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Mold-ripened cheese acquires a distinctive aroma and texture from mold cultures that mature on a fresh cheese wheel. Owing to its high moisture content (aw = 0.95) and pliability, soft cheese is prone to contamination. Many contaminating mold species are unable to grow at colder temperatures, and the lactic acid produced by the cheese bacteria inhibits further infiltration. Thus, Camembert cheese is generally well protected against contamination by a wide range of species. In this study, cocultures of Penicillium camemberti and widely distributed mycotoxin-producing mold species were incubated on different types of agars, and purchased Camembert samples were deliberately contaminated with mycotoxin-producing mold species capable of growing at both 25 °C and 4 °C. The production of mycotoxins was then monitored by the extraction of the metabolites and their subsequent measurement by means of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based targeted metabolite profiling approach. The production of cyclopiazonic acid (CPA) was highly dependent on the species cocultivated with Penicillium camemberti, the temperature and the substrate. Contamination of Camembert cheese with Penicillium chrysogenum, Mucor hiemalis, or Penicillium glabrum induced CPA production at 25 °C. Although mold growth on cheese was not always evident on biofilms for certain cultures, except for Penicillium citrinum, which stained the monosaccharide agar yellow, mycotoxins were detected in many agar and cheese samples, as in all monosaccharide agar samples. In conclusion, cheese should be immediately discarded upon the first appearance of mold. [ABSTRACT FROM AUTHOR]

Some Penicillium strains used in cheese ripening produce emerging mycotoxins, notably roquefortine C (ROQC) and cyclopiazonic acid (CPA), as well as enniatins (ENNs) and beauvericin (BEA). Co-occurrence of these mycotoxins in natural samples has been reported worldwide, however, most studies focus on the toxicity of a single mycotoxin. In the present study, the effects of ROQC and CPA alone and in combination with BEA and ENNs A, A1, B, and B1 were analysed in human neuroblastoma cells. ROQC and CPA reduced cell viability, with IC50 values of 49.5 and 7.3 µM, respectively, and induced caspase-8-mediated apoptosis. When ROQC and CPA were binary combined with ENNs, an enhancement of their individual effects was observed. Furthermore, a clear synergism was produced when ROQC and CPA were mixed with the four ENNs. An additive effect was also described for the combination of CPA + ENNs (A, A1, B, B1) + BEA. Finally, the effects of commercial cheese extracts containing the mentioned mycotoxins were evaluated, finding a strong reduction in cell viability. These results suggest that the co-occurrence of emerging mycotoxins in natural matrices could pose a potential health risk. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A novel co-bonded octyl and pyridine silica (OPS) sorbent was prepared and applied for the solid phase extraction (SPE) of cyclopiazonic acid (CPA, a type of mycotoxin) in feed and agricultural products for the first time. A simple mixed-ligand one-pot reaction strategy was employed for OPS sorbent preparation. Nitrogen adsorption–desorption measurements, elemental analysis (EI), thermal gravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FT-IR) analysis demonstrated the successful immobilization of octyl and quaternary ammonium groups onto the surface of silica gel. The large specific surface area, high-density functional groups, and mixed-mode anion-exchange characteristics of these silica particles made them the ideal material for the efficient extraction of CPA. Additionally, the OPS sorbents displayed excellent batch-to-batch reproducibility, satisfactory reusability, and low cost. The SPE parameters were optimized to explore the ionic and hydrophobic interactions between CPA and the functional groups, and the ultra-high performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UPLC-MS/MS) parameters were optimized to obtain a desirable extraction efficiency and high sensitivity to CPA. Meanwhile, the OPS sorbent presented a satisfactory extraction selectivity and low matrix effect. Under the optimized conditions, our developed CPA detection method was used to determine CPA level in rice, wheat flour, corn flour, peanut, and feed samples, exhibiting a lower detection limit, better linearity, higher sensitivity, and satisfactory extraction recovery rate than previously reported methods. Therefore, our method can be preferentially used as a method for the detection of CPA in agricultural products and feeds. [ABSTRACT FROM AUTHOR]

