previous information indicated that T-2 toxin could injure the function of the intestinal barrier, but the combined toxicity and mechanism of T-2 and HT-2 on the intestinal cells of porcines are still unknown. Therefore, we aimed to explore T-2 and HT-2 individually and combined on cellular viability, cell membrane integrity, the expression of tight junction-related proteins, and the generation of inflammatory factors in porcine intestinal epithelial cells (IPEC-J2). The results showed that T-2 and HT-2, individually or in combination, could induce a decrease in cell viability, an increase in LDH release and IL-1, IL-6, and TNF-α generation, and a decrease in the anti-inflammatory factor IL-10. Based on the analysis of immunofluorescence staining, real-time PCR, and western blotting, the tight junction protein expressions of Claudin-1, Occludin, and ZO-1 were significantly decreased in the T-2 and HT-2 individual or combination treated groups compared with the control. Furthermore, all the parameter changes in the T-2 + HT-2 combination group were much more serious than those in the individual dose groups. These results suggest that T-2 and HT-2, individually and in combination, could induce an intestinal function injury related to an inflammatory response and damage to the intestinal barrier function in porcine intestinal epithelial cells. Additionally, T-2 and HT-2 in combination showed a synergistic toxic effect, which will provide a theoretical basis to assess the risk of T-2 + HT-2 co-contamination in porcine feed.

Comparative Performance of Rapid Diagnostics for the Detection of T-2 and HT-2 Toxins in Oats.

The contamination of oat crops by trichothecene mycotoxins, T-2 and HT-2 is an ongoing threat to our food safety. Within the industry, there are increasing concerns about the continued and growing presence of these mycotoxins occurring in oat crops due to climate change, farming practices and the handling of crops post-harvest. To safeguard human health, monitoring these mycotoxins in foodstuffs is paramount to ensure human exposure is limited. To achieve this, effective testing regimes must be established within the industry, consisting not only of rapid, reliable, and accurate analytical methods but also efficient sampling strategies. Four commercial rapid diagnostic kits were assessed against liquid chromatography coupled to mass spectrometry and included three lateral flow devices and one enzyme-linked immunosorbent assay. One-way ANOVA showed a p-value of 0.45 indicating no significant difference between the methods assessed. Qualitative analysis revealed test kits 1, 2, 3, and 4 showed false negative/false positive rates of 1.1/2.2, 7.6/0, 2.2/0, and 6.5/0 percent, respectively. Test Kit 1, the Neogen Reveal® Q+ MAX for T-2/HT-2 Kit provided the most reliable, accurate and cost-effective results. Furthermore, its ease of use and no requirement for technical skill makes it applicable for on-site testing.

The Effect of Ustilago maydis and Delayed Harvesting on A- and B-Type Trichothecene Concentrations in Maize Grain.

The aim of this study was to investigate whether, in the context of a higher incidence of Ustilago maydis and Fusarium spp. at optimal and delayed harvest times, a higher incidence of mycotoxin contamination in maize grains could be expected. The field experiment was carried out at the Lithuanian Research Centre for Agriculture and Forestry experimental fields over three consecutive years (2020-2022). Two maize hybrids (Duxxbury and Lapriora) with different FAO numbers were used. The experimental design in the field was a randomized complete block design. Harvesting took place at three different times: first at physiological maturity, and then 10 (±2) and 20 (±2) days after the first harvest. Each hybrid had four repetitions at different harvest times. The U. maydis infection was only detected in 2021 and after the first harvest cobs were further divided into four different groups with four repetitions: healthy cobs, cobs visually infected with Fusarium spp., cobs visually infected with common smut, and cobs visually infected with both pathogens. No U. maydis-damaged maize cobs were found in 2020 and 2022. The levels of Fusarium microscopic fungi in maize grains were also from 4 to 16 times higher in 2021 than in 2020 and 2022. Harvest delays in 2020 led to a significant deoxynivalenol concentration increase in the Duxxbury hybrid and an HT-2 concentration increase in the Lapriora hybrid. In 2021, deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, and HT-2 concentrations significantly rose in both hybrids, but the T-2 concentration significantly increased only in the Lapriora hybrid. Deoxynivalenol concentrations were, respectively, 110 and 14.6 times higher than in cobs only infected with Fusarium spp. or U. maydis. Concentrations of 15-acetyl-deoxynivalenol were, respectively, 60, 67, and 43 times higher than in asymptomatic cobs and cobs only infected with Fusarium spp. or U. maydis. Cobs contaminated with both pathogens also had higher concentrations of 3-acetyl-deoxynivalenol. T-2 and HT-2 were detected in maize grains harvested from cobs infected only with Fusarium spp. The presence of U. maydis and Fusarium fungi in maize cobs, along with harvest delays, led to significant increases in mycotoxin concentrations, highlighting the importance of timely harvesting and pathogen management to mitigate mycotoxin contamination in maize grains.

