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Topical Collection "Aflatoxins"

A topical collection in Toxins (ISSN 2072-6651). This collection belongs to the section "Mycotoxins".

Editor

Collection Editor
Prof. Dr. Shohei Sakuda

Department of Biosciences, Faculty of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya City, Tochigi Prefecture 320-8551, Japan
E-Mail
Phone: +81-28-627-7213
Fax: +81-28-627-7187
Interests: bioorganic chemistry of bioactive natural compounds

Topical Collection Information

Dear Colleagues,

Aflatoxin contamination in important crops occurs worldwide. The health of around five hundred million people is being affected by aflatoxin contamination and many liver cancer cases are caused by aflatoxin intake. Economic loss due to aflatoxins is also very severe. These facts show that it is absolutely necessary to prevent and reduce aflatoxin contamination in food and feed. However, few practical methods are available for aflatoxin control at present. In this Special Issue, manuscripts that provide data useful for the prevention, reduction, and detoxification of aflatoxin contamination are invited. Surveys and analyses of aflatoxin contamination and aflatoxigenic fungi are important as basic information for aflatoxin control. Understanding the production mechanisms and biosyntheses of aflatoxins by aflatoxigenic fungi is important for determining the optimal targets of methods for controlling aflatoxin contamination. Microorganisms, which are effective for aflatoxin control, are useful as biocontrol agents. Chemicals and enzymes are useful for the prevention and detoxification of aflatoxin contamination. Natural products that are effective for aflatoxin control are important because they are kind to nature. All papers dealing with these objectives are welcome.

Prof. Dr. Shohei Sakuda
Collection Editor

Manuscript Submission Information

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Please visit the Instructions for Authors page before submitting a manuscript. The article processing charge (APC) for publication in this open access journal is 1800 CHF (Swiss Francs).

Related Special Issues

Aflatoxins

Keywords

  • contamination
  • analysis
  • production mechanism
  • biosynthesis
  • control
  • prevention
  • detoxification
  • microorganisms
  • natural products
  • inhibitors
  • toxicity

Published Papers (46 papers)

2019

Jump to: 2018, 2017, 2016, 2015

Open AccessArticle Multiple CH/π Interactions Maintain the Binding of Aflatoxin B1 in the Active Cavity of Human Cytochrome P450 1A2
Received: 27 January 2019 / Revised: 3 March 2019 / Accepted: 8 March 2019 / Published: 12 March 2019
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Abstract
Human cytochrome P450 1A2 (CYP1A2) is one of the key CYPs that activate aflatoxin B1 (AFB1), a notorious mycotoxin, into carcinogenic exo-8,9-epoxides (AFBO) in the liver. Although the structure of CYP1A2 is available, the mechanism of CYP1A2-specific binding to AFB [...] Read more.
Human cytochrome P450 1A2 (CYP1A2) is one of the key CYPs that activate aflatoxin B1 (AFB1), a notorious mycotoxin, into carcinogenic exo-8,9-epoxides (AFBO) in the liver. Although the structure of CYP1A2 is available, the mechanism of CYP1A2-specific binding to AFB1 has not been fully clarified. In this study, we used calculation biology to predict a model of CYP1A2 with AFB1, where Thr-124, Phe-125, Phe-226, and Phe-260 possibly participate in the specific binding. Site-directed mutagenesis was performed to construct mutants T124A, F125A, F226A, and F260A. Escherichia coli-expressed recombinant proteins T124A, F226A, and F260A had active structures, while F125A did not. This was evidenced by Fe2+∙Carbon monoxide (CO)-reduced difference spectra and circular dichroism spectroscopy. Mutant F125A was expressed in HEK293T cells. Steady kinetic assays showed that T124A had enhanced activity towards AFB1, while F125A, F226A, and F260A were significantly reduced in their ability to activate AFB1, implying that hydrogen bonds between Thr-124 and AFB1 were not important for substrate-specific binding, whereas Phe-125, Phe-226, and Phe-260 were essential for the process. The computation simulation and experimental results showed that the three key CH/π interactions between Phe-125, Phe-226, or Phe-260 and AFB1 collectively maintained the stable binding of AFB1 in the active cavity of CYP1A2. Full article
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Open AccessArticle Inhibition of Aflatoxin Production by Paraquat and External Superoxide Dismutase in Aspergillus flavus
Received: 15 January 2019 / Revised: 11 February 2019 / Accepted: 12 February 2019 / Published: 12 February 2019
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Abstract
Aflatoxin contamination of crops is a worldwide problem, and elucidation of the regulatory mechanism of aflatoxin production, for example relative to the oxidative–antioxidative system, is needed. Studies have shown that oxidative stress induced by reactive oxygen species promotes aflatoxin production. However, superoxide has [...] Read more.
Aflatoxin contamination of crops is a worldwide problem, and elucidation of the regulatory mechanism of aflatoxin production, for example relative to the oxidative–antioxidative system, is needed. Studies have shown that oxidative stress induced by reactive oxygen species promotes aflatoxin production. However, superoxide has been suggested to have the opposite effect. Here, we investigated the effects of the superoxide generator, paraquat, and externally added superoxide dismutase (SOD) on aflatoxin production in Aspergillus flavus. Paraquat with an IC50 value of 54.9 µM inhibited aflatoxin production without affecting fungal growth. It increased cytosolic and mitochondrial superoxide levels and downregulated the transcription of aflatoxin biosynthetic cluster genes, including aflR, a key regulatory protein. The addition of bovine Cu/ZnSOD to the culture medium suppressed the paraquat-induced increase in superoxide levels, but it did not fully restore paraquat-inhibited aflatoxin production because bovine Cu/ZnSOD with an IC50 value of 17.9 µg/mL itself inhibited aflatoxin production. Externally added bovine Cu/ZnSOD increased the SOD activity in fungal cell extracts and upregulated the transcription of genes encoding Cu/ZnSOD and alcohol dehydrogenase. These results suggest that intracellular accumulation of superoxide impairs aflatoxin production by downregulating aflR expression, and that externally added Cu/ZnSOD also suppresses aflatoxin production by a mechanism other than canonical superoxide elimination activity. Full article
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Open AccessArticle Biological System Responses of Dairy Cows to Aflatoxin B1 Exposure Revealed with Metabolomic Changes in Multiple Biofluids
Received: 7 November 2018 / Revised: 19 December 2018 / Accepted: 20 January 2019 / Published: 1 February 2019
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Abstract
Research on mycotoxins now requires a systematic study of post-exposure organisms. In this study, the effects of aflatoxin B1 (AFB1) on biofluids biomarkers were examined with metabolomics and biochemical tests. The results showed that milk concentration of aflatoxin M1 changed with the addition [...] Read more.
Research on mycotoxins now requires a systematic study of post-exposure organisms. In this study, the effects of aflatoxin B1 (AFB1) on biofluids biomarkers were examined with metabolomics and biochemical tests. The results showed that milk concentration of aflatoxin M1 changed with the addition or removal of AFB1. AFB1 significantly affected serum concentrations of superoxide dismutase (SOD) and malon dialdehyde (MDA), SOD/MDA, and the total antioxidant capacity. Significant differences of volatile fatty acids and NH3-N were detected in the rumen fluid. Eighteen rumen fluid metabolites, 11 plasma metabolites, and 9 milk metabolites were significantly affected by the AFB1. These metabolites are mainly involved in the pathway of amino acids metabolism. Our results suggest that not only is the study of macro-indicators (milk composition and production) important, but that more attention should be paid to micro-indicators (biomarkers) when assessing the risks posed by mycotoxins to dairy cows. Full article
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Open AccessArticle Lactobacillus bulgaricus or Lactobacillus rhamnosus Suppresses NF-κB Signaling Pathway and Protects against AFB1-Induced Hepatitis: A Novel Potential Preventive Strategy for Aflatoxicosis?
Received: 6 November 2018 / Revised: 22 December 2018 / Accepted: 24 December 2018 / Published: 4 January 2019
Cited by 1 | PDF Full-text (3785 KB) | HTML Full-text | XML Full-text
Abstract
Aflatoxin B1 (AFB1), a mycotoxin found in food and feed, is immunotoxic to animals and poses significant threat to the food industry and animal production. The primary target of AFB1 is the liver. To overcome aflatoxin toxicity, probiotic-mediated detoxification [...] Read more.
Aflatoxin B1 (AFB1), a mycotoxin found in food and feed, is immunotoxic to animals and poses significant threat to the food industry and animal production. The primary target of AFB1 is the liver. To overcome aflatoxin toxicity, probiotic-mediated detoxification has been proposed. In the present study, to investigate the protective effects and molecular mechanisms of Lactobacillus bulgaricus or Lactobacillus rhamnosus against liver inflammatory responses to AFB1, mice were administered with AFB1 (300 μg/kg) and/or Lactobacillus intragastrically for 8 weeks. AML12 cells were cultured and treated with AFB1, BAY 11-7082 (an NF-κB inhibitor), and different concentrations of L. bulgaricus or L. rhamnosus. The body weight, liver index, histopathological changes, biochemical indices, cytokines, cytotoxicity, and activation of the NF-κB signaling pathway were measured. AFB1 exposure caused changes in liver histopathology and biochemical functions, altered inflammatory response, and activated the NF-κB pathway. Supplementation of L. bulgaricus or L. rhamnosus significantly prevented AFB1-induced liver injury and alleviated histopathological changes and inflammatory response by decreasing NF-κB p65 expression. The results of in vitro experiments revealed that L. rhamnosus evidently protected against AFB1-induced inflammatory response and decreased NF-κB p65 expression when compared with L. bulgaricus. These findings indicated that AFB1 exposure can cause inflammatory response by inducing hepatic injury, and supplementation of L. bulgaricus or L. rhamnosus can produce significant protective effect against AFB1-induced liver damage and inflammatory response by regulating the activation of the NF-κB signaling pathway. Full article
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2018