Introduction: The loose-patch clamp technique was first developed and used in native amphibian skeletal muscle (SkM), offering useful features complementing conventional sharp micro-electrode, gap, or conventional patch voltage clamping. It demonstrated the feedback effects of pharmacological modification of ryanodine receptor (RyR)-mediated Ca2+ release on the Na+ channel (Nav1.4) currents, initiating excitation–contraction coupling in native murine SkM. The effects of the further RyR and Ca2+-ATPase (SERCA) antagonists, dantrolene and cyclopiazonic acid (CPA), additionally implicated background tubular-sarcoplasmic Ca2+ domains in these actions. Materials and methods: We extend the loose-patch clamp approach to ion current measurements in murine hippocampal brain slice cornu ammonis-1 (CA1) pyramidal neurons. We explored the effects on Na+ currents of pharmacologically manipulating RyR and SERCA-mediated intracellular store Ca2+ release and reuptake. We adopted protocols previously applied to native skeletal muscle. These demonstrated Ca2+-mediated feedback effects on the Na+ channel function. Results: Experiments applying depolarizing 15 ms duration loose-patch clamp steps to test voltages ranging from −40 to 120 mV positive to the resting membrane potential demonstrated that 0.5 mM caffeine decreased inward current amplitudes, agreeing with the previous SkM findings. It also decreased transient but not prolonged outward current amplitudes. However, 2 mM caffeine affected neither inward nor transient outward but increased prolonged outward currents, in contrast to its increasing inward currents in SkM. Furthermore, similarly and in contrast to previous SkM findings, both dantrolene (10 μM) and CPA (1 μM) pre-administration left both inward and outward currents unchanged. Nevertheless, dantrolene pretreatment still abrogated the effects of subsequent 0.5- and 2-mM caffeine challenges on both inward and outward currents. Finally, CPA abrogated the effects of 0.5 mM caffeine on both inward and outward currents, but with 2 mM caffeine, inward and transient outward currents were unchanged, but sustained outward currents increased. Conclusion: We, thus, extend loose-patch clamping to establish pharmacological properties of murine CA1 pyramidal neurons and their similarities and contrasts with SkM. Here, evoked though not background Ca2+-store release influenced Nav and Kv excitation, consistent with smaller contributions of background store Ca2+ release to resting [Ca2+]. This potential non-canonical mechanism could modulate neuronal membrane excitability or cellular firing rates. [ABSTRACT FROM AUTHOR]

Maize (Zea mays L.) is one of the most widely planted crops globally with Argentina leading world production and exportation. Santiago del Estero province, east of Tucumán and north of Córdoba encompasses eight agro-climatic zones in the Chaco Semi-arid region, agro-ecologically characterized by a wide temperature range and frequent drought periods that expose the crop to pathogens, particularly Aspergillus flavus. This pathogen is responsible for ear rot and grain contamination with mycotoxins such as aflatoxin B1 and cyclopiazonic acid. This study obtained fungal isolates from ears of maize and characterized them according to toxigenic capability and morphotype of sclerotia (S < 400 μm, associated with high levels of aflatoxins and L > 400 μm, related to variable levels of aflatoxins). In addition, those not producing aflatoxins were studied to determine phylogenetic relationships based on sequences of a segment of the CaM gene. Fifty-eight isolates were obtained in eight localities representing each agro-climatic zone, 30 of which were non-aflatoxigenic, 28 aflatoxigenic, and all producers of ciclopiazonic acid. Six isolates did not produce sclerotia, 51 were L and only one was S, the latter being a non-producer of aflatoxins. The number of sclerotia was positively correlated with the production of aflatoxin B1, while size was negatively correlated. The CaM gene sequences corroborated that the isolates belonged to the A. flavus clade and the high nucleotide similarity among them (99.4% to 100%) revealed almost zero genetic diversity in this geographic region. No significant differences were observed in the proportion of isolates between growing seasons or among agroclimatic districts. This research revealed characteristics of fungus populations in this agricultural region of north Argentina. Highlights: • Aspergillus flavus isolates from ears of maize were characterized according to toxigenic capability, morphotype of sclerotia and phylogenetic relationships. • Fifty-eight isolates were obtained, 30 non-aflatoxigenic, 28 aflatoxigenic, and all producers of ciclopiazonic acid. • Six isolates did not produce sclerotia, 51 were L and only one was S, the latter being a non-producer of aflatoxins. • All isolates belonged to the flavus clade. High nucleotide similarity among the isolates (99.4% to 100%) revealed almost zero genetic diversity in the region.