T-2 and HT-2 Toxins: Toxicity, Occurrence and Analysis: A Review.

One of the major classes of mycotoxins posing serious hazards to humans and animals and potentially causing severe economic impact to the cereal industry are the trichothecenes, produced by many fungal genera. As such, indicative limits for the sum of T-2 and HT-2 were introduced in the European Union in 2013 and discussions are ongoing as to the establishment of maximum levels. This review provides a concise assessment of the existing understanding concerning the toxicological effects of T-2 and HT-2 in humans and animals, their biosynthetic pathways, occurrence, impact of climate change on their production and an evaluation of the analytical methods applied to their detection. This study highlights that the ecology of F. sporotrichioides and F. langsethiae as well as the influence of interacting environmental factors on their growth and activation of biosynthetic genes are still not fully understood. Predictive models of Fusarium growth and subsequent mycotoxin production would be beneficial in predicting the risk of contamination and thus aid early mitigation. With the likelihood of regulatory maximum limits being introduced, increased surveillance using rapid, on-site tests in addition to confirmatory methods will be required. allowing the industry to be proactive rather than reactive.

Melatonin protects Leydig cells from HT-2 toxin-induced ferroptosis and apoptosis via glucose-6-phosphate dehydrogenase/glutathione -dependent pathway.

HT-2 toxin is a mycotoxin commonly found in food and water that can have adverse effects on male reproductive systems, including testosterone secretion. Ferroptosis and apoptosis are two types of programmed cell death that have been implicated in the regulation of cellular functions. Melatonin, a powerful antioxidant with various physiological functions, has been shown to regulate testosterone secretion. However, the mechanisms underlying the protective effects of melatonin against HT-2 toxin-induced damage in testosterone secretion are not fully understood. In this study, we investigated the effects of HT-2 toxin on sheep Leydig cells and the potential protective role of melatonin. We found that HT-2 toxin inhibited cell proliferation and testosterone secretion of Leydig cells in a dose-dependent manner and induced ferroptosis and apoptosis through intracellular reactive oxygen species accumulation, leading to lipid peroxidation. Exposure of Leydig cells to melatonin in vitro reversed the defective phenotypes caused by HT-2 toxin via a glucose-6-phosphate dehydrogenase/glutathione-dependent mechanism. Interference of glucose-6-phosphate dehydrogenase disrupted the beneficial effect of melatonin on ferroptosis and apoptosis in HT-2 toxin-treated Leydig cells. Furthermore, similar results were observed in vivo in the testes of male mice injected with HT-2 toxin with or without melatonin treatment for 30 days. Our findings suggest that melatonin inhibits ferroptosis and apoptosis by elevating the expression of glucose-6-phosphate dehydrogenase to eliminate reactive oxygen species accumulation in HT-2 toxin-treated Leydig cells. These results provide fundamental evidence for eliminating the adverse effects of HT-2 toxin on male reproduction.

Determination of mycotoxins by HPLC, LC-MS/MS and health risk assessment of the mycotoxins in bee products of Turkey.

