Maize Aspergillus section Flavi isolate diversity may be distinct from that of soil and subsequently the source of aflatoxin contamination.

Aspergillus section Flavi (Flavi) is a diverse group of fungal species whose common members include A. flavus and A. parasiticus. These are well-known for the production of aflatoxin (AF) B and G and other toxic metabolites, like cyclopiazonic acid (CPA). They are saprophytic soil dwellers and also become crop opportunistic epiphytes. The consequence is contamination of the crop with mycotoxins, such as carcinogenic AF. We investigated the Flavi community structure of maize and that of their surrounding soil, including their mycotoxigenicity. Furthermore, we investigated the link of the maize Flavi diversity with preharvest maize AF levels. The study was carried out in four selected districts of Zambia, in a low rainfall zone. The Flavi characterisation was triphasic, involving morphological (colony colour and sclerotia formation), metabolic (AF and CPA production) and genetic (calmodulin gene polymorphism) analyses. Flavi abundance was determined by dilution plate technique on modified rose Bengal agar. Results showed that Flavi communities on maize and in soil differed. Maize had a higher Flavi species diversity than soil. A. parasiticus dominated the soil community by frequency of field appearance (85%), while maize was dominated by A. minisclerotigenes (45%). CPA-producers with or without AF production dominated the maize (65%) while producers of only AF (B/G) dominated the soil (88%). The ratio between maize A. parasiticus and A. minisclerotigenes abundance seemed to have had a bearing on the levels of AF in maize, with a ratio close to 1:1 having higher levels than a pure community of either A. parasiticus or A. minisclerotigenes.

Origin of the 6/5/6/5 Tetracyclic Cyclopiazonic Acids.

The natural product α-cyclopiazonic acid (α-CPA) is a very potent Ca2+-ATPase inhibitor. The CPA family of compounds comprise over 80 chemical entities with at least five distinct skeletons. While α-CPA features a canonical 6/5/6/5/5 skeleton, the 6/5/6/5 skeleton is the most prevalent among the CPA family. However, the origin of the unique tetracyclic skeleton remains unknown. The 6/5/6/5-type CPAs may derive from a precursor of acetoacetyl-l-tryptophan (AATrp) generated from a hypothetic thioesterase-like pathway. Alternatively, cleavage of the tetramic acid ring would also result in the formation of the 6/5/6/5 scaffold. Aspergillus oryzae HMP-F28 is a marine sponge-associated filamentous fungus known to produce CPAs that act as primary neurotoxins. To elucidate the origin of this subfamily of CPAs, we performed homologous recombination and genetic engineering experiments on strain HMP-F28. Our results are supportive of the ring cleavage pathway through which the tetracyclic 6/5/6/5-type CPAs are generated from 6/5/6/5/5-type pentacyclic CPAs.

Establishment and evaluation of targeted molecular screening model for the ryanodine receptor or sarco/endoplasmic reticulum calcium ATPase.

BACKGROUD: Endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) is crucial for maintaining intracellular calcium homeostasis due to the calcium-signaling-related proteins on its membrane. While ryanodine receptors (RyR) on insect ER/SR membranes are well-known as targets for diamide insecticides, little is known about other calcium channels. Given the resistance of diamide insecticides, the establishment of molecular screening models targeting RyR or sarco/endoplasmic reticulum calcium ATPase (SERCA) is conducive to the discovery of new insecticidal molecules. RESULTS: The morphological features of Mythimna separata SR have closed vesicles with integrity and high density. The 282 proteins in the SR component contained RyR and SERCA. A measurement model for the release and uptake of calcium was successfully established by detecting calcium ions outside the SR membrane using a fluorescence spectrophotometer. In vitro testing systems using SR vesicles found that diamide insecticides could activate dose-dependently RyR, with EC50 values of 0.14 µM (Chlorantraniliprole), 0.21 µM (Flubendiamide), and 0.57 µM (Cyantraniliprole), respectively. However, dantrolene inhibited RyR-mediated calcium release with an IC50 value of 353.9 µM, suggesting that dantrolene can weakly antagonize RyR. Moreover, cyclopiazonic acid significantly reduced the enzyme activity and calcium uptake capacity of SERCA. On the contrary, CDN1163 markedly activated the enzyme activity and improved the calcium transport capacity of SERCA. CONCLUSIONS: SR vesicles can be used to study the function of unknown proteins on the SR membranes, as well as for high-throughput screening of highly active compounds targeting RyR or SERCA. © 2024 Society of Chemical Industry.