(1) Background: Food and feed safety legislation does not concern all the mycotoxins generated by Penicillium and Aspergillus spp. Certain mycotoxins, including mycophenolic acid (MPA), cyclopiazonic acid (CPA), penicillic acid (PA), roquefortine C (ROQ C), and gliotoxin (GLI), are regarded as having lower toxicity levels, and hence are not included in food and feed legislation. It is obvious that xenobiotics, including mycotoxins, exert synergistic harmful health effects on human and animal when exposed through food and feed. (2) Methods: The presence of these substances in maize and wheat grown in Albania across two consecutive harvesting seasons was investigated by liquid chromatography and mass spectrometry (LC-MS/MS). (3) Results: The findings indicated the presence of these mycotoxins in maize grain but not in wheat grain. In the 2014 season, they exhibited a higher contamination incidence than in the 2015 season. The most commonly detected mycotoxin was MPA, followed by CPA and ROQ C toxin, while PA and GLI were not detected. The MPA revealed a concentration range of 72.9–3447 μg/kg, with a mean value of 1064 μg/kg. Mycophenolic acid was detected in the maize samples collected during the 2015 season. (4) Conclusions: These findings suggest that focusing the investigation only on “controlled” mycotoxins will not produce a proper risk assessment and may not adequately address the possible harmful impacts of mycotoxins on human and animal health due to mycotoxins’ co-occurrence.

Cyclopiazonic acid (CPA) is an indole-tetramic acid neurotoxin produced by Aspergillus and Penicillium genera present mainly in fruit, cereals and nuts. This study compares the cytotoxicity produced by CPA after 24, 48 and 72 h of exposure using both monolayers and 3D spheroids in human neuroblastoma SH-SY5Y cells. Furthermore, CPA toxicokinetics was evaluated using in silico models. Cytotoxicity increased dose- and time-dependently, as shown by the MTT assay. The lowest CPA IC50 values were found in the monolayer study compared to the 3D spheroids at all exposure times (24 h: 864.01 vs. 1132; 48 h: 437 vs. 1069; 72 h: 392 vs. 567 nM). The CPA exposure on SH-SY5Y spheroid organization and morphology was also studied. Morphological changes, including spheroid disaggregation, were observed after mycotoxin exposure. The in silico methods, SwissADME and admetSAR, were used for short and full ADMEt profiles of CPA. The ADMEt predictive profile shows high gastrointestinal absorption and ability to penetrate the blood–brain barrier. Including in silico studies emphasizes the comprehensive approach to understanding mycotoxin toxicity and risk assessment. By combining in vitro 3D spheroid models with computational simulations, this study aims to provide a holistic perspective on the effects of CPA, enhancing the accuracy and relevance of our findings.

Mold-ripened cheese acquires a distinctive aroma and texture from mold cultures that mature on a fresh cheese wheel. Owing to its high moisture content (aw = 0.95) and pliability, soft cheese is prone to contamination. Many contaminating mold species are unable to grow at colder temperatures, and the lactic acid produced by the cheese bacteria inhibits further infiltration. Thus, Camembert cheese is generally well protected against contamination by a wide range of species. In this study, cocultures of Penicillium camemberti and widely distributed mycotoxin-producing mold species were incubated on different types of agars, and purchased Camembert samples were deliberately contaminated with mycotoxin-producing mold species capable of growing at both 25 °C and 4 °C. The production of mycotoxins was then monitored by the extraction of the metabolites and their subsequent measurement by means of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based targeted metabolite profiling approach. The production of cyclopiazonic acid (CPA) was highly dependent on the species cocultivated with Penicillium camemberti, the temperature and the substrate. Contamination of Camembert cheese with Penicillium chrysogenum, Mucor hiemalis, or Penicillium glabrum induced CPA production at 25 °C. Although mold growth on cheese was not always evident on biofilms for certain cultures, except for Penicillium citrinum, which stained the monosaccharide agar yellow, mycotoxins were detected in many agar and cheese samples, as in all monosaccharide agar samples. In conclusion, cheese should be immediately discarded upon the first appearance of mold.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Maize (Zea mays L.) is one of the most widely planted crops globally with Argentina leading world production and exportation. Santiago del Estero province, east of Tucumán and north of Córdoba encompasses eight agro-climatic zones in the Chaco Semi-arid region, agro-ecologically characterized by a wide temperature range and frequent drought periods that expose the crop to pathogens, particularly Aspergillus flavus. This pathogen is responsible for ear rot and grain contamination with mycotoxins such as aflatoxin B1 and cyclopiazonic acid. This study obtained fungal isolates from ears of maize and characterized them according to toxigenic capability and morphotype of sclerotia (S < 400 µm, associated with high levels of aflatoxins and L > 400 µm, related to variable levels of aflatoxins). In addition, those not producing aflatoxins were studied to determine phylogenetic relationships based on sequences of a segment of the CaM gene. Fifty-eight isolates were obtained in eight localities representing each agro-climatic zone, 30 of which were non-aflatoxigenic, 28 aflatoxigenic, and all producers of ciclopiazonic acid. Six isolates did not produce sclerotia, 51 were L and only one was S, the latter being a non-producer of aflatoxins. The number of sclerotia was positively correlated with the production of aflatoxin B1, while size was negatively correlated. The CaM gene sequences corroborated that the isolates belonged to the A. flavus clade and the high nucleotide similarity among them (99.4% to 100%) revealed almost zero genetic diversity in this geographic region. No significant differences were observed in the proportion of isolates between growing seasons or among agroclimatic districts. This research revealed characteristics of fungus populations in this agricultural region of north Argentina. [ABSTRACT FROM AUTHOR]