Jump to: 2019, 2017, 2016, 2015

Open AccessArticle Occurrence and Levels of Aflatoxins in Fish Feeds and Their Potential Effects on Fish in Nyeri, Kenya
Toxins 2018, 10(12), 543; https://doi.org/10.3390/toxins10120543
Received: 20 November 2018 / Revised: 9 December 2018 / Accepted: 10 December 2018 / Published: 17 December 2018
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Abstract
Aflatoxins are fungal metabolites that contaminate foods and feeds, causing adverse health effects in humans and animals. This study determined the occurrence of aflatoxins in fish feeds and their potential effects on fish. Eighty-one fish feeds were sampled from 70 farms and 8 [...] Read more.
Aflatoxins are fungal metabolites that contaminate foods and feeds, causing adverse health effects in humans and animals. This study determined the occurrence of aflatoxins in fish feeds and their potential effects on fish. Eighty-one fish feeds were sampled from 70 farms and 8 feed manufacturing plants in Nyeri, Kenya for aflatoxin analysis using competitive enzyme-linked immunosorbent assay. Fish were sampled from 12 farms for gross and microscopic pathological examination. Eighty-four percent of feeds sampled tested positive for aflatoxins, ranging from 1.8 to 39.7 µg/kg with a mean of 7.0 ± 8.3 µg/kg and the median of 3.6 µg/kg. Fifteen feeds (18.5%) had aflatoxins above the maximum allowable level in Kenya of 10 µg/kg. Homemade and tilapia feeds had significantly higher aflatoxin levels than commercial and trout feeds. Feeds containing maize bran and fish meal had significantly higher aflatoxin levels than those without these ingredients. Five trout farms (41.7%) had fish with swollen abdomens, and enlarged livers with white or yellow nodules, which microscopically had large dark basophilic hepatic cells with hyperchromatic nuclei in irregular cords. In conclusion, aflatoxin contamination of fish feeds is prevalent in Nyeri, and may be the cause of adverse health effects in fish in this region. Full article
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Open AccessArticle Chestnut Drying Is Critical in Determining Aspergillus flavus Growth and Aflatoxin Contamination
Toxins 2018, 10(12), 530; https://doi.org/10.3390/toxins10120530
Received: 26 October 2018 / Revised: 6 December 2018 / Accepted: 6 December 2018 / Published: 11 December 2018
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Abstract
Chestnut drying is used to prevent postharvest losses and microorganism contamination during storage. Several studies reported the contamination by aflatoxins (AFs) produced by Aspergillus spp. in chestnuts. The effect of drying temperatures (from 30 to 50 °C) was evaluated on the growth of [...] Read more.
Chestnut drying is used to prevent postharvest losses and microorganism contamination during storage. Several studies reported the contamination by aflatoxins (AFs) produced by Aspergillus spp. in chestnuts. The effect of drying temperatures (from 30 to 50 °C) was evaluated on the growth of A. flavus and the production of aflatoxins in chestnuts. The influence of the treatment on the proximate composition, the total phenol content and antioxidant activity of chestnuts was considered. Fungal colonization was observed on the nuts dried at 30, 35, and 40 °C; the incidence was lower at 40 °C. The highest concentrations of AFB1 and AFB2 were produced at 40 °C. No aflatoxins were detected at 45 or 50 °C. At 40 °C A. flavus was under suboptimal conditions for growth (aw 0.78), but the fungus was able to synthesize aflatoxins. As the temperatures applied increased, the total phenol content increased, while the antioxidant activity decreased. A drying treatment at 45 °C for seven days (aw 0.64) could be a promising method to effectively control both the growth of aflatoxigenic fungi and the production of aflatoxins. This study provides preliminary data useful to improve the current drying conditions used in chestnut mills, to reduce both fungal growth and aflatoxin production. Full article
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Open AccessArticle Aspergillus flavus NRRL 3251 Growth, Oxidative Status, and Aflatoxins Production Ability In Vitro under Different Illumination Regimes
Toxins 2018, 10(12), 528; https://doi.org/10.3390/toxins10120528
Received: 5 November 2018 / Revised: 5 December 2018 / Accepted: 6 December 2018 / Published: 10 December 2018
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Abstract
Aspergillus flavus is the most important mycotoxin-producing fungus involved in the global episodes of aflatoxin B1 contamination of crops at both the pre-harvest and post-harvest stages. However, in order to effectively control aflatoxin contamination in crops using antiaflatoxigenic and/or antifungal compounds, some [...] Read more.
Aspergillus flavus is the most important mycotoxin-producing fungus involved in the global episodes of aflatoxin B1 contamination of crops at both the pre-harvest and post-harvest stages. However, in order to effectively control aflatoxin contamination in crops using antiaflatoxigenic and/or antifungal compounds, some of which are photosensitive, a proper understanding of the photo-sensitive physiology of potential experimental strains need to be documented. The purpose of the study is therefore to evaluate the effect of visible (VIS) light illumination on growth and conidiation, aflatoxin production ability and modulation of A. flavus oxidative status during in vitro experiment. Aflatoxigenic A. flavus strain was inoculated in aflatoxin-inducing YES media and incubated under three different VIS illumination regimes during a 168 h growth period at 29 °C. VIS illumination reduced A. flavus mycelia biomass yield, both during growth on plates and in liquid media, promoted conidiation and increased the aflatoxin production. Furthermore, aflatoxin production increased with increased reactive oxidative species (ROS) levels at 96 h of growth, confirming illumination-driven oxidative stress modulation activity on A. flavus cells. Full article
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Open AccessArticle Alternative Splicing of the Aflatoxin-Associated Baeyer–Villiger Monooxygenase from Aspergillus flavus: Characterisation of MoxY Isoforms
Toxins 2018, 10(12), 521; https://doi.org/10.3390/toxins10120521
Received: 15 November 2018 / Revised: 1 December 2018 / Accepted: 3 December 2018 / Published: 5 December 2018
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Abstract
Aflatoxins are carcinogenic mycotoxins that are produced by the filamentous fungus Aspergillus flavus, a contaminant of numerous food crops. Aflatoxins are synthesised via the aflatoxin biosynthesis pathway, with the enzymes involved encoded by the aflatoxin biosynthesis gene cluster. MoxY is a type [...] Read more.
Aflatoxins are carcinogenic mycotoxins that are produced by the filamentous fungus Aspergillus flavus, a contaminant of numerous food crops. Aflatoxins are synthesised via the aflatoxin biosynthesis pathway, with the enzymes involved encoded by the aflatoxin biosynthesis gene cluster. MoxY is a type I Baeyer–Villiger monooxygenase (BVMO), responsible for the conversion of hydroxyversicolorone (HVN) and versicolorone (VN) to versiconal hemiacetal acetate (VHA) and versiconol acetate (VOAc), respectively. Using mRNA data, an intron near the C-terminus was identified that is alternatively spliced, creating two possible MoxY isoforms which exist in vivo, while analysis of the genomic DNA suggests an alternative start codon leading to possible elongation of the N-terminus. These four variants of the moxY gene were recombinantly expressed in Escherichia coli, and their activity evaluated with respect to their natural substrates HVN and VN, as well as surrogate ketone substrates. Activity of the enzyme is absolutely dependent on the additional 22 amino acid residues at the N-terminus. Two MoxY isoforms with alternative C-termini, MoxYAltN and MoxYAltNC, converted HVN and VN, in addition to a range of ketone substrates. Stability and flavin-binding data suggest that MoxYAltN is, most likely, the dominant isoform. MoxYAltNC is generated by intron splicing, in contrast to intron retention, which is the most prevalent type of alternative splicing in ascomycetes. The alternative C-termini did not alter the substrate acceptance profile, or regio- or enantioselectivity of the enzyme, but did significantly affect the solubility and stability. Full article
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Open AccessArticle Aspergillus texensis: A Novel Aflatoxin Producer with S Morphology from the United States
Toxins 2018, 10(12), 513; https://doi.org/10.3390/toxins10120513
Received: 31 October 2018 / Revised: 27 November 2018 / Accepted: 27 November 2018 / Published: 3 December 2018
Cited by 1 | PDF Full-text (1161 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Aflatoxins are carcinogenic metabolites produced primarily by fungi within Aspergillus section Flavi. These fungi infect a wide range of crops in warm regions. Molecular phylogenetic analyses of fungi with S morphology (average sclerotium size < 400 µm) within section Flavi collected from [...] Read more.