Effects of reversible SERCA inhibition on catecholamine exocytosis and intracellular [Ca2+] signaling in chromaffin cells from normotensive Wistar Kyoto rats and spontaneously hypertensive rats.

Intracellular Ca2+ ([Ca2+]i) signaling and catecholamine (CA) exocytosis from adrenal chromaffin cells (CCs) differ between mammalian species. These differences partly result from the different contributions of Ca2+-induced Ca2+-release (CICR) from internal stores, which boosts intracellular Ca2+ signals. Transient inhibition of the sarcoendoplasmic reticulum (SERCA) Ca2+ pump with cyclopiazonic acid (CPA) reduces CICR. Recently, Martínez-Ramírez et al. found that CPA had contrasting effects on catecholamine secretion and intracellular Ca2+ signals in mouse and bovine CCs, where it enhanced and inhibited exocytosis, respectively. After CPA withdrawal, exocytosis diminished in mouse CCs and increased in bovine CCs. These differences can be explained if mouse CCs have weak CICR and strong Ca2+ uptake, and the reverse is true for bovine CCs. Surprisingly, CPA slightly reduced the amplitude of Ca2+ signals in both mouse and bovine CCs. Here we examined the effects of CPA on stimulated CA exocytosis and Ca2+ signaling in rat CCs and investigated if it alters differently the responses of CCs from normotensive (WKY) or hypertensive (SHR) rats, which differ in the gain of CICR. Our results demonstrate that CPA application strongly inhibits voltage-gated exocytosis and Ca2+ transients in rat CCs, regardless of strain (SHR or WKY). Thus, despite the greater phylogenetic distance from the most recent common ancestors, suppression of endoplasmic reticulum (ER) Ca2+ uptake through CPA inhibits the CA secretion in rat CCs more similarly to bovine than mouse CCs, unveiling divergent evolutionary relationships in the mechanism of CA exocytosis of CCs between rodents. Agents that inhibit the SERCA pump, such as CPA, suppress catecholamine secretion equally well in WKY and SHR CCs and are not potential therapeutic agents for hypertension. Rat CCs display Ca2+ signals of varying widths. Some even show early and late Ca2+ components. Narrowing the Ca2+ transients by CPA and ryanodine suggests that the late component is mainly due to CICR. Simultaneous recordings of Ca2+ signaling and amperometry in CCs revealed the existence of a robust and predictable correlation between the kinetics of the whole-cell intracellular Ca2+ signal and the rate of exocytosis at the single-cell level.

Assessment of dietary exposure and levels of mycotoxins in sorghum from Niger State of Nigeria.

This study reports levels of mycotoxins in sorghum from Niger State, Nigeria, and provides a comprehensive assessment of their potential health risks by combining mycotoxin levels and dietary exposure assessment. A total of 240 samples of red and white sorghum were collected from both stores and markets across four microclimatic zones. Fungal species were identified using a dilution plate method. Aflatoxins (AFs), deoxynivalenol, nivalenol, and ochratoxin (OTA) were quantified using HPLC, whereas cyclopiazonic acid, fumonisins (FUMs) and zearalenone were quantified using ELISA. A. flavus and A. fumigatus were dominant fungal species followed by F. verticilloides, A. oryzae and P. verrucosum. Aflatoxins (mean: 29.97 µg/kg) were detected in all samples, whereas OTA (mean: 37.5 µg/kg) and FUMs (mean: 3269.8 µg/kg) were detected in 72% and 50% of the samples, respectively. Mycotoxins frequently co-occurred in binary mixtures of AFs + OTA and AFs + FUMs. Dietary exposure estimates were highest for FUMs at 230% of TDI and margin of exposures (MOEs) for both AFs and OTA (<10,000) indicating a potential risk associated with combined exposure to AFs and OTA. The Risk of hepatocellular carcinoma cases (HCC/year) attributable to AFs and OTA exposure from sorghum was estimated to be 5.99 × 105 and 0.24 × 105 cases for HBsAg + individuals based on 13.6% HBV incidence. Similarly, the HCC/year for AFs and OTA were assessed to be 3.59 × 105 and 0.14 × 105 at an 8.1% prevalence rate. Therefore, the results of this study demonstrate the high prevalence and dietary exposure to mycotoxins through sorghum consumption, raising public health and trade concerns.

