Development of Antioxidant Protein Extracts from Gilthead Sea Bream (Sparus aurata) Side Streams Assisted by Pressurized Liquid Extraction (PLE).

The pressurized liquid extraction (PLE) technique was used, for the first time, to obtain protein extracts with antioxidant activity from side streams (muscle, heads, viscera, skin, and tailfins) of gilthead sea bream (Sparus aurata) in order to give added value to these underutilized matrices. Extraction conditions previously optimized for sea bass (Dicentrarchus labrax) side streams were applied. Protein recovery percentages were 22% (muscle), 33% (heads), 78% (viscera), 24% (skin), and 26% (tailfins), which represented an increase of 1.2-4.5-fold compared to control samples (extraction by stirring). The SDS-PAGE profiles revealed that PLE-assisted extraction influenced protein molecular weight distribution of the obtained extracts. PLE conditions also allowed increasing the antioxidant capacity measured by both Trolox equivalent antioxidant capacity (TEAC; 1.3-2.4 fold) and oxygen radical absorbance capacity (ORAC; 1.9-6.4) assays for all fish extracts. Inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-qTOF-MS) were used to investigate the presence of toxic metals and mycotoxins in sea bream side streams. The levels of As, Hg, Cd, and Pb were below those established by authorities for fish muscle for human consumption (except for Cd in viscera samples). Through a nontargeted screening approach, no mycotoxins or related metabolites were detected for all sea bream side streams. This study contributes to the research on the valorization of fish processing side streams using environmentally friendly technology.

Exposure Biomarkers and Histopathological Analysis in Pig Liver After Exposure to Mycotoxins Under Field Conditions: Special Report on Fumonisin B1.

Liver samples from finisher pigs were collected at the slaughterhouses for the analysis of zearalenone (ZEA), alfa-/beta-zearalenone (α-ZE, ß-ZE), zearalanone (ZA), alfa-/beta-ZA (α-ZA, ß-ZA), aflatoxin B1 (AFB1) and aflatoxin M1, fumonisin B1 (FB1), ochratoxin A (OTA) and ochratoxin B, deoxynivalenol and deepoxi-deoxynivalenol (DOM-1). For the analysis liquid chromatography-triple quadrupole coupled with mass spectrometry was applied. Liver samples with detected FB1 were further histopathologically evaluated after hematoxylin and eosin staining. Various levels of liver mycotoxins were detected in all farms. Pig livers with 2.91-8.30 µg/kg of FB1 were detected in three farms, estimate of 850-2400 µg/kg of FB1 intake, whereas 0.54 µg/kg of OTA was detected in one farm, estimate of 75 µg/kg of OTA intake. Moreover, pig livers with 0.30 µg/kg of ZEA, 1.87 µg/kg of α-ZE, and 0.63 µg/kg of ß-ZE were detected in one farm, estimate with of 300 µg/kg of ZEA intake. The histopathological analysis revealed that the lesions' grading and necrosis grading were analogously increased when FB1 concentration increased from 2.91 to 4.36-8.30 µg/kg. The severity of megalocytosis was analogously increased with FB1 detection levels and particularly in levels of 4.36-8.3 µg/kg. However, the increased FB1 detection levels did not show analogous behavior with the severity of hepatic cell vacuolization. Results showed that FB1 remained the most critical risk factor in the Greek pig industry, whereas ZEA and AFB1 were also prevalent. The OTA contamination in pig farms raised a high risk for animal and human health.

Fast construction of core-shell structured magnetic covalent organic framework as sorbent for solid-phase extraction of zearalenone and its derivatives prior to their determination by UHPLC-MS/MS.

Magnetic covalent organic framework nanocomposite denoted as Fe3O4@TAPB-Tp with core-shell structure was fabricated via a simple template-mediated precipitation polymerization method at mild conditions. The polyimine network shell was created through the polymerization of 1,3,5-tris(4-aminophenyl)-benzene (TAPB) and 1,3,5-triformyl-phloroglucinol (Tp) in tetrahydrofuran (THF) by the Schiff-base reaction. Featuring with large specific surface area (163.19 m2 g-1), good solution dispersibility, and high stability, the obtained Fe3O4@TAPB-Tp exhibited high adsorption capacities and fast adsorption for zearalenone and its derivatives (ZEAs). The adsorption isotherms showed multilayer adsorption dominated at low concentration and monolayer adsorption at high concentration between the interface of ZEAs and Fe3O4@TAPB-Tp. With the Fe3O4@TAPB-Tp as sorbent, a magnetic solid-phase extraction-ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established for simultaneous adsorption and detection of five ZEAs in complex samples. The proposed method displayed favorable linearity, low limits of detection (0.003 ~ 0.018 µg kg-1), and good repeatability (2.37~10.4%). The developed method has been applied for real sample analysis, with recoveries of 81.27~90.26%. These results showed that Fe3O4@TAPB-Tp has a good application potential for the adsorption of ZEAs in food samples. Magnetic covalent organic framework nanocomposite (Fe3O4@TAPB-Tp) were quickly fabricated at mild conditions and used as effective adsorbent for magnetic solid-phase extraction of zearalenone and its derivatives (ZEAs) from food samples prior to ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis.