Aflatoxins are carcinogenic metabolites produced primarily by fungi within Aspergillus section Flavi. These fungi infect a wide range of crops in warm regions. Molecular phylogenetic analyses of fungi with S morphology (average sclerotium size < 400 µm) within section Flavi collected from across the United States (US) resulted in the discovery of a novel aflatoxin-producing species, Aspergillus texensis. Aspergillus texensis was isolated from maize grown in Arkansas, Louisiana, and Texas, and from soils cropped to maize in Texas. Aspergillus texensis produces sparse conidia and abundant sclerotia on various culture media, and on maize. Physiological studies have revealed optimal growth on culture media at 35 °C. All isolates of A. texensis produced B and G aflatoxins, cyclopiazonic acid and aspergillic acid. Aspergillus texensis and A. flavus S strain morphotypes produced similar concentrations of total aflatoxins on maize (p > 0.05). Phylogenetic analyses of aflatoxin-producers based on partial gene sequences of the β-tubulin (0.9 kb), calmodulin (1.2 kb), and nitrate reductase (2.1 kb) genes placed A. texensis in a highly supported monophyletic clade closely related to A. minisclerotigenes and a previously reported unnamed lineage designated Lethal Aflatoxicosis Fungus. Full article
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Open AccessArticle Streptomyces roseolus, A Promising Biocontrol Agent Against Aspergillus flavus, the Main Aflatoxin B1 Producer
Toxins 2018, 10(11), 442; https://doi.org/10.3390/toxins10110442
Received: 1 October 2018 / Revised: 26 October 2018 / Accepted: 27 October 2018 / Published: 30 October 2018
Cited by 1 | PDF Full-text (2362 KB) | HTML Full-text | XML Full-text
Abstract
Crop contamination by aflatoxin B1 is a current problem in tropical and subtropical regions. In the future, this contamination risk may be expanded to European countries due to climate change. The development of alternative strategies to prevent mycotoxin contamination that further contribute [...] Read more.
Crop contamination by aflatoxin B1 is a current problem in tropical and subtropical regions. In the future, this contamination risk may be expanded to European countries due to climate change. The development of alternative strategies to prevent mycotoxin contamination that further contribute to the substitution of phytopharmaceutical products are thus needed. For this, a promising method resides in the use of biocontrol agents. Several actinobacteria strains have demonstrated to effectively reduce the aflatoxin B1 concentration. Nevertheless, the molecular mechanism of action by which these biological agents reduce the mycotoxin concentration has not been determined. The aim of the present study was to test the potential use of Streptomyces roseolus as a biocontrol agent against aflatoxin B1 contamination. Co-cultures with Aspergillus flavus were conducted, and the molecular fungal response was investigated through analyzing the q-PCR expression of 65 genes encoding relevant fungal functions. Moreover, kojic and cyclopiazonic acid concentrations, as well as morphological fungal changes were also analyzed. The results demonstrated that reduced concentrations of aflatoxin B1 and kojic acid were respectively correlated with the down-regulation of the aflatoxin B1 gene cluster and kojR gene expression. Moreover, a fungal hypersporulated phenotype and a general over-expression of genes involved in fungal development were observed in the co-culture condition. Full article
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Open AccessArticle Evaluation of Aflatoxin M1 Effects on the Metabolomic and Cytokinomic Profiling of a Hepatoblastoma Cell Line
Toxins 2018, 10(11), 436; https://doi.org/10.3390/toxins10110436
Received: 26 September 2018 / Revised: 26 October 2018 / Accepted: 26 October 2018 / Published: 28 October 2018
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Abstract
Hepatoblastoma incidence has been associated with different environmental factors even if no data are reported about a correlation between aflatoxin exposure and hepatoblastoma initiation. Considering that hepatoblastoma develops in infants and children and aflatoxin M1 (AFM1), the aflatoxin B1 (AFB1) hydroxylated metabolite, can [...] Read more.
Hepatoblastoma incidence has been associated with different environmental factors even if no data are reported about a correlation between aflatoxin exposure and hepatoblastoma initiation. Considering that hepatoblastoma develops in infants and children and aflatoxin M1 (AFM1), the aflatoxin B1 (AFB1) hydroxylated metabolite, can be present in mothers’ milk and in marketed milk products, in this study we decided to test the effects of AFM1 on a hepatoblastoma cell line (HepG2). Firstly, we evaluated the effects of AFM1 on the cell viability, apoptosis, cell cycle, and metabolomic and cytokinomic profile of HepG2 cells after treatment. AFM1 induced: (1) a decrease of HepG2 cell viability, reaching IC50 at 9 µM; (2) the blocking of the cell cycle in the G0/G1 phase; (3) the decrease of formiate levels and incremented level of some amino acids and metabolites in HepG2 cells after treatment; and (4) the increase of the concentration of three pro-inflammatory cytokines, IL-6, IL-8, and TNF-α, and the decrease of the anti-inflammatory interleukin, IL-4. Our results show that AFM1 inhibited the growth of HepG2 cells, inducing both a modulation of the lipidic, glycolytic, and amino acid metabolism and an increase of the inflammatory status of these cells. Full article
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Open AccessArticle The PHD Transcription Factor Rum1 Regulates Morphogenesis and Aflatoxin Biosynthesis in Aspergillus flavus
Received: 8 June 2018 / Revised: 16 July 2018 / Accepted: 18 July 2018 / Published: 20 July 2018
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Abstract
Aspergillus flavus produces mycotoxins especially aflatoxin B1 and infects crops worldwide. As a PHD transcription factor, there is no report on the role of Rum1 in the virulence of Aspergillus spp. yet. This study explored the biological function of Rum1 in A. [...] Read more.
Aspergillus flavus produces mycotoxins especially aflatoxin B1 and infects crops worldwide. As a PHD transcription factor, there is no report on the role of Rum1 in the virulence of Aspergillus spp. yet. This study explored the biological function of Rum1 in A. flavus through the construction of rum1 deletion mutants and rum1 complementation strains with the method of homologous recombination. It was found, in the study, that Rum1 negatively regulates conidiation through abaA and brlA, positively regulates sclerotia formation through nsdC, nsdD, and sclR, triggers aflatoxin biological synthesis, and enhances the activity of amylase. Our findings suggested that Rum1 plays a major role in the growth of mycelia, conidia, and sclerotia production along with aflatoxin biosynthesis in A. flavus. Full article
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Open AccessArticle Detection of Aflatoxigenic and Atoxigenic Mexican Aspergillus Strains by the Dichlorvos–Ammonia (DV–AM) Method
Received: 2 June 2018 / Revised: 24 June 2018 / Accepted: 25 June 2018 / Published: 27 June 2018
Cited by 1 | PDF Full-text (3363 KB) | HTML Full-text | XML Full-text
Abstract
The dichlorvos–ammonia (DV–AM) method is a sensitive method for distinguishing aflatoxigenic fungi by detecting red (positive) colonies. In this study, the DV–AM method was applied for the isolation of aflatoxigenic and atoxigenic fungi from soil samples from a maize field in Mexico. In [...] Read more.
The dichlorvos–ammonia (DV–AM) method is a sensitive method for distinguishing aflatoxigenic fungi by detecting red (positive) colonies. In this study, the DV–AM method was applied for the isolation of aflatoxigenic and atoxigenic fungi from soil samples from a maize field in Mexico. In the first screening, we obtained two isolates from two soil subsamples of 20 independent samples and, in the second screening, we obtained two isolates from one subsample of these. Morphological and phylogenic analyses of the two isolates (MEX-A19-13, MEX-A19-2nd-5) indicated that they were Aspergillus flavus located in the A. flavus clade. Chemical analyses demonstrated that one isolate could produce B-type aflatoxins, while the other produced no aflatoxins. These results demonstrate that the DV–AM method is useful for the isolation of both aflatoxigenic and atoxigenic Aspergilli. Full article
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Open AccessArticle Analysis of the Relationship between Alternative Respiration and Sterigmatocystin Formation in Aspergillus nidulans
Received: 1 April 2018 / Revised: 16 April 2018 / Accepted: 17 April 2018 / Published: 20 April 2018
Cited by 2 | PDF Full-text (2715 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Aspergillus nidulans has one gene for alternative oxidase (EC 1.10.3.11). To investigate the relationship between this mitochondrial terminal oxidase and the formation of the mycotoxin sterigmatocystin, the encoding aodA gene was both deleted and overexpressed. Relative to the wild-type, the cyanide-resistant fraction of [...] Read more.