Lupenone attenuates thapsigargin-induced endoplasmic reticulum stress and apoptosis in pancreatic beta cells possibly through inhibition of protein tyrosine kinase 2 activity.

AIMS: Prolonged high levels of cytokines, glucose, or free fatty acids are associated with diabetes, elevation of cytosolic Ca2+ concentration ([Ca2+]C), and depletion of Ca2+ concentration in the endoplasmic reticulum (ER) of pancreatic beta cells. This Ca2+ imbalance induces ER stress and apoptosis. Lupenone, a lupan-type triterpenoid, is beneficial in diabetes; however, its mechanism of action is yet to be clarified. This study evaluated the protective mechanism of lupenone against thapsigargin-induced ER stress and apoptosis in pancreatic beta cells. MATERIALS AND METHODS: MIN6, INS-1, and native mouse islet cells were used. Western blot for protein expressions, measurement of [Ca2+]C, and in vivo glucose tolerance test were mainly performed. KEY FINDINGS: Thapsigargin increased the protein levels of cleaved caspase 3, cleaved PARP, and the phosphorylated form of JNK, ATF4, and CHOP. Thapsigargin increased the interaction between stromal interaction molecule1 (Stim1) and Orai1, enhancing store-operated calcium entry (SOCE). SOCE is further activated by protein tyrosine kinase 2 (Pyk2), which is Ca2+-dependent and phosphorylates the tyrosine residue at Y361 in Stim1. Lupenone inhibited thapsigargin-mediated Pyk2 activation, suppressed [Ca2+]C, ER stress, and apoptosis. Lupenone restored impaired glucose-stimulated insulin secretion effectuated by thapsigargin and glucose intolerance in a low-dose streptozotocin-induced diabetic mouse model. SIGNIFICANCE: These results suggested that lupenone attenuated thapsigargin-induced ER stress and apoptosis by inhibiting SOCE; this may be due to the hindrance of Pyk2-mediated Stim1 tyrosine phosphorylation. In beta cells that are inevitably exposed to frequent [Ca2+]C elevation, the attenuation of abnormally high SOCE would be beneficial for their survival.

Are Current Aspergillus sojae Strains Originated from a Native Aflatoxigenic Aspergillus Species Population Also Present in California?

Aspergillus sojae has long been considered a domesticated strain of Aspergillus parasiticus. This study delineated relationships among the two species and an Aspergillus PWE36 isolate. Of 25 examined clustered aflatoxin genes of PWE36, 20 gene sequences were identical to those of A. sojae, but all had variations to those of A. parasiticus. Additionally, PWE36 developmental genes of conidiation and sclerotial formation, overall, shared higher degrees of nucleotide sequence identity with A. sojae genes than with A. parasiticus genes. Examination of defective cyclopiazonic acid gene clusters revealed that the PWE36 deletion pattern was identical only to those of A. sojae. Using A. sojae SMF134 genome sequence as a reference, visualization of locally collinear blocks indicated that PWE36 shared higher genome sequence homologies with A. sojae than with A. parasiticus. Phylogenetic inference based on genome-wide single nucleotide polymorphisms (SNPs) and total SNP counts showed that A. sojae strains formed a monophyletic clade and were clonal. Two (Argentinian and Ugandan) A. parasiticus isolates but not including an Ethiopian isolate formed a monophyletic clade, which showed that A. parasiticus population is genetically diverse and distant to A. sojae. PWE36 and A. sojae shared a most recent common ancestor (MRCA). The estimated divergence time for PWE36 and A. sojae was about 0.4 mya. Unlike Aspergillus oryzae, another koji mold that includes genetically diverse populations, the findings that current A. sojae strains formed a monophyletic group and shared the MRCA with PWE36 allow A. sojae to be continuously treated as a species for food safety reasons.

Homogeneous immunoassay for cyclopiazonic acid based upon mimotopes and upconversion-resonance energy transfer.