Current Applications of Magnetic Nanomaterials for Extraction of Mycotoxins, Pesticides, and Pharmaceuticals in Food Commodities.

Environmental pollutants, such as mycotoxins, pesticides, and pharmaceuticals, are a group of contaminates that occur naturally, while others are produced from anthropogenic sources. With increased research on the adverse ecological and human health effects of these pollutants, there is an increasing need to regularly monitor their levels in food and the environment in order to ensure food safety and public health. The application of magnetic nanomaterials in the analyses of these pollutants could be promising and offers numerous advantages relative to conventional techniques. Due to their ability for the selective adsorption, and ease of separation as a result of magnetic susceptibility, surface modification, stability, cost-effectiveness, availability, and biodegradability, these unique magnetic nanomaterials exhibit great achievement in the improvement of the extraction of different analytes in food. On the other hand, conventional methods involve longer extraction procedures and utilize large quantities of environmentally unfriendly organic solvents. This review centers its attention on current applications of magnetic nanomaterials and their modifications in the extraction of pollutants in food commodities.

Myco-DES: Enabling Remote Extraction of Mycotoxins for Robust and Reliable Quantification by Stable Isotope Dilution LC-MS/MS.

Mycotoxins remain a global threat to human and animal health, especially in countries lacking effective measures to detect and control contaminated commodities. As the quantification of mycotoxins usually relies on complex and expensive techniques, the availability of suitable instrumentation is often a bottleneck in reliable mycotoxin detection. As part of our research toward strategies offering widespread access to mycotoxin analysis while cutting down on costs, we present a new extraction and quantification protocol combining materials originally designed for dried blood spot analysis with stable isotope dilution analysis. Its key benefits are that extraction of mycotoxins can be carried out at remote sites and by minimally trained personnel, while quantification will take place in specialized central laboratories simply connected by regular, paper-based mail. As a proof of concept, aflatoxins, ochratoxin A, and deoxynivalenol were extracted from cereal-based foodstuffs, fixed on paper cards for transport, and successfully quantified after re-extraction by stable isotope dilution LC-MS/MS analysis. Several materials (cellulose/polyethylene terephthalate/glass fiber, nontreated/chemically treated) as well as possible transport and storage conditions (temperature, humidity) were evaluated. The final myco-DES (dried extract spots) protocol allows quantification of mycotoxin levels currently recognized as safe (aflatoxin B1: 2 µg/kg, ochratoxin A: 3 µg/kg, deoxynivalenol: 500 µg/kg) after a storage of up to 4 weeks under tropical climate conditions (40 °C, 75% relative humidity).

Analysis of multiple mycotoxins-contaminated wheat by a smart analysis platform.

This study describes a smart analysis platform capable of quantitative measurements using a multiplex lateral flow strip. Using the multi-mycotoxin strip, five fungal toxins were simultaneously and quantitatively detected in naturally contaminated wheat. First, a matrix-based standard curve was established for the detection of aflatoxin B1 (AFB1), fumonisin B1 (FB1), T-2, deoxynivalenol (DON), and zearalenone (ZEN). Established on an open android system, the platform is able to read 6 lines on the strip simultaneously. The platform is equipped with a Quick Response code scanning model, which reads the established standard curves, and then rapidly quantify mycotoxins in naturally contaminated wheat. All the data and sample information are stored on a central server through the platform which is linked to the cloud. The limits of detection (LOD) for AFB1, FB1, T-2, DON, and ZEN in wheat were 4, 20, 10, 200, and 40 µg/kg and the visual cut off values was 20, 1000, 200, 4000, and 400 µg/kg, separately. To validate the platform and the multi-mycotoxin detection method, 10 wheat samples were analyzed and the results were in a good agreement with those obtained by LC-MS/MS. The platform will be a powerful tool for crop monitoring services.