Aspergillus nidulans has one gene for alternative oxidase (EC 1.10.3.11). To investigate the relationship between this mitochondrial terminal oxidase and the formation of the mycotoxin sterigmatocystin, the encoding aodA gene was both deleted and overexpressed. Relative to the wild-type, the cyanide-resistant fraction of respiration in the late stationary stage—when sterigmatocystin production occurs—doubled in the overexpressing mutant carrying three aodA gene copies, but decreased to 10% in the deletant. Essentially identical results were obtained regardless whether the cultures were illuminated or protected from light. In contrast, sterigmatocystin yield in the aodA deletant was about half of that in the control when grown in the dark, while aodA overexpression resulted in up to 70% more sterigmatocystin formed, the yield increasing with alternative oxidase activity. Results were quite different when cultures were illuminated: under those conditions, sterigmatocystin volumetric yields were considerably lower, and statistically unvarying, regardless of the presence, absence, or the copy number of aodA. We conclude that the copy number of aodA, and hence, the balance between alternative- and cytochrome C-mediated respiration, appears to correlate with sterigmatocystin production in A. nidulans, albeit only in the absence of light. Full article
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Open AccessArticle G Protein α Subunit GpaB is Required for Asexual Development, Aflatoxin Biosynthesis and Pathogenicity by Regulating cAMP Signaling in Aspergillus flavus
Received: 5 February 2018 / Revised: 5 March 2018 / Accepted: 7 March 2018 / Published: 10 March 2018
Cited by 2 | PDF Full-text (4978 KB) | HTML Full-text | XML Full-text
Abstract
The heterotrimeric G proteins are critical for signal transduction and function in numerous biological processes including vegetative growth, asexual development and fungal virulence in fungi. Here, we identified four G protein alpha subunits (GanA, GpaB, FadA and GaoC) in the notorious Aflatoxin-producing fungus [...] Read more.
The heterotrimeric G proteins are critical for signal transduction and function in numerous biological processes including vegetative growth, asexual development and fungal virulence in fungi. Here, we identified four G protein alpha subunits (GanA, GpaB, FadA and GaoC) in the notorious Aflatoxin-producing fungus Aspergillus flavus. GanA, GpaB and FadA have homologues in other fungal species, while GaoC is a novel one. Here, we showed that the loss function of gpaB displayed a defect in conidiophore formation and considerably reduced expression levels of conidia-specific genes brlA and abaA. A decreased viability of cell wall integrity stress and oxidative stress were also found in the ∆gpaB mutant. More importantly, aflatoxin (AF) biosynthesis and infection on crop seeds were severely impaired in the gpaB-deficient mutant. Further analyses demonstrated that the intracellular cAMP levels significantly reduced in the gpaB-deficient mutant compared to wildtype strains. Additionally, an alteration of PKA activities in the ∆gpaB mutant was also found. Overall, our results indicated that GpaB played diverse roles in asexual sporulation, AF biosynthesis and virulence by regulating cAMP signaling in Aspergillus flavus. Full article
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Open AccessArticle Survey of Candidate Genes for Maize Resistance to Infection by Aspergillus flavus and/or Aflatoxin Contamination
Received: 21 December 2017 / Revised: 20 January 2018 / Accepted: 24 January 2018 / Published: 31 January 2018
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Abstract
Many projects have identified candidate genes for resistance to aflatoxin accumulation or Aspergillus flavus infection and growth in maize using genetic mapping, genomics, transcriptomics and/or proteomics studies. However, only a small percentage of these candidates have been validated in field conditions, and their [...] Read more.
Many projects have identified candidate genes for resistance to aflatoxin accumulation or Aspergillus flavus infection and growth in maize using genetic mapping, genomics, transcriptomics and/or proteomics studies. However, only a small percentage of these candidates have been validated in field conditions, and their relative contribution to resistance, if any, is unknown. This study presents a consolidated list of candidate genes identified in past studies or in-house studies, with descriptive data including genetic location, gene annotation, known protein identifiers, and associated pathway information, if known. A candidate gene pipeline to test the phenotypic effect of any maize DNA sequence on aflatoxin accumulation resistance was used in this study to determine any measurable effect on polymorphisms within or linked to the candidate gene sequences, and the results are published here. Full article
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Open AccessArticle Comparative Response of the Hepatic Transcriptomes of Domesticated and Wild Turkey to Aflatoxin B1
Received: 29 November 2017 / Revised: 8 January 2018 / Accepted: 10 January 2018 / Published: 13 January 2018
Cited by 2 | PDF Full-text (1405 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
The food-borne mycotoxin aflatoxin B1 (AFB1) poses a significant risk to poultry, which are highly susceptible to its hepatotoxic effects. Domesticated turkeys (Meleagris gallopavo) are especially sensitive, whereas wild turkeys (M. g. silvestris) are more resistant. [...] Read more.
The food-borne mycotoxin aflatoxin B1 (AFB1) poses a significant risk to poultry, which are highly susceptible to its hepatotoxic effects. Domesticated turkeys (Meleagris gallopavo) are especially sensitive, whereas wild turkeys (M. g. silvestris) are more resistant. AFB1 toxicity entails bioactivation by hepatic cytochrome P450s to the electrophilic exo-AFB1-8,9-epoxide (AFBO). Domesticated turkeys lack functional hepatic GST-mediated detoxification of AFBO, and this is largely responsible for the differences in resistance between turkey types. This study was designed to characterize transcriptional changes induced in turkey livers by AFB1, and to contrast the response of domesticated (susceptible) and wild (more resistant) birds. Gene expression responses to AFB1 were examined using RNA-sequencing. Statistically significant differences in gene expression were observed among treatment groups and between turkey types. Expression analysis identified 4621 genes with significant differential expression (DE) in AFB1-treated birds compared to controls. Characterization of DE transcripts revealed genes dis-regulated in response to toxic insult with significant association of Phase I and Phase II genes and others important in cellular regulation, modulation of apoptosis, and inflammatory responses. Constitutive expression of GSTA3 was significantly higher in wild birds and was significantly higher in AFB1-treated birds when compared to controls for both genetic groups. This pattern was also observed by qRT-PCR in other wild and domesticated turkey strains. Results of this study emphasize the differential response of these genetically distinct birds, and identify genes and pathways that are differentially altered in aflatoxicosis. Full article
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Open AccessArticle Light-Irradiation Wavelength and Intensity Changes Influence Aflatoxin Synthesis in Fungi
Received: 8 December 2017 / Revised: 25 December 2017 / Accepted: 3 January 2018 / Published: 5 January 2018
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Abstract
Fungi respond to light irradiation by forming conidia and occasionally synthesizing mycotoxins. Several light wavelengths, such as blue and red, affect the latter. However, the relationship between light irradiation and mycotoxin synthesis varies depending on the fungal species or strain. This study focused [...] Read more.
Fungi respond to light irradiation by forming conidia and occasionally synthesizing mycotoxins. Several light wavelengths, such as blue and red, affect the latter. However, the relationship between light irradiation and mycotoxin synthesis varies depending on the fungal species or strain. This study focused on aflatoxin (AF), which is a mycotoxin, and the types of light irradiation that increase AF synthesis. Light-irradiation tests using the visible region indicated that blue wavelengths in the lower 500 nm region promoted AF synthesis. In contrast, red wavelengths of 660 nm resulted in limited significant changes compared with dark conditions. Irradiation tests with different intensity levels indicated that a low light intensity increased AF synthesis. For one fungal strain, light irradiation decreased the AF synthesis under all wavelength conditions. However, the decrease was mitigated by 525 nm low intensity irradiation. Thus, blue-green low intensity irradiation may increase AF synthesis in fungi. Full article
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2017