The aim of this study is to determine the levels of deoxynivalenol (DON), HT-2 toxin (HT2), T-2 toxin (T2), and ochratoxin A (OTA) in bee products (bee pollen, propolis, honey and royal jelly) available in Turkey. In addition, exposure and health risk assessments were performed to identify the potential health risk of these mycotoxins. The mycotoxins were analyzed by high-performance liquid chromatography (HPLC) with a UV detector and positive samples were confirmed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The most common mycotoxins in all bee products were DON and T-2 toxin, with mean concentrations of 1.601 and 0.704 µg/per kg dry sample, respectively, followed by OTA and HT-2 toxin. It was determined that the mycotoxins taken as a result of consuming bee products in specified amounts do not pose a risk to health.

Abnormal expression of TSG-6 disturbs extracellular matrix homeostasis in chondrocytes from endemic osteoarthritis.

Background and aims: Kashin-Beck disease (KBD) is a unique endemic osteochondropathy with unclear pathogenesis in China. T-2 toxin exposure has been identified as a significant risk factor of KBD. However, the mechanism of articular cartilage damage induced by T-2 toxin is a conundrum. We explored the role of the extracellular matrix-related gene TSG-6 in the articular chondrocyte damage process under the exposure of HT-2 toxin. Methods: TSG-6 was identified as a candidate gene by mining our previous gene expression profiling of KBD and verified by qRT-PCR and immunohistochemistry. Then, TSG-6 was silenced by RNA interference technology and overexpressed induction by TNF-α. Gradient concentrations of HT-2 toxin were added to intervene with C28/I2 chondrocytes. MTT was used to observe the proliferation and cell viability of chondrocytes, and qRT-PCR was utilized to detect the expression changes of MMP1, MMP3, MMP13, COL2A1, and proteoglycan before and after treatments for verification. Results: TSG-6 was upregulated in KBD chondrocytes at the mRNA level and upregulated in the superficial, middle, and deep zones of KBD cartilage. After TSG-6 silencing, the expression of MMP1, MMP3, MMP13, and proteoglycan was significantly decreased while COL2A1 expression was significantly increased, which was reversed after the overexpression of TSG-6 induced by TNF-α (p < 0.05). The survival rate of chondrocytes was correspondingly reduced with an increase in the HT-2 toxin concentration. Compared with the blank control group, the expression of MMPs was increased in the intervention group of HT-2 toxin, while the expression of proteoglycan and COL2A1 decreased (p < 0.05). Conclusion: The upregulation of the TSG-6 gene may play a role in promoting the damage and degradation of the extracellular matrix in KBD chondrocytes under the exposure of HT-2 toxin.

Assessment of information as regards the toxicity of T-2 and HT-2 toxin for ruminants.

In 2011, the EFSA Panel on Contaminants in the Food Chain (CONTAM) adopted a Scientific Opinion on the risks for animal health related to the presence of T-2 (T2) and HT-2 (HT2) toxin in food and feed. No observed adverse effect levels (NOAELs) and lowest observed adverse effect levels (LOAELs) were derived for different animal species. In ruminants a LOAEL was established for the sum of T2 and HT2 of 0.3 mg/kg body weight (bw) per day, based on studies with calves and lambs. The CONTAM Panel noted that the effects observed in nutritionally challenged heifers and ewes give rise to the assumption that rumen detoxification of T2 may not always be complete and therefore effective to prevent adverse effects in ruminants. However, the limited data on the effects of T2 on adult ruminants did not allow a conclusion. The European Commission requested EFSA to review the information regarding the toxicity of T2 and HT2 for ruminants and to revise, if necessary, the established Reference Point (RP). Adverse effect levels of 0.001 and 0.01 mg T2/kg bw per day for, respectively, sheep and cows, were derived from case studies, estimated to correspond to feed concentrations of 0.035 mg T2/kg for sheep and 0.6 mg T2/kg for cows. RPs for adverse animal health effects of 0.01 mg/kg feed for sheep and 0.2 mg/kg feed for cows were established. For goats, the RP for cows was selected, in the absence of data that they are more sensitive. Based on mean exposure estimates performed in the previous Opinion, the risk of adverse health effects of feeds containing T2 and HT2 was considered a concern for lactating sheep. For milking goats, a comparison performed between dietary exposure and the RP derived for cows, indicates a potential risk for adverse health effects. For dairy cows and fattening beef, the risk is considered low.