Two Phytotoxins Isolated from the Pathogenic Fungus of the Invasive Weed Xanthium italicum.

Alternariol and altenuisol were isolated as the major phytotoxins produced by an Alternaria sp. pathogenic fungus of the invasive weed Xanthium italicum. Altenuisol exhibited stronger phytotoxic effect compared with alternariol. At 10 µg/mL, alternariol and altenuisol promoted root growth of the monocot plant Pennisetum alopecuroides by 11.1 % and 75.2 %, respectively, however, inhibitory activity was triggered by the increase of concentration, with root elongation being suppressed by 35.5 % and 52.0 % with alternariol and altenuisol at 1000 µg/mL, respectively. Alternariol slightly inhibited root length of the dicot plant Medicago sativa at 10-1000 µg/mL, whereas altenuisol stimulated root growth by 51.0 % at 10 µg/mL and inhibited root length by 43.4 % at 200 µg/mL. Alternariol and altenuisol did not exert strong regulatory activity on another dicot plant, Amaranthus retroflexus, when tested concentration was low, however, when the concentration reached 1000 µg/mL, they reduced root length by 68.1 % and 51.0 %, respectively. Alternariol and altenuisol exerted similar effect on shoot growth of three tested plants but to a lesser extent. It is noteworthy to mention that this is the first report on the phytotoxicity of altenuisol.

Dilute-and-Shoot HPLC-UV Method for Determination of Urinary Creatinine as a Normalization Tool in Mycotoxin Biomonitoring in Pigs.

A simple, rapid, and accurate HPLC-UV method was developed for the determination of creatinine in pig urine. Usually, it is determined in urine in biomonitoring of xenobiotics to correct for variations in dilutions of urine samples. The colorimetric method (based on Jaffe reaction), which was mainly used for this purpose in mycotoxin biomonitoring, is not a reliable approach for pig urine. Therefore, a novel and accurate HPLC method for creatinine determination was developed. The sample preparation was based on the dilute and shoot approach. An HPLC separation was performed with a porous graphitic carbon column with an aqueous mobile phase to achieve satisfactory retention time for creatinine. The method has been successfully validated, applied for the determination of creatinine in pig urine, and compared with other methods commonly used for that purpose-a colorimetric method based on Jaffe reaction and commercial ELISA test. The developed HPLC method shows the highest precision and accuracy for pig urine samples. Finally, the method was applied as a normalization tool in LC-MS/MS mycotoxin biomarkers analysis. The standardization to a constant creatinine level (0.5 mg/mL) enables similar matrix effects for eleven mycotoxin biomarkers for pig urine samples with different creatinine levels.

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.

Mycotoxin contamination of foods in Southern Africa: A 10-year review (2007-2016).

Major staple foods in Southern Africa are prone to mycotoxin contamination, posing health risks to consumers and consequent economic losses. Regional climatic zones favor the growth of one or more main mycotoxin producing fungi, Aspergillus, Fusarium and Penicillium. Aflatoxin contamination is mainly reported in maize, peanuts and their products, fumonisin contamination in maize and maize products and patulin in apple juice. Lack of awareness of occurrence and risks of mycotoxins, poor agricultural practices and undiversified diets predispose populations to dietary mycotoxin exposure. Due to a scarcity of reports in Southern Africa, reviews on mycotoxin contamination of foods in Africa have mainly focused on Central, Eastern and Western Africa. However, over the last decade, a substantial number of reports of dietary mycotoxins in South Africa have been documented, with fewer reports documented in Botswana, Lesotho, Malawi, Mozambique, Zambia and Zimbabwe. Despite the reported high dietary levels of mycotoxins, legislation for their control is absent in most countries in the region. This review presents an up-to-date documentation of the epidemiology of mycotoxins in agricultural food commodities and discusses the implications on public health, current and recommended mitigation strategies, legislation, and challenges of mycotoxin research in Southern Africa.

Macrocyclic Trichothecene Mycotoxins from a Deadly Poisonous Mushroom, Podostroma cornu-damae.