Strains of Penicillium spp. are used for fungi-ripened cheeses and Aspergillus spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin α-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel epitope mimicking peptide, or mimotope, selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved, and the analysis of individual phage clones revealed four different mimotope peptide sequences. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, surface plasmon resonance binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion nanoparticles (UCNP, type NaYF4:Yb3+, Er3+) were used as energy donors and coated with streptavidin to anchor the synthetic biotinylated mimotope. Alexa Fluor 555, used as an energy acceptor, was conjugated to the anti-CPA antibody fragment. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg mL-1 (1.5 µg kg-1) and an IC50 value of 0.36 ng mL-1. No significant cross-reactivity was observed with other co-produced mycotoxins. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) as a reference method.

Pegriseofamines A-E: Five cyclopiazonic acid related indole alkaloids from the fungus Penicillium griseofulvum.

Five new cyclopiazonic acid related indole alkaloids, pegriseofamines A-E (1-5), were isolated from the fungus Penicillium griseofulvum. Their structures and absolute configurations were determined by NMR, HRESIMS, quantum-chemical calculation, and X-ray diffraction experiments. Among them, pegriseofamine A (1) possesses an undescribed 6/5/6/7 tetracyclic ring system generated by the fusion of an azepine and an indole unit via a cyclohexane, and the postulated biosynthetic origin of 1 was discussed. Compound 4 could relieve liver injury and prevent hepatocyte apoptosis in ConA-induced autoimmune liver disease.

Putative Core Transcription Factors Affecting Virulence in Aspergillus flavus during Infection of Maize.

Aspergillus flavus is an opportunistic pathogen responsible for millions of dollars in crop losses annually and negative health impacts on crop consumers globally. A. flavus strains have the potential to produce aflatoxin and other toxic secondary metabolites, which often increase during plant colonization. To mitigate the impacts of this international issue, we employ a range of strategies to directly impact fungal physiology, growth and development, thus requiring knowledge on the underlying molecular mechanisms driving these processes. Here we utilize RNA-sequencing data that are obtained from in situ assays, whereby Zea mays kernels are inoculated with A. flavus strains, to select transcription factors putatively driving virulence-related gene networks. We demonstrate, through growth, sporulation, oxidative stress-response and aflatoxin/CPA analysis, that three A. flavus strains with knockout mutations for the putative transcription factors AFLA_089270, AFLA_112760, and AFLA_031450 demonstrate characteristics such as reduced growth capacity and decreased aflatoxin/CPA accumulation in kernels consistent with decreased fungal pathogenicity. Furthermore, AFLA_089270, also known as HacA, eliminates CPA production and impacts the fungus's capacity to respond to highly oxidative conditions, indicating an impact on plant colonization. Taken together, these data provide a sound foundation for elucidating the downstream molecular pathways potentially contributing to fungal virulence.

Cyclopiazonic acid in soft-ripened and blue cheeses marketed in the USA.

Strains of Penicillium camemberti and P. roqueforti are used in the production of soft-ripened and blue-veined cheeses. However, some strains can produce toxic secondary metabolites (mycotoxins), including α-cyclopiazonic acid (CPA), a neurotoxin. Data on the levels of CPA in cheeses marketed in the USA are extremely limited. An enzyme-linked immunosorbent assay was adapted for measuring CPA in soft-ripened and blue-veined cheeses. Recoveries from cheese curds were 103 ± 27% (n = 30). A total of 254 samples of soft-ripened, blue and miscellaneous cheeses were examined. CPA was detected in 36/79 (45.6%) of soft-ripened cheeses and in 41/168 (24.4%) of blue-veined cheeses. Median levels in positive samples were 48.5 µg/kg and 30 µg/kg, respectively. The highest levels found were 3,820 µg/kg (in a Brie), 1,250 µg/kg (in a blue) and 7,900 µg/kg (in a Monte Enebro). The implication of such exposures is unknown, as a consensus on acceptable intake remains to be established.

The Vapor Phase of Selected Essential Oils and Their Antifungal Activity In Vitro and In Situ against Penicillium commune, a Common Contaminant of Cheese.