Searching for the Fusarium spp. Which Are Responsible for Trichothecene Contamination in Oats Using Metataxonomy to Compare the Distribution of Toxigenic Species in Fields from Spain and the UK.

The contamination of oats with Fusarium toxins poses a high risk for food safety. Among them, trichothecenes are the most frequently reported in European oats, especially in northern countries. The environmental conditions related to the climate change scenario might favour a distribution shift in Fusarium species and the presence of these toxins in Southern European countries. In this paper, we present an ambitious work to determine the species responsible for trichothecene contamination in Spanish oats and to compare the results in the United Kingdom (UK) using a metataxonomic approach applied to both oat grains and soil samples collected from both countries. Regarding T-2 and HT-2 toxin producers, F. langsethiae was detected in 38% and 25% of the oat samples from the UK and Spain, respectively, and to the best of our knowledge, this is the first report of the detection of this fungus in oats from Spain. The relevant type B trichothecene producer, F. poae, was the most frequently detected Fusarium species in oats from both origins. Other important trichothecene producers, such as the Fusarium tricinctum species complex or Fusarium cerealis, were also frequently detected in oat fields. Many Fusarium toxins, including T-2 and HT-2 toxins, deoxynivalenol, or nivalenol, were detected in oat samples. The results obtained in this work revealed a clear change in the distribution of trichothecene producers and the necessity to establish the potential of these species to colonize oats and their ability to produce mycotoxins.

Determination of T-2 and HT-2 Toxins in Seed of Milk Thistle [Silybum marianum (L.) Gaertn.] Using Immunoaffinity Column by UPLC-MS/MS.

Milk thistle [Silybum marianum (L.) Gaertn.] achieved a significant increase in interest over the past few years from local and foreign pharmaceutical corporations. The silymarin complex of constituents extracted from milk thistle achenes provides compelling health benefits primarily thanks to antioxidant activities and hepatoprotective effects. However, consuming mycotoxin-contaminated plant material can cause immunosuppression and hepatotoxic problems. The aim of this study was to develop and validate a method for the determination of mycotoxin content in milk thistle. Fusarium toxins as T-2 and HT-2 toxins in grown milk thistle harvested from a breeding station in the Czech Republic during 2020-2021 were studied. The analysis of T-2 and HT-2 toxins was performed by UPLC-MS/MS after immunoaffinity columns EASI-EXTRACT® T-2 & HT-2 clean up. All analysed samples of milk thistle were contaminated with T-2 toxin and HT-2 toxin. The content of T-2 toxin in the samples from 2020 was in the range of 122.7-290.2 µg/kg and HT-2 toxin 157.0-319.0 µg/kg. In 2021, the content of T-2 toxin was in the range of 28.8-69.9 µg/kg and HT-2 toxin was 24.2-75.4 µg/kg. The results show that the climatic conditions of the year of harvesting have a highly statistically significant effect on the content of T-2 and HT-2 toxins in milk thistle.

Potential Role of Individual and Combined Effects of T-2 Toxin, HT-2 Toxin and Neosolaniol on the Apoptosis of Porcine Leydig Cells.

T-2 toxin usually co-occurs with HT-2 toxin and neosolaniol (NEO) in the grains and feed. Our previous studies found that T-2 toxin and its metabolites' binary or ternary combination exposure to porcine Leydig cells (LCs) displayed synergism in certain range of dosage and cannot be predicted based on individual toxicity. However, the possible mechanism of these mycotoxins' combined exposure to cell lesions remains unknown. Based on 50% cell viability, the mechanism of apoptosis in porcine Leydig cells was investigated after exposure to T-2, HT-2, NEO individual and binary or ternary combinations. Compared with control, the adenosine triphosphate (ATP) content decreased, reactive oxygen species (ROS) level increased, and mitochondrial membrane potential (MMP) decreased in all treated groups. Additionally, the cell apoptosis rates were significantly increased in test groups (p < 0.05), and the B-cell lymphoma 2 (Bcl-2) Associated X (Bax)/Bcl-2 ratio and the expression of caspase 3, caspase 8, cytochrome c (Cytc) in the treated group are all significantly higher than the control group. Moreover, the expression of Cytc and caspase 8 gene in NEO and T-2+NEO groups was significantly higher than that in other individual and combined groups. It can be concluded that the toxicities of T-2, HT-2, and NEO individually and in combination can induce apoptosis related to the oxidative stress and mitochondrial damage, and the synergistic effect between toxins may be greater than a single toxin effect, which is beneficial for assessing the possible risk of the co-occurrences in foodstuffs to human and animal health.