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Open AccessArticle Carbon Dioxide Mediates the Response to Temperature and Water Activity Levels in Aspergillus flavus during Infection of Maize Kernels
Received: 26 September 2017 / Revised: 17 November 2017 / Accepted: 14 December 2017 / Published: 22 December 2017
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Abstract
Aspergillus flavus is a saprophytic fungus that may colonize several important crops, including cotton, maize, peanuts and tree nuts. Concomitant with A. flavus colonization is its potential to secrete mycotoxins, of which the most prominent is aflatoxin. Temperature, water activity (aw) [...] Read more.
Aspergillus flavus is a saprophytic fungus that may colonize several important crops, including cotton, maize, peanuts and tree nuts. Concomitant with A. flavus colonization is its potential to secrete mycotoxins, of which the most prominent is aflatoxin. Temperature, water activity (aw) and carbon dioxide (CO2) are three environmental factors shown to influence the fungus-plant interaction, which are predicted to undergo significant changes in the next century. In this study, we used RNA sequencing to better understand the transcriptomic response of the fungus to aw, temperature, and elevated CO2 levels. We demonstrate that aflatoxin (AFB1) production on maize grain was altered by water availability, temperature and CO2. RNA-Sequencing data indicated that several genes, and in particular those involved in the biosynthesis of secondary metabolites, exhibit different responses to water availability or temperature stress depending on the atmospheric CO2 content. Other gene categories affected by CO2 levels alone (350 ppm vs. 1000 ppm at 30 °C/0.99 aw), included amino acid metabolism and folate biosynthesis. Finally, we identified two gene networks significantly influenced by changes in CO2 levels that contain several genes related to cellular replication and transcription. These results demonstrate that changes in atmospheric CO2 under climate change scenarios greatly influences the response of A. flavus to water and temperature when colonizing maize grain. Full article
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Open AccessEditor’s ChoiceArticle Aspergillus korhogoensis, a Novel Aflatoxin Producing Species from the Côte d’Ivoire
Toxins 2017, 9(11), 353; https://doi.org/10.3390/toxins9110353
Received: 26 September 2017 / Revised: 13 October 2017 / Accepted: 26 October 2017 / Published: 31 October 2017
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Abstract
Several strains of a new aflatoxigenic species of Aspergillus, A. korhogoensis, were isolated in the course of a screening study involving species from section Flavi found contaminating peanuts (Arachis hypogaea) and peanut paste in the Côte d’Ivoire. Based on [...] Read more.
Several strains of a new aflatoxigenic species of Aspergillus, A. korhogoensis, were isolated in the course of a screening study involving species from section Flavi found contaminating peanuts (Arachis hypogaea) and peanut paste in the Côte d’Ivoire. Based on examination of four isolates, this new species is described using a polyphasic approach. A concatenated alignment comprised of nine genes (ITS, benA, cmdA, mcm7, amdS, rpb1, preB, ppgA, and preA) was subjected to phylogenetic analysis, and resulted in all four strains being inferred as a distinct clade. Characterization of mating type for each strain revealed A. korhogoensis as a heterothallic species, since three isolates exhibited a singular MAT1-1 locus and one isolate exhibited a singular MAT1-2 locus. Morphological and physiological characterizations were also performed based on their growth on various types of media. Their respective extrolite profiles were characterized using LC/HRMS, and showed that this new species is capable of producing B- and G-aflatoxins, aspergillic acid, cyclopiazonic acid, aflavarins, and asparasones, as well as other metabolites. Altogether, our results confirm the monophyly of A. korhogoensis, and strengthen its position in the A. flavus clade, as the sister taxon of A. parvisclerotigenus. Full article
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Open AccessEditor’s ChoiceArticle Palladium Nanoparticles-Based Fluorescence Resonance Energy Transfer Aptasensor for Highly Sensitive Detection of Aflatoxin M1 in Milk
Toxins 2017, 9(10), 318; https://doi.org/10.3390/toxins9100318
Received: 12 September 2017 / Revised: 5 October 2017 / Accepted: 6 October 2017 / Published: 13 October 2017
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Abstract
A highly sensitive aptasensor for aflatoxin M1 (AFM1) detection was constructed based on fluorescence resonance energy transfer (FRET) between 5-carboxyfluorescein (FAM) and palladium nanoparticles (PdNPs). PdNPs (33 nm) were synthesized through a seed-mediated growth method and exhibited broad and strong [...] Read more.
A highly sensitive aptasensor for aflatoxin M1 (AFM1) detection was constructed based on fluorescence resonance energy transfer (FRET) between 5-carboxyfluorescein (FAM) and palladium nanoparticles (PdNPs). PdNPs (33 nm) were synthesized through a seed-mediated growth method and exhibited broad and strong absorption in the whole ultraviolet-visible (UV-Vis) range. The strong coordination interaction between nitrogen functional groups of the AFM1 aptamer and PdNPs brought FAM and PdNPs in close proximity, which resulted in the fluorescence quenching of FAM to a maximum extent of 95%. The non-specific fluorescence quenching caused by PdNPs towards fluorescein was negligible. After the introduction of AFM1 into the FAM-AFM1 aptamer-PdNPs FRET system, the AFM1 aptamer preferentially combined with AFM1 accompanied by conformational change, which greatly weakened the coordination interaction between the AFM1 aptamer and PdNPs. Thus, fluorescence recovery of FAM was observed and a linear relationship between the fluorescence recovery and the concentration of AFM1 was obtained in the range of 5–150 pg/mL in aqueous buffer with the detection limit of 1.5 pg/mL. AFM1 detection was also realized in milk samples with a linear detection range from 6 pg/mL to 150 pg/mL. The highly sensitive FRET aptasensor with simple configuration shows promising prospect in detecting a variety of food contaminants. Full article
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Open AccessArticle The Putative Histone Methyltransferase DOT1 Regulates Aflatoxin and Pathogenicity Attributes in Aspergillus flavus
Received: 4 July 2017 / Revised: 20 July 2017 / Accepted: 20 July 2017 / Published: 24 July 2017
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Abstract
Lysine methyltransferases transfer methyl groups in specific lysine sites, which regulates a variety of important biological processes in eukaryotes. In this study, we characterized a novel homolog of the yeast methyltransferase DOT1 in A. flavus, and observed the roles of dot1 [...] Read more.
Lysine methyltransferases transfer methyl groups in specific lysine sites, which regulates a variety of important biological processes in eukaryotes. In this study, we characterized a novel homolog of the yeast methyltransferase DOT1 in A. flavus, and observed the roles of dot1 in A. flavus. Deletion of dot1 showed a significant decrease in conidiation, but an increase in sclerotia formation. A change in viability to multiple stresses was also found in the Δdot1 mutant. Additionally, aflatoxin (AF) production was found severely impaired in the Δdot1 mutant. Further analysis by qRT-PCR revealed that the transcription of AF structural genes and their regulator gene aflS were prominently suppressed in the Δdot1 mutant. Furthermore, our data revealed that Dot1 is important for colonizing maize seeds in A. flavus. Our research indicates that Dot1 is involved in fungal development, aflatoxin biosynthesis and fungal virulence in A. flavus, which might provide a potential target for controlling A. flavus with new strategies. Full article
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Open AccessEditor’s ChoiceArticle The Mode of Action of Cyclo(l-Ala-l-Pro) in Inhibiting Aflatoxin Production of Aspergillus flavus
Received: 21 June 2017 / Accepted: 11 July 2017 / Published: 12 July 2017
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Abstract
Cyclo(l-Ala-l-Pro) inhibits aflatoxin production in aflatoxigenic fungi without affecting fungal growth. The mode of action of cyclo(l-Ala-l-Pro) in inhibiting aflatoxin production of Aspergillus flavus was investigated. A glutathione S-transferase (GST) of the fungus, designated [...] Read more.
Cyclo(l-Ala-l-Pro) inhibits aflatoxin production in aflatoxigenic fungi without affecting fungal growth. The mode of action of cyclo(l-Ala-l-Pro) in inhibiting aflatoxin production of Aspergillus flavus was investigated. A glutathione S-transferase (GST) of the fungus, designated AfGST, was identified as a binding protein of cyclo(l-Ala-l-Pro) in an experiment performed using cyclo(l-Ala-l-Pro)-immobilized Sepharose beads. Cyclo(l-Ala-l-Pro) specifically bound to recombinant AfGST and inhibited its GST activity. Ethacrynic acid, a known GST inhibitor, inhibited the GST activity of recombinant AfGST and aflatoxin production of the fungus. Ethacrynic acid reduced the expression level of AflR, a key regulatory protein for aflatoxin production, similar to cyclo(l-Ala-l-Pro). These results suggest that cyclo(l-Ala-l-Pro) inhibits aflatoxin production by affecting GST function in A. flavus, and that AfGST inhibitors are possible candidates as selective aflatoxin production inhibitors. Full article