This study aimed to determine the in vitro and in situ antifungal activity of (14) selected essential oils (EOS), namely clove, thyme, red thyme, litsea, eucalyptus, niaouli, fennel, anise, cumin, basil, rosemary, sage, bergamot mint, and marjoram, by vapor contact against the growth of two strains of Penicillium commune (KMi-183 and KMi-402). Furthermore, to exclude the negative effect of EOs on the lactic acid bacteria (LABs) (Streptococcus spp.) on cheeses, their influence was monitored. Next, the sensory evaluation of cheese treated by EOs was evaluated. The results show that litsea and clove EOs were the most effective in the vapor phase against both tested strains. These EOs were characterized by the highest amount of α- (40.00%) and ß-Citral (34.35%) in litsea and eugenol (85.23%) in clove. The antitoxicogenic activity of less effective (in growth inhibition) EOs on cyclopiazonic acid (CPA) production by the tested strains was also observed. The growth of Streptococcus spp. (ranging from 8.11 to 9.69 log CFU/g) was not affected by the EOs in treated cheese. Even though the evaluators recognized some EOs in sensory evaluation by the triangle test, they did not have a negative effect on the taste and smell of the treated cheeses and were evaluated as edible. The antifungal activity of EOs against several types of microscopic fungi and their effect on the sensory properties of treated foods needs to be further tested to achieve the most effective protection of foods from their direct contaminants.

Interaction of the Emerging Mycotoxins Beauvericin, Cyclopiazonic Acid, and Sterigmatocystin with Human Serum Albumin.

Beauvericin (BEA), cyclopiazonic acid (CPA), and sterigmatocystin (STC) are emerging mycotoxins. They appear as contaminants in food and animal feed, leading to economic losses and health risks. Human serum albumin (HSA) forms stable complexes with certain mycotoxins, including ochratoxins, alternariol, citrinin, and zearalenone. HSA binding can influence the toxicokinetics of xenobiotics, and albumin can also be considered and applied as a relatively cheap affinity protein. Therefore, we examined the potential interactions of BEA, CPA, and STC with HSA employing fluorescence spectroscopy, ultracentrifugation, ultrafiltration, and molecular modeling. Spectroscopic and ultracentrifugation studies demonstrated the formation of low-affinity BEA-HSA (Ka ≈ 103 L/mol) and moderately strong CPA-HSA and STC-HSA complexes (Ka ≈ 104 L/mol). In ultrafiltration experiments, CPA slightly displaced each site marker (warfarin, naproxen, and camptothecin) tested, while BEA and STC did not affect significantly the albumin binding of these drugs. Modeling studies suggest that CPA occupies Sudlow's site I, while STC binds to the Heme site (FA1) on HSA. Considering the interactions of CPA with the site markers, the CPA-HSA interaction may have toxicological importance.

The Occurrence of Five Unregulated Mycotoxins Most Important for Traditional Dry-Cured Meat Products.

This study investigated the occurrence of 5 unregulated mycotoxins in a total of 250 traditional dry-cured meat products sampled in 2020 and 2021 in five Croatian regions (eastern, northern, central, western, and southern). Aflatoxin B1 (AFB1), ochratoxin A (OTA), sterigmatocystin (STC), citrinin (CIT), and cyclopiazonic acid (CPA) concentrations were related to the geographical region of the product's origin and to local weather. The results revealed the contamination of 27% of samples, namely, STC in 4% of samples in concentrations of up to 3.93 µg/kg, OTA in 10% of samples in concentrations of up to 4.81 µg/kg, and CPA in 13% of samples in concentrations of up to 335.5 µg/kg. No AFB1 or CIT contamination was seen. Although no statistically significant differences in concentrations of individual mycotoxins across the production regions were found, differences in mycotoxin occurrence were revealed. The eastern and western regions, with moderate climate, delivered the largest number of contaminated samples, while the southern region, often compared with subtropics, delivered the smallest, so that the determined mycotoxins were probably mainly produced by the Penicillium rather than the Aspergillus species. Due to the interaction of various factors that may affect mycotoxin biosynthesis during production, the detected concentrations cannot be related solely to the weather.

'Kodo poisoning': cause, science and management.