IntroductionThe loose-patch clamp technique was first developed and used in native amphibian skeletal muscle (SkM), offering useful features complementing conventional sharp micro-electrode, gap, or conventional patch voltage clamping. It demonstrated the feedback effects of pharmacological modification of ryanodine receptor (RyR)-mediated Ca2+ release on the Na+ channel (Nav1.4) currents, initiating excitation–contraction coupling in native murine SkM. The effects of the further RyR and Ca2+-ATPase (SERCA) antagonists, dantrolene and cyclopiazonic acid (CPA), additionally implicated background tubular-sarcoplasmic Ca2+ domains in these actions.Materials and methodsWe extend the loose-patch clamp approach to ion current measurements in murine hippocampal brain slice cornu ammonis-1 (CA1) pyramidal neurons. We explored the effects on Na+ currents of pharmacologically manipulating RyR and SERCA-mediated intracellular store Ca2+ release and reuptake. We adopted protocols previously applied to native skeletal muscle. These demonstrated Ca2+-mediated feedback effects on the Na+ channel function.ResultsExperiments applying depolarizing 15 ms duration loose-patch clamp steps to test voltages ranging from −40 to 120 mV positive to the resting membrane potential demonstrated that 0.5 mM caffeine decreased inward current amplitudes, agreeing with the previous SkM findings. It also decreased transient but not prolonged outward current amplitudes. However, 2 mM caffeine affected neither inward nor transient outward but increased prolonged outward currents, in contrast to its increasing inward currents in SkM. Furthermore, similarly and in contrast to previous SkM findings, both dantrolene (10 μM) and CPA (1 μM) pre-administration left both inward and outward currents unchanged. Nevertheless, dantrolene pretreatment still abrogated the effects of subsequent 0.5- and 2-mM caffeine challenges on both inward and outward currents. Finally, CPA abrogated the effects of 0.5 mM caffeine on both inward and outward currents, but with 2 mM caffeine, inward and transient outward currents were unchanged, but sustained outward currents increased.ConclusionWe, thus, extend loose-patch clamping to establish pharmacological properties of murine CA1 pyramidal neurons and their similarities and contrasts with SkM. Here, evoked though not background Ca2+-store release influenced Nav and Kv excitation, consistent with smaller contributions of background store Ca2+ release to resting [Ca2+]. This potential non-canonical mechanism could modulate neuronal membrane excitability or cellular firing rates.

AbstractAspergillus 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.

A novel co-bonded octyl and pyridine silica (OPS) sorbent was prepared and applied for the solid phase extraction (SPE) of cyclopiazonic acid (CPA, a type of mycotoxin) in feed and agricultural products for the first time. A simple mixed-ligand one-pot reaction strategy was employed for OPS sorbent preparation. Nitrogen adsorption–desorption measurements, elemental analysis (EI), thermal gravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FT-IR) analysis demonstrated the successful immobilization of octyl and quaternary ammonium groups onto the surface of silica gel. The large specific surface area, high-density functional groups, and mixed-mode anion-exchange characteristics of these silica particles made them the ideal material for the efficient extraction of CPA. Additionally, the OPS sorbents displayed excellent batch-to-batch reproducibility, satisfactory reusability, and low cost. The SPE parameters were optimized to explore the ionic and hydrophobic interactions between CPA and the functional groups, and the ultra-high performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UPLC-MS/MS) parameters were optimized to obtain a desirable extraction efficiency and high sensitivity to CPA. Meanwhile, the OPS sorbent presented a satisfactory extraction selectivity and low matrix effect. Under the optimized conditions, our developed CPA detection method was used to determine CPA level in rice, wheat flour, corn flour, peanut, and feed samples, exhibiting a lower detection limit, better linearity, higher sensitivity, and satisfactory extraction recovery rate than previously reported methods. Therefore, our method can be preferentially used as a method for the detection of CPA in agricultural products and feeds.