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Open AccessArticle Probing the Characterization of the Interaction of Aflatoxins B1 and G1 with Calf Thymus DNA In Vitro
Received: 19 April 2017 / Revised: 20 June 2017 / Accepted: 28 June 2017 / Published: 1 July 2017
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Abstract
The binding characterization of aflatoxins with calf thymus DNA (ctDNA) under physiological conditions was investigated. Multispectroscopic techniques, ctDNA melting, viscosity measurements, and molecular docking techniques were employed to elucidate the binding mechanism of the aflatoxins with DNA. The fluorescence results indicated that both [...] Read more.
The binding characterization of aflatoxins with calf thymus DNA (ctDNA) under physiological conditions was investigated. Multispectroscopic techniques, ctDNA melting, viscosity measurements, and molecular docking techniques were employed to elucidate the binding mechanism of the aflatoxins with DNA. The fluorescence results indicated that both aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) bound to the ctDNA, forming complexes through hydrogen bonding. The binding constants of AFB1 and AFG1 with ctDNA reached up to 103 L·mol−1 and 104 L·mol−1, respectively, and AFG1 exhibited a higher binding propensity than that of AFB1. Furthermore, both AFB1 and AFG1 bound to the ctDNA through groove binding, as evidenced by the results of the spectroscopic, iodide quenching effect, viscosity, and ctDNA melting measurements. Changes in the circular dichroism signal manifested that both AFB1 and AFG1 induced an increase in the right-handed helicity, but only minimally influenced the base stacking of the DNA. A molecular docking study of the aflatoxin’s binding with the DNA revealed a groove binding mode, which was driven mainly by hydrogen bonding. This study of aflatoxin–ctDNA interaction may provide novel insights into the toxicological effect of the mycotoxins. Full article
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Open AccessArticle Impact of Mycotoxins Secreted by Aspergillus Molds on the Inflammatory Response of Human Corneal Epithelial Cells
Received: 30 March 2017 / Revised: 17 June 2017 / Accepted: 19 June 2017 / Published: 22 June 2017
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Abstract
Exposure to molds and mycotoxins not only contributes to the onset of respiratory disease, it also affects the ocular surface. Very few published studies concern the evaluation of the effect of mycotoxin exposure on ocular cells. The present study investigates the effects of [...] Read more.
Exposure to molds and mycotoxins not only contributes to the onset of respiratory disease, it also affects the ocular surface. Very few published studies concern the evaluation of the effect of mycotoxin exposure on ocular cells. The present study investigates the effects of aflatoxin B1 (AFB1) and gliotoxin, two mycotoxins secreted by Aspergillus molds, on the biological activity of the human corneal epithelial (HCE) cells. After 24, 48, and 72 h of exposure, cellular viability and inflammatory response were assessed. Both endpoint cell viability colorimetric assays and continuous cell impedance measurements, providing noninvasive real-time assessment of the effect on cells, were performed. Cytokine gene expression and interleukin-8 release were quantified. Gliotoxin appeared more cytotoxic than AFB1 but, at the same time, led to a lower increase of the inflammatory response reflecting its immunosuppressive properties. Real-time cell impedance measurement showed a distinct profile of cytotoxicity for both mycotoxins. HCE cells appeared to be a well-suited in vitro model to study ocular surface reactivity following biological contaminant exposure. Low, but persistent inflammation, caused by environmental factors, such as fungal toxins, leads to irritation and sensitization, and could be responsible for allergic manifestations which, in turn, could lead to mucosal hyper-reactivity. Full article
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Open AccessArticle Aflatoxin B1 Tolerance and Accumulation in Black Soldier Fly Larvae (Hermetia illucens) and Yellow Mealworms (Tenebrio molitor)
Received: 8 May 2017 / Revised: 26 May 2017 / Accepted: 29 May 2017 / Published: 2 June 2017
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Abstract
Crops contaminated with fungal mycotoxins such as aflatoxin B1 (AFB1) are often downgraded or removed from the food chain. This study aimed to evaluate the tolerance and accumulation of AFB1 in two insect species to determine whether they could be used to retain [...] Read more.
Crops contaminated with fungal mycotoxins such as aflatoxin B1 (AFB1) are often downgraded or removed from the food chain. This study aimed to evaluate the tolerance and accumulation of AFB1 in two insect species to determine whether they could be used to retain condemned mycotoxin contaminated crops in the food chain. First, instar black soldier fly larvae (Hermetia illucens, BSF) and yellow mealworm (Tenebrio molitor, YMW) were fed poultry feed spiked with AFB1 and formulated to contain levels of 0.01, 0.025, 0.05, 0.10, 0.25, and up to 0.5 mg/kg dry feed. Poultry feed without any additions and feed with only the solvent added served as controls. The AFB1 in the feed did not affect survival and body weight in the BSF and YMW larvae (p > 0.10), indicating a high tolerance to aflatoxin B1 in both species. Furthermore, AFB1 and aflatoxin M1 (AFM1) were below the detection limit (0.10 µg/kg) in BSF larvae, whereas the YMW had AFB1 levels that were approximately 10% of the European Union’s legal limit for feed materials and excreted AFM1. It is concluded that both BSF larvae and YMW have a high AFB1 tolerance and do not accumulate AFB1. Full article
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Open AccessArticle Inhibitory Activities of Blasticidin S Derivatives on Aflatoxin Production by Aspergillus Flavus
Received: 11 April 2017 / Revised: 22 May 2017 / Accepted: 22 May 2017 / Published: 26 May 2017
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Abstract
Blasticidin S (BcS) is a protein synthesis inhibitor which shows strong growth inhibitory activity against a number of microorganisms. However, BcS inhibited aflatoxin production by Aspergillus flavus without affecting its growth. In order to obtain information about the structure–activity relationship of BcS as [...] Read more.
Blasticidin S (BcS) is a protein synthesis inhibitor which shows strong growth inhibitory activity against a number of microorganisms. However, BcS inhibited aflatoxin production by Aspergillus flavus without affecting its growth. In order to obtain information about the structure–activity relationship of BcS as an aflatoxin production inhibitor, BcS derivatives were prepared and their aflatoxin production inhibitory activities were evaluated. Among five derivatives, blasticidin S carboxymethyl ester, deaminohydroxyblasticidin S, and pyrimidinoblasticidin S showed inhibitory activity, while the others did not. The IC50 value for aflatoxin production of the carboxymethyl ester derivative was one-fifth of that of BcS although their antimicrobial activities were almost the same. These results indicate that the inhibitory activity of BcS against aflatoxin production was enhanced by esterification of its carboxyl group and that the carboxymethyl ester derivative might be more suitable for practical use than BcS because of the specificity of the carboxymethyl ester derivative, which inhibited aflatoxin production more than BcS. Full article
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Open AccessArticle Doses of Immunogen Contribute to Specificity Spectrums of Antibodies against Aflatoxin
Received: 28 February 2017 / Revised: 3 May 2017 / Accepted: 17 May 2017 / Published: 19 May 2017
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Abstract
Research about antibody specificity spectra was conducted to develop single-specific antibodies or broad-specific antibodies. Aflatoxins, as one class of high-toxicity mycotoxins, were selected as the research targets to investigate the effect of the immunogen dose on antibody specificity spectra. For this aim, 16 [...] Read more.
Research about antibody specificity spectra was conducted to develop single-specific antibodies or broad-specific antibodies. Aflatoxins, as one class of high-toxicity mycotoxins, were selected as the research targets to investigate the effect of the immunogen dose on antibody specificity spectra. For this aim, 16 monoclonal antibodies were induced by low or high doses of aflatoxin B1-BSA, and 34 monoclonal antibodies were induced by low or high doses of aflatoxin M1-BSA. The specificities of the antibodies induced, whether by aflatoxin B1 conjugate or aflatoxin M1 conjugate, indicated that the low dose of the immunogen induced a narrow spectrum of antibody specificity, while the high dose of the immunogen showed an advantage to form a broad spectrum of antibody specificity. Therefore, this report provides important information for the development of new antibodies against small molecules like aflatoxins. Full article
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2016