Molecular super-gluing: a straightforward tool for antibody labelling and its application to mycotoxin biosensing.

Mycotoxins are low molecular weight toxic compounds, which can cause severe health problems in animals and humans. Immunoassays allow rapid, simple and cost-effective screening of mycotoxins. Sandwich assays with a direct readout provide great improvement in terms of selectivity and sensitivity, compared to the widely used competitive assay formats, for the analysis of low molecular weight molecules. In this work, we report a non-competitive fluorescence anti-immune complex (IC) immunoassay, based on the specific recognition of HT-2 toxin with a pair of recombinant antibody fragments, namely antigen-binding fragment (Fab) (anti-HT-2 (10) Fab) and single-chain variable fragment (scFv) (anti-IC HT-2 (10) scFv). The SpyTag and SpyCatcher glue proteins were applied for the first time as a bioconjugation tool for the analysis of mycotoxins. To this aim, a SpyTag-mScarlet-I (fluorescent protein) and scFv-SpyCatcher fusion proteins were constructed, produced and fused in situ during the assay by spontaneous Tag-Catcher binding. The assay showed an excellent sensitivity with an EC50 of 4.8 ± 0.4 ng mL-1 and a dynamic range from 1.7 ± 0.3 to 13 ± 2 ng mL-1, an inter-day reproducibility of 8.5% and a high selectivity towards HT-2 toxin without cross-reactivity with other Fusarium toxins. The bioassay was applied to the analysis of the toxin in an oat reference material and in oat samples, with a LOD of 0.6 µg kg-1, and the results were validated by analysing a certificate reference material and by HPLC-MS/MS.

Fusarium chaquense, sp. nov, a novel type A trichothecene-producing species from native grasses in a wetland ecosystem in Argentina.

The Chaco wetland is among the most biologically diverse regions in Argentina. In collections of fungi from asymptomatic native grasses (Poaceae) from the wetlands, we identified isolates of Fusarium that were morphologically similar to F. armeniacum, but distinct from it by their production of abundant microconidia. All the isolates had identical, or nearly identical, partial sequences of TEF1 and RPB2. But they were distinct from reference sequences from F. armeniacum and Fusarium species closely related to it. Phylogenetic analysis of 34 full-length housekeeping gene sequences retrieved from whole genome sequences of three Chaco wetland isolates, 29 genes resolved the isolates as an exclusive clade within the F. sambucinum species complex. Based on results of the morphological and phylogenetic analysis, we concluded that the Chaco wetland isolates are a distinct and novel species, herein described as Fusarium chaquense, sp. nov., which is closely related to F. armeniacum. F. chaquense in culture can produce the trichothecenes T-2 and HT-2 toxin, neosolaniol, diacetoxyscirpenol, and monoacetoxyscirpenol, as well as beauvericin and the pigment aurofusarin. Genome sequence analysis also revealed the presence of three previously described loci required for trichothecene biosynthesis. This research represents the first study of Fusarium in a natural ecosystem in Argentina.

Free and conjugated Alternaria and Fusarium mycotoxins during Pilsner malt production and double-mash brewing.