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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

To investigate the metabolic transformation of cyclopiazonic acid (CPA) in the liver of different species and to supplement accurate risk assessment information, the metabolism of CPA in liver microsomes from four animals and humans was studied using the ultra–high–performance liquid chromatography–quadrupole/time–of–flight method. The results showed that a total of four metabolites were obtained, and dehydrogenation, hydroxylation, methylation, and glucuronidation were identified as the main metabolic pathways of CPA. Rat liver microsomes exhibited the highest metabolic capacity for CPA, with dehydrogenated (C 20 H 18 N 2 O 3) and glucuronic acid-conjugated (C 26 H 28 N 2 O 10) metabolites identified in all liver microsomes except chicken, indicating significant species metabolic differences. Moreover, C 20 H 18 N 2 O 3 was only detected in the incubation system with cytochromes P450 3A4 (CYP3A4). The hydroxylated (C 20 H 20 N 2 O 4) and methylated (C 21 H 22 N 2 O 3) metabolites were detected in all incubation systems except for the CYP2C9, with CYP3A4 demonstrating the strongest metabolic capacity. The "cocktail" probe drug method showed that CPA exhibited a moderate inhibitory effect on the CYP3A4 (IC 50 value = 8.658 μM), indicating that the substrate had a negative effect on enzyme activity. Our results provide new insights to understand the biotransformation profile of CPA in animals and humans. [Display omitted] • Four metabolites of cyclopiazonic acid (CPA) were first identified in liver microsome system. • Liver microsomes from mice has the highest capacity for CPA metabolism. • CYP3A4 is the main metabolic enzyme of CPA. • CPA exhibited a moderate inhibitory effect on the CYP3A4 enzyme. [ABSTRACT FROM AUTHOR]

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 β-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. [ABSTRACT FROM AUTHOR]

A new oxygenated tricyclic cyclopiazonic acid (CPA) alkaloid, asperorydine Q (1), along with seven known compounds, namely, asperorydines O (2) and J (3), speradine H (4), cyclopiamides A (5) and H (6), saadamysin (7), and pyrazinemethanol (8), were isolated from the coral-associated Aspergillus flavus GXIMD 02503. The structures were elucidated by physicochemical properties and comprehensive spectroscopic data analysis. Compounds 1–5 and 7–8 exhibited potent inhibition of lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) with the IC50 values ranging from 6.5 to 21.8 µmol L−1. In addition, the most potent one, pyrazinemethanol (8), dose-dependently suppressed receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation without obvious cytotoxicity in bone marrow macrophages cells (BMMCs), suggesting it is a promising lead compound for the treatment of osteolytic diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Mycotoxins may be present in traditional meat products (TMPs) due to production of moulds overgrowing their surface during ripening or due to contaminated spices or meat used in their production. The aim of this study was to evaluate the human exposure to the mycotoxins, including aflatoxins (AFs) B 1 , B 2 , G 1 and G 2 (AFB 1 , AFB 2 , AFG 1 , AFG 2), ochratoxin A (OTA), cyclopiazonic acid (CPA), citrinin (CIT) and sterigmatocystin (STC), which may be present in TMPs, using 288 samples available on the market in relation to the consumption data of Croatian citizens. AFs and CIT weren't detected in any sample, so the exposure to these mycotoxins in TMP can be considered negligible. The determined exposure to STC was 0.011–0.020 ng/kg bw/day far below the exposure of low health concern set by the European Food Safety Authority (EFSA). The determined exposure to CPA was up to 5.75 ng/kg bw/day, but there isn't yet a health-based guideline value (HBGV) for the consumer risk associated with CPA in food. The determined exposure to OTA was 0.043–0.121 ng/kg bw/day, and according to the margin of exposure (MoE) approach, in the case of the upper bound (UB) scenario, for neoplastic effects a low concern was identified in 0.12% of consumers, while for non-neoplastic effects a potential health concern was identified in 4.15% of consumers. Among the TMP categories, dry-fermented sausages contributed the most to consumer exposure for all three mycotoxins detected. Results indicate that mycotoxins presence should be monitored over a longer period of time where different data can be expected in order to observe the trend of human exposure. • Human exposure to eight mycotoxins from traditional meat products were determined. • Exposure to citrinin and aflatoxins from meat products can be found negligible. • Ochratoxin A contributed to the exposure of consumers of traditional meat products. • The group of sausages contributed the most to consumer exposure to mycotoxins. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Twelve indole alkaloids, including α-cyclopiazonic acid (CPA) (1), nine 2-oxo indole CPA derivatives (2–10), and two open-ring indole CPA derivatives (11 and 12), were isolated from the fermentation broth of a deep-sea derived fungus Aspergillus sp. SCSIO 41024. Their structures and absolute configurations were elucidated mainly by using extensive NMR spectroscopic, mass spectrometric and single crystal X-ray diffraction analysis. To the best of our knowledge, the crystallographic data of 3 and 7 were firstly reported, and the absolute configuration of 3 was confirmed for the first time by the single crystal X-ray diffraction analysis. Most isolated compounds were tested for their antimicrobial, antitumor and radical scavenging activities. In addition, compounds 1, 2 and 11 showed moderate antioxidative activity against DPPH with IC50 values of 190.1, 31.9, 228.4 μg/mL, respectively. [ABSTRACT FROM AUTHOR]