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Open AccessArticle Aflatoxin B1-Induced Developmental and DNA Damage in Caenorhabditis elegans
Received: 17 October 2016 / Revised: 6 December 2016 / Accepted: 22 December 2016 / Published: 26 December 2016
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Abstract
Aflatoxin B1 (AFB1) is a ubiquitous mycotoxin produced by toxicogenic Aspergillus species. AFB1 has been reported to cause serious adverse health effects, such as cancers and abnormal development and reproduction, in animals and humans. AFB1 is also a [...] Read more.
Aflatoxin B1 (AFB1) is a ubiquitous mycotoxin produced by toxicogenic Aspergillus species. AFB1 has been reported to cause serious adverse health effects, such as cancers and abnormal development and reproduction, in animals and humans. AFB1 is also a potent genotoxic mutagen that causes DNA damage in vitro and in vivo. However, the link between DNA damage and abnormal development and reproduction is unclear. To address this issue, we examined the DNA damage, germline apoptosis, growth, and reproductive toxicity following exposure to AFB1, using Caenorhabditis elegans as a study model. Results found that AFB1 induced DNA damage and germline apoptosis, and significantly inhibited growth and reproduction of the nematodes in a concentration-dependent manner. Exposure to AFB1 inhibited growth or reproduction more potently in the DNA repair-deficient xpa-1 nematodes than the wild-type N2 strain. According to the relative expression level of pathway-related genes measured by real-time PCR, the DNA damage response (DDR) pathway was found to be associated with AFB1-induced germline apoptosis, which further played an essential role in the dysfunction of growth and reproduction in C. elegans. Full article
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Open AccessArticle Growth-Phase Sterigmatocystin Formation on Lactose Is Mediated via Low Specific Growth Rates in Aspergillus nidulans
Toxins 2016, 8(12), 354; https://doi.org/10.3390/toxins8120354
Received: 23 October 2016 / Revised: 20 November 2016 / Accepted: 23 November 2016 / Published: 28 November 2016
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Abstract
Seed contamination with polyketide mycotoxins such as sterigmatocystin (ST) produced by Aspergilli is a worldwide issue. The ST biosynthetic pathway is well-characterized in A. nidulans, but regulatory aspects related to the carbon source are still enigmatic. This is particularly true for lactose, [...] Read more.
Seed contamination with polyketide mycotoxins such as sterigmatocystin (ST) produced by Aspergilli is a worldwide issue. The ST biosynthetic pathway is well-characterized in A. nidulans, but regulatory aspects related to the carbon source are still enigmatic. This is particularly true for lactose, inasmuch as some ST production mutant strains still synthesize ST on lactose but not on other carbon substrates. Here, kinetic data revealed that on d-glucose, ST forms only after the sugar is depleted from the medium, while on lactose, ST appears when most of the carbon source is still available. Biomass-specified ST production on lactose was significantly higher than on d-glucose, suggesting that ST formation may either be mediated by a carbon catabolite regulatory mechanism, or induced by low specific growth rates attainable on lactose. These hypotheses were tested by d-glucose limited chemostat-type continuous fermentations. No ST formed at a high growth rate, while a low growth rate led to the formation of 0.4 mg·L−1 ST. Similar results were obtained with a CreA mutant strain. We concluded that low specific growth rates may be the primary cause of mid-growth ST formation on lactose in A. nidulans, and that carbon utilization rates likely play a general regulatory role during biosynthesis. Full article
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Open AccessArticle RNA Sequencing of Contaminated Seeds Reveals the State of the Seed Permissive for Pre-Harvest Aflatoxin Contamination and Points to a Potential Susceptibility Factor
Toxins 2016, 8(11), 317; https://doi.org/10.3390/toxins8110317
Received: 7 September 2016 / Revised: 26 October 2016 / Accepted: 28 October 2016 / Published: 3 November 2016
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Abstract
Pre-harvest aflatoxin contamination (PAC) is a major problem facing peanut production worldwide. Produced by the ubiquitous soil fungus, Aspergillus flavus, aflatoxin is the most naturally occurring known carcinogen. The interaction between fungus and host resulting in PAC is complex, and breeding for [...] Read more.
Pre-harvest aflatoxin contamination (PAC) is a major problem facing peanut production worldwide. Produced by the ubiquitous soil fungus, Aspergillus flavus, aflatoxin is the most naturally occurring known carcinogen. The interaction between fungus and host resulting in PAC is complex, and breeding for PAC resistance has been slow. It has been shown that aflatoxin production can be induced by applying drought stress as peanut seeds mature. We have implemented an automated rainout shelter that controls temperature and moisture in the root and peg zone to induce aflatoxin production. Using polymerase chain reaction (PCR) and high performance liquid chromatography (HPLC), seeds meeting the following conditions were selected: infected with Aspergillus flavus and contaminated with aflatoxin; and not contaminated with aflatoxin. RNA sequencing analysis revealed groups of genes that describe the transcriptional state of contaminated vs. uncontaminated seed. These data suggest that fatty acid biosynthesis and abscisic acid (ABA) signaling are altered in contaminated seeds and point to a potential susceptibility factor, ABR1, as a repressor of ABA signaling that may play a role in permitting PAC. Full article
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Open AccessArticle Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B1
Received: 29 June 2016 / Accepted: 5 September 2016 / Published: 12 September 2016
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Abstract
Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B1 (AFB1) and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB1 in pigs remains to be clarified, especially [...] Read more.
Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B1 (AFB1) and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB1 in pigs remains to be clarified, especially the principal cytochrome P450 oxidases responsible for its activation. In this study, we cloned CYP3A29 from pig liver and expressed it in Escherichia coli, and its activity has been confirmed with the typical P450 CO-reduced spectral characteristic and nifedipine-oxidizing activity. The reconstituted membrane incubation proved that the recombinant CYP3A29 was able to oxidize AFB1 to form AFB1-exo-8,9-epoxide in vitro. The structural basis for the regioselective epoxidation of AFB1 by CYP3A29 was further addressed. The T309A mutation significantly decreased the production of AFBO, whereas F304A exhibited an enhanced activation towards AFB1. In agreement with the mutagenesis study, the molecular docking simulation suggested that Thr309 played a significant role in stabilization of AFB1 binding in the active center through a hydrogen bond. In addition, the bulk phenyl group of Phe304 potentially imposed steric hindrance on the binding of AFB1. Our study demonstrates the bioactivation of pig CYP3A29 towards AFB1 in vitro, and provides the insight for understanding regioselectivity of CYP3A29 to AFB1. Full article
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Open AccessArticle Confirmation and Fine Mapping of a Major QTL for Aflatoxin Resistance in Maize Using a Combination of Linkage and Association Mapping
Received: 22 July 2016 / Revised: 19 August 2016 / Accepted: 26 August 2016 / Published: 2 September 2016
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Abstract
Maize grain contamination with aflatoxin from Aspergillus flavus (A. flavus) is a serious health hazard to animals and humans. To map the quantitative trait loci (QTLs) associated with resistance to A. flavus, we employed a powerful approach that differs from [...] Read more.
Maize grain contamination with aflatoxin from Aspergillus flavus (A. flavus) is a serious health hazard to animals and humans. To map the quantitative trait loci (QTLs) associated with resistance to A. flavus, we employed a powerful approach that differs from previous methods in one important way: it combines the advantages of the genome-wide association analysis (GWAS) and traditional linkage mapping analysis. Linkage mapping was performed using 228 recombinant inbred lines (RILs), and a highly significant QTL that affected aflatoxin accumulation, qAA8, was mapped. This QTL spanned approximately 7 centi-Morgan (cM) on chromosome 8. The confidence interval was too large for positional cloning of the causal gene. To refine this QTL, GWAS was performed with 558,629 single nucleotide polymorphisms (SNPs) in an association population comprising 437 maize inbred lines. Twenty-five significantly associated SNPs were identified, most of which co-localised with qAA8 and explained 6.7% to 26.8% of the phenotypic variation observed. Based on the rapid linkage disequilibrium (LD) and the high density of SNPs in the association population, qAA8 was further localised to a smaller genomic region of approximately 1500 bp. A high-resolution map of the qAA8 region will be useful towards a marker-assisted selection (MAS) of A. flavus resistance and a characterisation of the causal gene. Full article
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Open AccessArticle The Stress Response Regulator AflSkn7 Influences Morphological Development, Stress Response, and Pathogenicity in the Fungus Aspergillus flavus
Received: 12 March 2016 / Revised: 22 June 2016 / Accepted: 23 June 2016 / Published: 5 July 2016
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Abstract
This study focused on AflSkn7, which is a stress response regulator in the aflatoxin-producing Aspergillus flavus. The ΔAflSkn7 mutants exhibited partially defective conidial formation and a complete inability to generate sclerotia, indicating AflSkn7 affects A. flavus asexual and sexual development. The [...] Read more.
This study focused on AflSkn7, which is a stress response regulator in the aflatoxin-producing Aspergillus flavus. The ΔAflSkn7 mutants exhibited partially defective conidial formation and a complete inability to generate sclerotia, indicating AflSkn7 affects A. flavus asexual and sexual development. The mutants tolerated osmotic stress but were partially susceptible to the effects of cell wall stress. Additionally, the ΔAflSkn7 mutants were especially sensitive to oxidative stress. These observations confirmed that AflSkn7 influences oxidative stress responses rather than osmotic stress responses. Additionally, AflSkn7 was observed to increase aflatoxin biosynthesis and seed infection rates. These results indicate AflSkn7 affects A. flavus morphological development, stress response, aflatoxin production, and pathogenicity. The results of this study may facilitate the development of new methods to manage A. flavus infections. Full article
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Open AccessArticle Inhibitory Activities of Alkyl Syringates and Related Compounds on Aflatoxin Production
Received: 21 April 2016 / Revised: 27 May 2016 / Accepted: 1 June 2016 / Published: 7 June 2016
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Abstract
Inhibitors of aflatoxin production of aflatoxigenic fungi are useful for preventing aflatoxin contamination in crops. As methyl syringate weakly inhibits aflatoxin production, aflatoxin production inhibitory activities of additional alkyl syringates with alkyl chains from ethyl to octyl were examined. Inhibitory activity toward aflatoxin [...] Read more.