Many mycotoxigenic fungi infect the food crops and affect the quality of the produce due to production of mycotoxins. Kodo millet is one of the important minor millets cultivated in India, mostly confined to marginal lands and tribal regions but has high yield potential under good management. The grains are nutritious and have anti-oxidant properties besides having many medicinal properties. However, the consumption is often hindered by the condition called 'kodo poisoning' resulting from fungal contamination producing cyclopiazonic acid, a toxic fungal secondary metabolite. An attempt has been made here to review the limited information available on kodo poisoning, its causes and effects, and proposed management practices by which the contamination can be checked. Further research efforts are essential for identifying sources of natural resistance to fungal metabolite, induction of host resistance through antimicrobial compounds or microbial antagonism to the pathogens to achieve cleaner grains from this crop even under high humid and rainy conditions. By effective adoption of both pre- and post-harvest management the kodo millet grains can be made safe for human consumption and can be popularized as a nutritious grain.

Chicken heterophils extracellular traps act as early effectors against cyclopiazonic acid dependent upon NADPH oxidase, ROS and glycolysis.

Cyclopiazonic acid (CPA) is a secondary metabolite produced by Aspergillus and Penicillium, which is present in contaminated crops and food, causing severe toxicity to humans and animals. Heterophil extracellular traps (HETs) are a novel host innate immune mechanism of chicken heterophils against pathogen infection. However, whether CPA can cause immunotoxicity of heterophils on HETs release remains unclear. Here, we attempt to detect the effects of CPA on HETs release, and further investigate the molecular mechanisms underlying these processes. We exposed heterophils to 2.5, 5, 10 µM CPA for 90 min. The results showed that CPA induced the release of HETs in heterophils, consisting of DNA-modified citrullinated histone 3 and elastase. The quantitative analysis of HETs content was positively correlated with CPA concentration. CPA also promoted reactive oxygen species production and phosphorylation of ERK1/2 and p38. In addition, CPA-triggered HETs formation was reduced by NADPH oxidase, ERK1/2, and p38 signaling pathway and glycolysis inhibitors, indicating that CPA-induced HETs were related to the production of ROS dependent on NADPH oxidase, ERK1/2, and p38 signaling pathways, as well as glycolysis. Our study describes the underlying mechanism of CPA-induced HETs release, which may provide a further understanding of the immunotoxicology of CPA poisoning.

Cumulative Effects of Non-Aflatoxigenic Aspergillus flavus Volatile Organic Compounds to Abate Toxin Production by Mycotoxigenic Aspergilli.

Previously, authors reported that individual volatile organic compounds (VOCs) emitted by non-aflatoxigenic Aspergillus flavus could act as a mechanism of biocontrol to significantly reduce aflatoxins and cyclopiazonic acid (CPA) produced by toxigenic strains. In this study, various combinations and volumes of three mycotoxin-reductive VOCs (2,3-dihydrofuran, 3-octanone and decane) were assessed for their cumulative impacts on four Aspergillus strains (LA1-LA4), which were then analyzed for changes in growth, as well as the production of mycotoxins, including aflatoxins, CPA and multiple indole diterpenes. Fungal growth remained minimally inhibited when exposed to various combinations of VOCs. No single combination was able to consistently, or completely, inhibit aflatoxin or CPA across all toxigenic strains tested. However, the combination of 2,3-dihydrofuran and 3-octanone offered the greatest overall reductions in aflatoxin and CPA production. Despite no elimination of their production, findings showed that combining VOCs produced solely by non-aflatoxigenic A. flavus still inhibited several agriculturally important mycotoxins, including B and G aflatoxins and CPA. Therefore, other VOC combinations are worth testing as post-harvest biocontrol treatments to ensure the prolonged effectiveness of pre-harvest biocontrol efforts.

Coexposure of Cyclopiazonic Acid with Aflatoxin B1 Involved in Disrupting Amino Acid Metabolism and Redox Homeostasis Causing Synergistic Toxic Effects in Hepatocyte Spheroids.