Three new macrocyclic trichothecenes (1-3) and five known related compounds (4-8) were isolated from the MeOH extract of a plate culture of the fungus Podostroma cornu-damae, a deadly poisonous mushroom. Miophytocen D (1) is a rearranged macrocyclic type D trichothecene, featuring a bicyclo-[6.5]dodecahydrocyclopenta[ b]chromene scaffold, and the structures of new compounds (1-3) were delineated by the combination of 1D and 2D NMR spectroscopic experiments and HRESIMS, modified Mosher's esterification, and quantum chemical ECD calculations. The isolated compounds (1-8) were evaluated for cytotoxicity against four human breast cancer cell lines (Bt549, HCC70, MDA-MB-231, and MDA-MB-468). Compounds 4, 6, and 8 exhibited significant cytotoxic effects against the breast cancer cell lines, with IC50 values in the range of 0.02-80 nM, which is stronger than doxorubicin, the positive control, and a structure-activity relationship was suggested.

Naturally occurring mixtures of Alternaria toxins: anti-estrogenic and genotoxic effects in vitro.

Alternaria molds can produce a variety of different mycotoxins, often resulting in food contamination with chemical mixtures, posing a challenge for risk assessment. Some of these metabolites possess estrogenic properties, an effect whose toxicological relevance is questioned in the light of the strong genotoxic and cytotoxic properties of co-occurring toxins. Thus, we tested a complex extract from A. alternata for estrogenic properties in Ishikawa cells. By assessing alkaline phosphatase activity, we did not observe estrogen receptor (ER) activation at non-cytotoxic concentrations (≤ 10 µg/ml). Furthermore, an extract stripped of highly genotoxic perylene quinones also did not mediate estrogenic effects, despite diminished genotoxic properties in the comet assay (≥ 10 µg/ml). Interestingly, both extracts impaired the estrogenicity of 17ß-estradiol (E2) at non-cytotoxic concentrations (5-10 µg/ml), indicating anti-estrogenic effects which could not be explained by the presence of known mycoestrogens. A mechanism for this unexpected result might be the activation of the aryl hydrocarbon receptor (AhR) by Alternaria metabolites, as indicated by the induction of CYP1A1 transcription. While a direct influence on the metabolism of E2 could not be confirmed by LC-MS/MS, literature describing a direct interplay of the AhR with estrogenic pathways points to a corresponding mode of action. Taken together, the present study indicates AhR-mediated anti-estrogenic effects as a novel mechanism of naturally co-occurring Alternaria toxin mixtures. Furthermore, our results confirm their genotoxic activity and raise questions about the contribution of still undiscovered metabolites to toxicological properties.

Bioavailability, metabolism, and excretion of a complex Alternaria culture extract versus altertoxin II: a comparative study in rats.

Despite the frequent infection of agricultural crops by Alternaria spp., their toxic secondary metabolites and potential food contaminants lack comprehensive metabolic characterization. In this study, we investigated their bioavailability, metabolism, and excretion in vivo. A complex Alternaria culture extract (50 mg/kg body weight) containing 11 known toxins and the isolated lead toxin altertoxin II (0.7 mg/kg body weight) were administered per gavage to groups of 14 Sprague Dawley rats each. After 3 h and 24 h, plasma, urine and feces were collected to determine toxin recoveries. For reliable quantitation, an LC-MS/MS method for the simultaneous detection of 20 Alternaria toxins and metabolites was developed and optimized for either biological matrix. The obtained results demonstrated efficient excretion of alternariol (AOH) and its monomethyl ether (AME) via feces (> 89%) and urine (> 2.6%) after 24 h, while the majority of tenuazonic acid was recovered in urine (20 and 87% after 3 and 24 h, respectively). Moreover, modified forms of AOH and AME were identified in urine and fecal samples confirming both, mammalian phase-I (4-hydroxy-AOH) and phase-II (sulfates) biotransformation in vivo. Despite the comparably high doses, perylene quinones were recovered only at very low levels (altertoxin I, alterperylenol, < 0.06% in urine and plasma, < 5% in feces) or not at all (highly genotoxic, epoxide-holding altertoxin II, stemphyltoxin III). Interestingly, altertoxin I was detected in all matrices of rats receiving altertoxin II and suggests enzymatic de-epoxidation in vivo. In conclusion, the present study contributes valuable information to advance our understanding of the emerging Alternaria mycotoxins and their relevance on food safety.

Fungal mycobiota and mycotoxin risk for traditional artisan Italian cave cheese.