Malting and brewing have previously been demonstrated to be risky procedures in terms of mycotoxins contamination. The goal of the study was to describe the fate of less investigated Fusarium and Alternaria mycotoxins, together with their conjugates, during these processes. The Pilsner malt producing process, together with double-mash brewing, were performed in a pilot-scale malting and brewery plants to simulate production of lager - the most popular type of central European beer. In addition, changes in temperature during barley germination were investigated to assess the influence of this critical step. QuEChERS-like extraction followed by UHPLC-HRMS/MS were utilized to quantify the mass balance of 13 mycotoxins and four of their conjugates. The results confirmed germination as the most determining malting step, followed by mashing of malt during brewing. Occurrence of type A trichothecenes, Alternaria mycotoxins and their conjugates in the final beer product indicates the need to take mitigation measures.

Dysregulation of Cells Cycle and Apoptosis in Human Induced Pluripotent Stem Cells Chondrocytes Through p53 Pathway by HT-2 Toxin: An in vitro Study.

Kashin-Beck disease (KBD) mainly damages growth plate of adolescents and is susceptible to both gene and gene-environmental risk factors. HT-2 toxin, which is a primary metabolite of T-2 toxin, was regarded as one of the environmental risk factors of KBD. We used successfully generated KBD human induced pluripotent stem cells (hiPSCs) and control hiPSCs, which carry different genetic information. They have potential significance in exploring the effects of HT-2 toxin on hiPSC chondrocytes and interactive genes with HT-2 toxin for the purpose of providing a cellular disease model for KBD. In this study, we gave HT-2 toxin treatment to differentiating hiPSC chondrocytes in order to investigate the different responses of KBD hiPSC chondrocytes and control hiPSC chondrocytes to HT-2 toxin. The morphology of HT-2 toxin-treated hiPSC chondrocytes investigated by transmission electron microscope clearly showed that the ultrastructure of organelles was damaged and type II collagen expression in hiPSC chondrocytes was downregulated by HT-2 treatment. Moreover, dysregulation of cell cycle was observed; and p53, p21, and CKD6 gene expressions were dysregulated in hiPSC chondrocytes after T-2 toxin treatment. Flow cytometry also demonstrated that there were significantly increased amounts of late apoptotic cells in KBD hiPSC chondrocytes and that the mRNA expression level of Fas was upregulated. In addition, KBD hiPSC chondrocytes presented stronger responses to HT-2 toxin than control hiPSC chondrocytes. These findings confirmed that HT-2 is an environmental risk factor of KBD and that p53 pathway interacted with HT-2 toxin, causing damaged ultrastructure of organelles, accelerating cell cycle in G1 phase, and increasing late apoptosis in KBD hiPSC chondrocytes.

Comparative Analysis of Machine Learning Methods to Predict Growth of F. sporotrichioides and Production of T-2 and HT-2 Toxins in Treatments with Ethylene-Vinyl Alcohol Films Containing Pure Components of Essential Oils.

The efficacy of ethylene-vinyl alcohol copolymer films (EVOH) incorporating the essential oil components cinnamaldehyde (CINHO), citral (CIT), isoeugenol (IEG), or linalool (LIN) to control growth rate (GR) and production of T-2 and HT-2 toxins by Fusarium sporotrichioides cultured on oat grains under different temperature (28, 20, and 15 °C) and water activity (aw) (0.99 and 0.96) regimes was assayed. GR in controls/treatments usually increased with increasing temperature, regardless of aw, but no significant differences concerning aw were found. Toxin production decreased with increasing temperature. The effectiveness of films to control fungal GR and toxin production was as follows: EVOH-CIT > EVOH-CINHO > EVOH-IEG > EVOH-LIN. With few exceptions, effective doses of EVOH-CIT, EVOH-CINHO, and EVOH-IEG films to reduce/inhibit GR by 50%, 90%, and 100% (ED50, ED90, and ED100) ranged from 515 to 3330 µg/culture in Petri dish (25 g oat grains) depending on film type, aw, and temperature. ED90 and ED100 of EVOH-LIN were >3330 µg/fungal culture. The potential of several machine learning (ML) methods to predict F. sporotrichioides GR and T-2 and HT-2 toxin production under the assayed conditions was comparatively analyzed. XGBoost and random forest attained the best performance, support vector machine and neural network ranked third or fourth depending on the output, while multiple linear regression proved to be the worst.