Species belonging to Aspergillus section Flavi occur naturally in crops and can cause food spoilage and/or toxin production. The aim of this study was to determine the occurrence and diversity of the species of Aspergillus section Flavi found in wheat and sorghum at harvest time and during silage storage, and to evaluate the toxigenic potential of the isolates to determine the contamination risk of mycotoxins in grains. Strains from Aspergillus flavus and Aspergillus parasiticus were found based on multi-gene phylogenetic analyses. This is the first report on the presence of A. parasiticus in wheat from Uruguay. Of the 80 isolates Aspergillus section Flavi, 30% produced aflatoxins (AFs), mainly type B1, and 25% produced cyclopiazonic acid (CPA). Within the isolates from wheat samples, 35% were AFs producers and 27.5% were CPA producers. Among the Aspergillus section Flavi isolates from sorghum, 25% were AFs producers while 22.5% were CPA producers. This work contributes to the knowledge of the species in crops and helps define appropriate strategies for the prevention and control of contamination with AFs and CPA by Aspergillus section Flavi fungi.

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.

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. [ABSTRACT FROM AUTHOR]

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.

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 β-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.

Aspergillus section Flavi includes some of the most famous mycotoxin producing filamentous fungi known to mankind. In recent years a number of new species have been included in section Flavi, however these species have been much less studied from a chemical point of view. In this study, we explored one representative strain of a total of 28 fungal species in section Flavi by systematically evaluating the relationship between taxonomy and secondary metabolites with LC-MS/MS analysis for the first time and dereplication through an in-house database and the Global Natural Product Social Molecular Networking (GNPS) platform. This approach allowed rapid identification of two new cyclopiazonic acid producers (A. alliaceus and A. arachidicola) and two new tenuazonic acid producers (A. arachidicola and A. leporis). Moreover, for the first time we report species from section Flavi to produce fumifungin and sphingofungins B-D. Altogether, this study emphasizes that the chemical diversity of species in genus Aspergillus section Flavi is larger than previously recognized, and especially that understudied species are prolific producers of important mycotoxins such as fumi- and sphingofungins not previously reported from this section. Furthermore, our work demonstrates Global Natural Product Social (GNPS) Molecular Networking as a powerful tool for large-scale chemotaxonomic analysis of closely related species in filamentous fungi.

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.

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.

Purposes: Since the role of store-operated calcium entry (SOCE) in endothelium-dependent hyperpolarization (EDH)-mediated vasorelaxation of mesenteric arteries in health and colitis is not fully understood, cyclopiazonic acid (CPA), a specific inhibitor of the sarco(endo) plasmic reticulum calcium-ATPases (SERCA), was used as a SOCE activator to investigate its role in normal mice and its alteration in colitis mice.Methods: The changes in Ca2+ signaling in vascular endothelial cells (VEC) were examined by single cell Ca2+ imaging and tension of mesenteric arteries in response to CPA were examined using Danish DMT520A microvascular measuring system.Results: CPA activated the SOCE through depletion of the endoplasmic reticulum (ER) Ca2+ in endothelial cells. CPA had a concentration-dependent vasorelaxing effect in endothelium-intact mesenteric arteries, which was lost after endothelial removal. Both nitric oxide (NO) and prostacyclin (PGI2) inhibitors did not affect CPA-induced vasorelaxation; however, after both NO and PGI2 were inhibited, KCa channel blocker [10 mM tetraethylammonium chloride (TEA)] inhibited CPA-induced vasorelaxation while KCa channel activator (0.3 μM SKA-31) promoted it. Two SOCE blockers [30 μM SKF96365 and 100 μM flufenamic acid (FFA)], and an Orai channel blocker (30 μM GSK-7975A) inhibited this vasorelaxation. The inhibition of both Na+/K+-ATPase (NKA) and Na+/Ca2+-exchange (NCX) also inhibited CPA-induced vasorelaxation. Finally, the CPA involved in EDH-induced vasorelaxation by the depletion of ER Ca2+ of mesenteric arteries was impaired in colitis mice.Conclusion: Depletion of ER Ca2+ by CPA induces a vasorelaxation of mesenteric arteries that is mediated through EDH mechanism and invokes the activation of SOCE. The CPA-induced endothelium-dependent dilation is impaired in colitis which may limit blood perfusion to the intestinal mucosa.