Cyclopiazonic acid (CPA), an emerging toxin, has been found in various foods such as corn, peanuts, and figs. Aspergillus flavus can produce CPA, leading to coexposure with highly toxic aflatoxin B1 (AFB1), but the mechanism of their combined action is not clear. In this study, cocultured hepatocyte spheroids were used as the evaluation model, and two concentration settings of isotoxicity and different toxicity ratios were used to investigate the combined toxic effects. Metabolomics was subsequently used to analyze the potential mechanisms underlying the effects of their exposure. AFB1 and CPA might exhibit stronger cytotoxicity, with significant combined effects on mitochondrial morphology, activity, and reactive oxygen levels. The gene expression analysis revealed that the overexpression of AKT genes could mitigate the combined effects of AFB1 and CPA to some extent. Metabolomics analysis indicated that AFB1 and CPA significantly downregulated the metabolism of l-aspartate and antioxidant substances (e.g., penicillamine, myricetin, and ethanolamine). The pathway enrichment analysis also revealed a large impact on amino acid metabolism, likely affecting intracellular redox homeostasis. In addition, the presence of CPA affects intracellular glucose metabolism and lipid metabolism pathways. This study suggested a direction for future research on relevant toxic pathways and provided possible ideas for inhibiting or mitigating toxicity.

Aspergillus flavus La3279, a component strain of the Aflasafe™ biocontrol product, contains a partial aflatoxin biosynthesis gene cluster followed by a genomic region highly variable among A. flavus isolates.

Aspergillus flavus communities in agricultural fields consist of isolates with varying abilities to produce aflatoxins, which are highly toxic and carcinogenic to humans and animals. Biological control using multiple non-aflatoxigenic strains as a formulation to outcompete aflatoxigenic A. flavus has become a mainstream strategy. Aflasafe™ is a biocontrol product composed of four strains, Ka16127, La3279, La3304 and Og0222. It was first developed in Nigeria and is now widely used on maize and groundnut. In this study, phylogenetic analyses based on genome-wide single nucleotide polymorphisms showed that Ka16127 and La3304 were more closely related to each other than both were to La3279, and the three were distantly related to Og0222. Detailed molecular characterization of La3279 indicated that its genome, contradictory to the published report, lacked approximately half of the aflatoxin gene cluster as well as the entire cyclopiazonic acid gene cluster. La3279 was a member of the previously known "pattern E" group, which includes A. flavus and Aspergillus oryzae isolates that have the aforementioned deletion followed by a 3.8-kb "E block" sequence insertion. In comparison to the E block, corresponding regions in typical aflatoxigenic S-morphotype/genotype isolates as well as Ka16127 and La3304 were found to lack 1.1 kb of the 5' portion whereas L-morphotype/genotype isolates contained a complete nonhomologous region characterized by 2.5 copies of A. flavus telomeric repeat sequence at one end. Regions corresponding to the E block were highly variable and were useful for classifying A. flavus isolates into groups that mostly contained both mating types. The presence of both mating-type genes in genetically closely related A. flavus suggests a previously active sexual cycle. It could facilitate the development of a refined biocontrol strategy such as deploying biocontrol strains with the same mating-type that is predominant in a field A. flavus population.

Nematicidal Activity of Cyclopiazonic Acid Derived From Penicillium commune Against Root-Knot Nematodes and Optimization of the Culture Fermentation Process.

Among 200 fungal strains isolated from the soil, only one culture filtrate of Aspergillus flavus JCK-4087 showed strong nematicidal activity against Meloidogyne incognita. The nematicidal metabolite isolated from the culture filtrate of JCK-4087 was identified as cyclopiazonic acid (CPA). Because JCK-4087 also produced aflatoxins, six strains of Penicillium commune, which have been reported to be CPA producers, were obtained from the bank and then tested for their CPA productivity. CPA was isolated from the culture filtrate of P. commune KACC 45973. CPA killed the second-stage juveniles of M. incognita, M. hapla, and M. arearia with EC50-3 days 4.50, 18.82, and 60.51 µg mL-1, respectively. CPA also significantly inhibited egg hatch of M. incognita and M. hapla after a total of 28 days of treatment with the concentrations > 25 µg mL-1. The enhancement of CPA production by P. commune KACC 45973 was explored using an optimized medium based on Plackett-Burman design (PBD) and central composite design (CCD). The highest CPA production (381.48 µg mL-1) was obtained from the optimized medium, exhibiting an increase of 7.88 times when compared with that from potato dextrose broth culture. Application of the wettable power-type formulation of the ethyl acetate extract of the culture filtrate of KACC 45973 reduced gall formation and nematode populations in tomato roots and soils under greenhouse conditions. These results suggest that CPA produced by P. commune KACC 45973 can be used as either a biochemical nematicide or a lead molecule for developing chemical nematicides to control root-knot nematodes.