Cave cheese is a surface mold-ripened variety of cheese produced also in South of Italy, exploiting fungal population naturally occurring on cave walls, as part of secondary microbiota for ripening. In this study, 148 fungal strains were isolated from 22 independent cave cheese samples, collected in 13 Italian geographical locations, mostly in Apulian area. DNA-based identification showed the presence of twenty-four fungal species in the outer part of the cheese ripened in caves. Aspergillus westerdijkiae and Penicillium biforme resulted the most frequently isolated species, followed by Penicillium roqueforti and Penicillium solitum. The 86% of cheese sample presented at least one toxigenic species and the 45% revealed the presence of ochratoxigenic species, A. westerdijkiae and A. steynii, suggesting possible mycotoxin risk during ripening stage in caves, confirmed by the presence of ochratoxin A (OTA) in the rind of 36% of samples. In conclusion, cave cheese is a susceptible product for toxigenic mold growth and in particular OTA contamination, therefore adeguate scientific tools for matching organolectic consumer expectations and complete safety of food should be developed, as well as spontaneously molded and not monitored cheeses should not be consumed to avoid mycotoxin risk.

Mycotoxin Incidence in Some Fish Products: QuEChERS Methodology and Liquid Chromatography Linear Ion Trap Tandem Mass Spectrometry Approach.

The inclusion of vegetal raw materials in feed for fish farming has increased the risk of mycotoxin occurrence in feed, as well as in edible tissues from fish fed with contaminated feed, due to the carry-over to muscle portions. Therefore, the objective of this study was to evaluate the occurrence of 15 mycotoxins in processed fish products, which are commonly consumed, such as smoked salmon and trout, different types of sushi, and gula substitutes. A QuEChERS method was employed to perform the mycotoxin extraction from fish samples. For mycotoxin identification and quantitation, the selected technique was the liquid chromatography-tandem mass spectrometry linear ion trap (LC-MS/MS-LIT). Smoked fish and sushi samples results were negative regarding the presence of all 15 mycotoxins studied. In contrast, small amounts of fusarenon-X and enniatin B were found in gula substitute samples.

Dirhamnolipid Produced by the Pathogenic Fungus Colletotrichum gloeosporioides BWH-1 and Its Herbicidal Activity.

Fungal phytotoxins used as ecofriendly bioherbicides are becoming efficient alternatives to chemical herbicides for sustainable weed management. Previous study found that cultures of the pathogenic fungus Colletotrichum gloeosporioides BWH-1 showed phytotoxic activity. This study further isolated the major phytotoxin from cultures of the strain BWH-1 using bioactivity-guided isolation, by puncturing its host plant for an activity test and analyzing on the HPLC-DAD-3D mode for a purity check. Then, the active and pure phytotoxin was characterized as a dirhamnolipid (Rha-Rha-C10-C10) using the NMR, ESIMS, IR and UV methods. The herbicidal activity of dirhamnolipid was evaluated by the inhibition rate on the primary root length and the fresh plant weight of nine test plants, and the synergistic effect when combining with commercial herbicides. Dirhamnolipid exhibited broad herbicidal activity against eight weed species with IC50 values ranging from 28.91 to 217.71 mg L-1 and no toxicity on Oryza sativa, and the herbicidal activity could be synergistically improved combining dirhamnolipid with commercial herbicides. Thus, dirhamnolipid that originated from C. gloeosporioides BWH-1 displayed the potential to be used as a bioherbicide alone, or as an adjuvant added into commercial herbicides, leading to a decrease in herbicides concentration and increased control efficiency.

Identification of Patulin from Penicillium coprobium as a Toxin for Enteric Neurons.

The identification and characterization of fungal commensals of the human gut (the mycobiota) is ongoing, and the effects of their various secondary metabolites on the health and disease of the host is a matter of current research. While the neurons of the central nervous system might be affected indirectly by compounds from gut microorganisms, the largest peripheral neuronal network (the enteric nervous system) is located within the gut and is exposed directly to such metabolites. We analyzed 320 fungal extracts and their effect on the viability of a human neuronal cell line (SH-SY5Y), as well as their effects on the viability and functionality of the most effective compound on primary enteric neurons of murine origin. An extract from P. coprobium was identified to decrease viability with an EC50 of 0.23 ng/µL in SH-SY5Y cells and an EC50 of 1 ng/µL in enteric neurons. Further spectral analysis revealed that the effective compound was patulin, and that this polyketide lactone is not only capable of evoking ROS production in SH-SY5Y cells, but also diverse functional disabilities in primary enteric neurons such as altered calcium signaling. As patulin can be found as a common contaminant on fruit and vegetables and causes intestinal injury, deciphering its specific impact on enteric neurons might help in the elaboration of preventive strategies.

Gramillin A and B: Cyclic Lipopeptides Identified as the Nonribosomal Biosynthetic Products of Fusarium graminearum.