Cyclopiazonic acid (CPA) is an indole tetrameric acid mycotoxin. This study carried out to investigate the potential effects of CPA on male reproductive system. In the current study, 40 adult male mice were divided into five groups (n = 8). The control group did not expose to CPA, while animals in vehicle‐received group; received the CPA‐solvent (0.05% dimethyl sulfoxide) and the animals of third, fourth, and fifth groups received CPA 0.03, 0.06, and 0.12 mg/kg, body weight, respectively for 28 days. Morphometric and morphological deviations, spermatogenesis indices, malondialdehyde (MDA) content, total thiol molecules (TTM) concentration, total antioxidant capacity (TAC), protein carbonylation rate (CO), and nitric oxide (NO) concentration were examined. The expression changes of apoptotic genes (P53, Bcl‐2, and Caspase III) at mRNA level were also evaluated by qPCR technique. Reduction in the Leydig and Sertoli cells population, diameter of seminiferous tubules, and spermatogenesis parameters was significant only in the group that received the highest dose of CPA. An increase in the level of MDA, NO, and CO in testicular tissue and reduction of TAC and TTM were observed in the CPA‐exposed groups. Significant up‐regulation (p <.05) in the expression of P53 and Caspase III genes and down‐regulation of Bcl‐2 gene were found in the CPA‐received groups. These results are indicating the detrimental effects of CPA on the testicles, which may attribute to the CPA‐induced oxidative stress and apoptosis. Moreover, results also help to understand a serious concern about the presence of CPA in foods as a potential risk factor in male‐related infertility. [ABSTRACT FROM AUTHOR]

Purposes : Since the role of store-operated calcium entry (SOCE) in endothelium-dependent hyperpolarization (EDH)-mediated vasorelaxation of mesenteric arteries in health and colitis is not fully understood, cyclopiazonic acid (CPA), a specific inhibitor of the sarco(endo) plasmic reticulum calcium-ATPases (SERCA), was used as a SOCE activator to investigate its role in normal mice and its alteration in colitis mice. Methods : The changes in Ca2+ signaling in vascular endothelial cells (VEC) were examined by single cell Ca2+ imaging and tension of mesenteric arteries in response to CPA were examined using Danish DMT520A microvascular measuring system. Results : CPA activated the SOCE through depletion of the endoplasmic reticulum (ER) Ca2+ in endothelial cells. CPA had a concentration-dependent vasorelaxing effect in endothelium-intact mesenteric arteries, which was lost after endothelial removal. Both nitric oxide (NO) and prostacyclin (PGI2) inhibitors did not affect CPA-induced vasorelaxation; however, after both NO and PGI2 were inhibited, KCa channel blocker [10 mM tetraethylammonium chloride (TEA)] inhibited CPA-induced vasorelaxation while KCa channel activator (0.3 μM SKA-31) promoted it. Two SOCE blockers [30 μM SKF96365 and 100 μM flufenamic acid (FFA)], and an Orai channel blocker (30 μM GSK-7975A) inhibited this vasorelaxation. The inhibition of both Na+/K+-ATPase (NKA) and Na+/Ca2+-exchange (NCX) also inhibited CPA-induced vasorelaxation. Finally, the CPA involved in EDH-induced vasorelaxation by the depletion of ER Ca2+ of mesenteric arteries was impaired in colitis mice. Conclusion : Depletion of ER Ca2+ by CPA induces a vasorelaxation of mesenteric arteries that is mediated through EDH mechanism and invokes the activation of SOCE. The CPA-induced endothelium-dependent dilation is impaired in colitis which may limit blood perfusion to the intestinal mucosa. [ABSTRACT FROM AUTHOR]

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.