The virulence and broad host range of Fusarium graminearum is associated with its ability to secrete an arsenal of phytotoxic secondary metabolites, including the regulated mycotoxins belonging to the deoxynivalenol family. The TRI genes responsible for the biosynthesis of deoxynivalenol and related compounds are usually expressed during fungal infection. However, the F. graminearum genome harbors an array of unexplored biosynthetic gene clusters that are also co-induced with the TRI genes, including the nonribosomal peptide synthetase 8 ( NRPS8) gene cluster. Here, we identify two bicyclic lipopeptides, gramillin A (1) and B (2), as the biosynthetic end products of NRPS8. Structural elucidation by high-resolution LC-MS and NMR, including 1H-15N-13C HNCO and HNCA on isotopically enriched compounds, revealed that the gramillins possess a fused bicyclic structure with ring closure of the main peptide macrocycle occurring via an anhydride bond. Through targeted gene disruption, we characterized the GRA1 biosynthetic gene and its transcription factor GRA2 in the NRPS8 gene cluster. Further, we show that the gramillins are produced in planta on maize silks, promoting fungal virulence on maize but have no discernible effect on wheat head infection. Leaf infiltration of the gramillins induces cell death in maize, but not in wheat. Our results show that F. graminearum deploys the gramillins as a virulence agent in maize, but not in wheat, thus displaying host-specific adaptation.

Characterization of an antimicrobial and phytotoxic ribonuclease secreted by the fungal wheat pathogen Zymoseptoria tritici.

The fungus Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB) disease of wheat leaves. Zymoseptoria tritici secretes many functionally uncharacterized effector proteins during infection. Here, we characterized a secreted ribonuclease (Zt6) with an unusual biphasic expression pattern. Transient expression systems were used to characterize Zt6, and mutants thereof, in both host and non-host plants. Cell-free protein expression systems monitored the impact of Zt6 protein on functional ribosomes, and in vitro assays of cells treated with recombinant Zt6 determined toxicity against bacteria, yeasts and filamentous fungi. We demonstrated that Zt6 is a functional ribonuclease and that phytotoxicity is dependent on both the presence of a 22-amino-acid N-terminal 'loop' region and its catalytic activity. Zt6 selectively cleaves both plant and animal rRNA species, and is toxic to wheat, tobacco, bacterial and yeast cells, but not to Z. tritici itself. Zt6 is the first Z. tritici effector demonstrated to have a likely dual functionality. The expression pattern of Zt6 and potent toxicity towards microorganisms suggest that, although it may contribute to the execution of wheat cell death, it is also likely to have an important secondary function in antimicrobial competition and niche protection.

Discovery and Characterization of 4-Hydroxy-2-pyridone Derivative Sambutoxin as a Potent and Promising Anticancer Drug Candidate: Activity and Molecular Mechanism.

Sambutoxin, a representative derivative of 4-hydroxy-2-pyridone, was isolated from Hericium alpestre for the first time in this study. The possible correlation between the sambutoxin-induced suppression of tumor growth and its influence on cell-cycle arrest and apoptosis was investigated. The effects of sambutoxin on reactive oxygen species (ROS) production, DNA damage, mitochondrial transmembrane potential, cell apoptosis, and the expression of related proteins were evaluated. An in vitro cell viability study demonstrated that sambutoxin could inhibit the proliferation of various cancer cells. Treatment with sambutoxin induced the production of ROS, which caused DNA damage. Furthermore, the subsequent sambutoxin-induced activation of ATM and Chk2 resulted in G2/M arrest, accompanied by decreased expression of cdc25C, cdc2, and cyclin B1. Sambutoxin induced apoptosis by activating the mitochondrial apoptosis pathway through an increased Bax/Bcl-2 ratio, loss of mitochondrial membrane potential (ΔΨm), cytochrome (Cyt) c release, caspase-9 and caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) degradation. The ROS elevation induced the sustained phosphorylation of c-Jun N-terminal kinase (JNK), while SP600125, a JNK inhibitor, nearly completely reversed sambutoxin-induced apoptosis. Accordingly, an in vivo study showed that sambutoxin exhibited potential antitumor activity in a BALB/c nude mouse xenograft model without significant systemic toxicity. Moreover, the expression changes in proteins related to the G2/M phase, DNA damage, and apoptosis in vivo were consistent with those in vitro. Importantly, sambutoxin has remarkable antiproliferative effects and is a promising anticarcinogen candidate for cancer treatment.