Pre-blockade of the sarco-endoplasmic reticulum (ER) calcium ATPase (SERCA) with irreversible thapsigargin depresses exocytosis in adrenal bovine chromaffin cells (BCCs). Distinct expression of voltage-dependent Ca2+-channel subtypes and of the Ca2+-induced Ca2+ release (CICR) mechanism in BCCs versus mouse chromaffin cells (MCCs) has been described. We present a parallel study on the effects of the acute SERCA blockade with reversible cyclopizonic acid (CPA), to repeated pulsing with acetylcholine (ACh) at short (15 s) and long intervals (60 s) at 37 °C, allowing the monitoring of the initial size of a ready-release vesicle pool (RRP) and its depletion and recovery in subsequent stimuli. We found (i) strong depression of exocytosis upon ACh pulsing at 15-s intervals and slower depression at 60-s intervals in both cell types; (ii) facilitation of exocytosis upon acute SERCA inhibition, with back to depression upon CPA washout in MCCs; (iii) blockade of exocytosis upon acute SERCA inhibition and pronounced rebound of exocytosis upon CPA washout in BCCs; (iv) basal [Ca2+]c elevation upon stimulation with ACh at short intervals (but not at long intervals) in both cell types; and (v) augmentation of basal [Ca2+]c and inhibition of peak [Ca2+]c amplitude upon CPA treatment in both cell types, with milder effects upon stimulation at 60-s intervals. These results are compatible with the view that while in MCCs the uptake of Ca2+ via SERCA contributes to the mitigation of physiological ACh triggered secretion, in BCCs the uptake of Ca2+ into the ER facilitates such responses likely potentiating a Ca2+-induced Ca2+ release mechanism. These drastic differences in the regulation of ACh-triggered secretion at 37 °C may help to understand different patterns of the regulation of exocytosis by the circulation of Ca2+ at a functional ER Ca2+ store. [ABSTRACT FROM AUTHOR]

Species belonging to Aspergillus section Flavi occur naturally in crops and can cause food spoilage and/or toxin production. The aim of this study was to determine the occurrence and diversity of the species of Aspergillus section Flavi found in wheat and sorghum at harvest time and during silage storage, and to evaluate the toxigenic potential of the isolates to determine the contamination risk of mycotoxins in grains. Strains from Aspergillus flavus and Aspergillus parasiticus were found based on multi-gene phylogenetic analyses. This is the first report on the presence of A. parasiticus in wheat from Uruguay. Of the 80 isolates Aspergillus section Flavi, 30% produced aflatoxins (AFs), mainly type B1, and 25% produced cyclopiazonic acid (CPA). Within the isolates from wheat samples, 35% were AFs producers and 27.5% were CPA producers. Among the Aspergillus section Flavi isolates from sorghum, 25% were AFs producers while 22.5% were CPA producers. This work contributes to the knowledge of the species in crops and helps define appropriate strategies for the prevention and control of contamination with AFs and CPA by Aspergillus section Flavi fungi. [ABSTRACT FROM AUTHOR]

• CPA show a reduction action on SAN automaticity and block I f directly at high concentration of 10 μM. • A direct inhibitory effect of CPA on I f in SAN cells is concentration- and time-dependent. • CPA slow down the steady-state activation current and downregulate the protein expression of I f. • CPA may be a new potential agent to improve heart rate. Objective: The spontaneous action potential of isolated sinoatrial node (SAN) cells is regulated by a coupled-clock system of two clocks: the calcium clock and membrane clock. However, it remains unclear whether calcium clock inhibitors have a direct effect on the membrane clock. The purpose of this study was to investigate the direct effect of cyclopiazonic acid (CPA), a selective calcium clock inhibitor, on the function of the membrane clock of SAN cells. Methods: at SAN cells were isolated by trypsinization and identified based on morphology and electrophysiology. I f and HCN currents were recorded via patch clamp technique. The expression of the HCN channel protein was determined by Western blotting analysis. Results: The diastolic depolarization rate of spontaneous action potentials and the current densities of I f were reduced by exposure to 10 μM CPA. The inhibitory effect of CPA was concentration-dependent with an IC 50 value of 16.3 μM and a Hill coefficient of 0.98. The effect of CPA on I f current was also time-dependent, and the I f current amplitude was partially restored after washout. Furthermore, the steady-state activation curve of the I f current was shifted to a negative potential, indicating that channel activation slowed down. Finally, the protein expression of HCN4 in HEK293 cells was markedly downregulated by CPA. Conclusions: These results indicate that the direct inhibition effect of CPA on the I f current in SAN cells is both concentration- and time-dependent. The underlying mechanisms may involve slowing down steady-state activation and the downregulation of pacemaker channel protein expression. [ABSTRACT FROM AUTHOR]

Aspergillus flavus Link is frequently isolated fungi in environmental studies. This species produces sclerotia, a phenotypic feature that has been related to the production of aflatoxins and cyclopiazonic acid. The objective of this study was to analyze the characteristics of the sclerotia of environmental strains of A. flavus due to their relationship with mycotoxin biogenesis. The sclerotia of 14 strains from the Microbial Culture Collection of the Faculty of Biology were measured and the qualitative detection of aflatoxins and cyclopiazonic acid was performed. All sclerotia produced were of the L morphotype (> 400 µm) and the presence of aflatoxins or cyclopiazonic acid was not detected. Knowledge of the morphological characteristics of these resistance structures and their relationship to the biogenesis of mycotoxins in populations of this species contributes to knowledge of their diversity in the neotropics. [ABSTRACT FROM AUTHOR]