Inhibitors of aflatoxin production of aflatoxigenic fungi are useful for preventing aflatoxin contamination in crops. As methyl syringate weakly inhibits aflatoxin production, aflatoxin production inhibitory activities of additional alkyl syringates with alkyl chains from ethyl to octyl were examined. Inhibitory activity toward aflatoxin production of Aspergillus flavus became stronger as the length of the alkyl chains on the esters became longer. Pentyl, hexyl, heptyl, and octyl syringates showed strong activity at 0.05 mM. Heptyl and octyl parabens, and octyl gallate also inhibited aflatoxin production as strongly as octyl syringate. Alkyl parabens and alkyl gallates inhibit the complex II activity of the mitochondrial respiration chain; thus, whether alkyl syringates inhibit complex II activity was examined. Inhibitory activities of alkyl syringates toward complex II also became stronger as the length of the alkyl chains increased. The complex II inhibitory activity of octyl syringate was comparable to that of octyl paraben and octyl gallate. These results suggest that alkyl syringates, alkyl parabens, and alkyl gallates, including commonly used food additives, are useful for aflatoxin control. Full article
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Open AccessArticle Effects of Zinc Chelators on Aflatoxin Production in Aspergillus parasiticus
Received: 31 March 2016 / Revised: 25 May 2016 / Accepted: 27 May 2016 / Published: 2 June 2016
Cited by 2 | PDF Full-text (1608 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Zinc concentrations strongly influence aflatoxin accumulation in laboratory media and in food and feed crops. The presence of zinc stimulates aflatoxin production, and the absence of zinc impedes toxin production. Initial studies that suggested a link between zinc and aflatoxin biosynthesis were presented [...] Read more.
Zinc concentrations strongly influence aflatoxin accumulation in laboratory media and in food and feed crops. The presence of zinc stimulates aflatoxin production, and the absence of zinc impedes toxin production. Initial studies that suggested a link between zinc and aflatoxin biosynthesis were presented in the 1970s. In the present study, we utilized two zinc chelators, N,N,N′,N′-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and 2,3-dimercapto-1-propanesulfonic acid (DMPS) to explore the effect of zinc limitation on aflatoxin synthesis in Aspergillus parasiticus. TPEN but not DMPS decreased aflatoxin biosynthesis up to six-fold depending on whether A. parasiticus was grown on rich or minimal medium. Although we observed significant inhibition of aflatoxin production by TPEN, no detectable changes were observed in expression levels of the aflatoxin pathway gene ver-1 and the zinc binuclear cluster transcription factor, AflR. Treatment of growing A. parasiticus solid culture with a fluorescent zinc probe demonstrated an increase in intracellular zinc levels assessed by increases in fluorescent intensity of cultures treated with TPEN compared to controls. These data suggest that TPEN binds to cytoplasmic zinc therefore limiting fungal access to zinc. To investigate the efficacy of TPEN on food and feed crops, we found that TPEN effectively decreases aflatoxin accumulation on peanut medium but not in a sunflower seeds-derived medium. From an application perspective, these data provide the basis for biological differences that exist in the efficacy of different zinc chelators in various food and feed crops frequently contaminated by aflatoxin. Full article
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Open AccessArticle Use of Cold Atmospheric Plasma to Detoxify Hazelnuts from Aflatoxins
Received: 8 March 2016 / Revised: 15 April 2016 / Accepted: 18 April 2016 / Published: 26 April 2016
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Abstract
Aflatoxins, produced by Aspergillus flavus and A. parasiticus, can contaminate different foodstuffs, such as nuts. Cold atmospheric pressure plasma has the potential to be used for mycotoxin detoxification. In this study, the operating parameters of cold atmospheric pressure plasma were optimized to [...] Read more.
Aflatoxins, produced by Aspergillus flavus and A. parasiticus, can contaminate different foodstuffs, such as nuts. Cold atmospheric pressure plasma has the potential to be used for mycotoxin detoxification. In this study, the operating parameters of cold atmospheric pressure plasma were optimized to reduce the presence of aflatoxins on dehulled hazelnuts. First, the effect of different gases was tested (N2, 0.1% O2 and 1% O2, 21% O2), then power (400, 700, 1000, 1150 W) and exposure time (1, 2, 4, and 12 min) were optimized. In preliminary tests on aflatoxin standard solutions, this method allowed to obtain a complete detoxification using a high power for a few minutes. On hazelnuts, in similar conditions (1000 W, 12 min), a reduction in the concentration of total aflatoxins and AFB1 of over 70% was obtained. Aflatoxins B1 and G1 were more sensitive to plasma treatments compared to aflatoxins B2 and G2, respectively. Under plasma treatment, aflatoxin B1 was more sensitive compared to aflatoxin G1. At the highest power, and for the longest time, the maximum temperature increment was 28.9 °C. Cold atmospheric plasma has the potential to be a promising method for aflatoxin detoxification on food, because it is effective and it could help to maintain the organoleptic characteristics. Full article
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Open AccessFeature PaperArticle Deciphering the Anti-Aflatoxinogenic Properties of Eugenol Using a Large-Scale q-PCR Approach
Received: 31 March 2016 / Revised: 13 April 2016 / Accepted: 18 April 2016 / Published: 26 April 2016
Cited by 12 | PDF Full-text (1763 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Produced by several species of Aspergillus, Aflatoxin B1 (AFB1) is a carcinogenic mycotoxin contaminating many crops worldwide. The utilization of fungicides is currently one of the most common methods; nevertheless, their use is not environmentally or economically sound. Thus, [...] Read more.
Produced by several species of Aspergillus, Aflatoxin B1 (AFB1) is a carcinogenic mycotoxin contaminating many crops worldwide. The utilization of fungicides is currently one of the most common methods; nevertheless, their use is not environmentally or economically sound. Thus, the use of natural compounds able to block aflatoxinogenesis could represent an alternative strategy to limit food and feed contamination. For instance, eugenol, a 4-allyl-2-methoxyphenol present in many essential oils, has been identified as an anti-aflatoxin molecule. However, its precise mechanism of action has yet to be clarified. The production of AFB1 is associated with the expression of a 70 kB cluster, and not less than 21 enzymatic reactions are necessary for its production. Based on former empirical data, a molecular tool composed of 60 genes targeting 27 genes of aflatoxin B1 cluster and 33 genes encoding the main regulatory factors potentially involved in its production, was developed. We showed that AFB1 inhibition in Aspergillus flavus following eugenol addition at 0.5 mM in a Malt Extract Agar (MEA) medium resulted in a complete inhibition of the expression of all but one gene of the AFB1 biosynthesis cluster. This transcriptomic effect followed a down-regulation of the complex composed by the two internal regulatory factors, AflR and AflS. This phenomenon was also influenced by an over-expression of veA and mtfA, two genes that are directly linked to AFB1 cluster regulation. Full article
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Open AccessArticle Functional Genomic Analysis of Aspergillus flavus Interacting with Resistant and Susceptible Peanut
Received: 30 December 2015 / Revised: 30 January 2016 / Accepted: 5 February 2016 / Published: 15 February 2016
Cited by 10 | PDF Full-text (1299 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
In the Aspergillus flavus (A. flavus)–peanut pathosystem, development and metabolism of the fungus directly influence aflatoxin contamination. To comprehensively understand the molecular mechanism of A. flavus interaction with peanut, RNA-seq was used for global transcriptome profiling of A. flavus during interaction with [...] Read more.
In the Aspergillus flavus (A. flavus)–peanut pathosystem, development and metabolism of the fungus directly influence aflatoxin contamination. To comprehensively understand the molecular mechanism of A. flavus interaction with peanut, RNA-seq was used for global transcriptome profiling of A. flavus during interaction with resistant and susceptible peanut genotypes. In total, 67.46 Gb of high-quality bases were generated for A. flavus-resistant (af_R) and -susceptible peanut (af_S) at one (T1), three (T2) and seven (T3) days post-inoculation. The uniquely mapped reads to A. flavus reference genome in the libraries of af_R and af_S at T2 and T3 were subjected to further analysis, with more than 72% of all obtained genes expressed in the eight libraries. Comparison of expression levels both af_R vs. af_S and T2 vs. T3 uncovered 1926 differentially expressed genes (DEGs). DEGs associated with mycelial growth, conidial development and aflatoxin biosynthesis were up-regulated in af_S compared with af_R, implying that A. flavus mycelia more easily penetrate and produce much more aflatoxin in susceptible than in resistant peanut. Our results serve as a foundation for understanding the molecular mechanisms of aflatoxin production differences between A. flavus-R and -S peanut, and offer new clues to manage aflatoxin contamination in crops. Full article
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Open AccessFeature PaperArticle The Master Transcription Factor mtfA Governs Aflatoxin Production, Morphological Development and Pathogenicity in the Fungus Aspergillus flavus
Received: 7 December 2015 / Revised: 23 December 2015 / Accepted: 6 January 2016 / Published: 20 January 2016
Cited by 21 | PDF Full-text (5515 KB) | HTML Full-text | XML Full-text
Abstract
Aspergillus flavus produces a variety of toxic secondary metabolites; among them, the aflatoxins (AFs) are the most well known. These compounds are highly mutagenic and carcinogenic, particularly AFB1. A. flavus is capable of colonizing a number of economically-important crops, such as [...] Read more.
Aspergillus flavus produces a variety of toxic secondary metabolites; among them, the aflatoxins (AFs) are the most well known. These compounds are highly mutagenic and carcinogenic, particularly AFB1. A. flavus is capable of colonizing a number of economically-important crops, such as corn, cotton, peanut and tree nuts, and contaminating them with AFs. Molecular genetic studies in A. flavus could identify novel gene targets for use in strategies to reduce AF contamination and its adverse impact on food and feed supplies worldwide. In the current study, we investigated the role of the master transcription factor gene mtfA in A. flavus. Our results revealed that forced overexpression of mtfA results in a drastic decrease or elimination of several secondary metabolites, among them AFB1. The reduction in AFB1 was accompanied by a decrease in aflR expression. Furthermore, mtfA also regulates development; conidiation was influenced differently by this gene depending on the type of colonized substrate. In addition to its effect on conidiation, mtfA is necessary for the normal maturation of sclerotia. Importantly, mtfA positively affects the pathogenicity of A. flavus when colonizing peanut seeds. AF production in colonized seeds was decreased in the deletion mtfA strain and particularly in the overexpression strain, where only trace amounts were detected. Interestingly, a more rapid colonization of the seed tissue occurred when mtfA was overexpressed, coinciding with an increase in lipase activity and faster maceration of the oily part of the seed. Full article
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Open AccessFeature PaperArticle Reduction of Aflatoxins in Apricot Kernels by Electronic and Manual Color Sorting
Received: 30 December 2015 / Revised: 12 January 2016 / Accepted: 14 January 2016 / Published: 19 January 2016
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Abstract
The efficacy of color sorting on reducing aflatoxin levels in shelled apricot kernels was assessed. Naturally-contaminated kernels were submitted to an electronic optical sorter or blanched, peeled, and manually sorted to visually identify and sort discolored kernels (dark and spotted) from healthy ones. [...] Read more.
The efficacy of color sorting on reducing aflatoxin levels in shelled apricot kernels was assessed. Naturally-contaminated kernels were submitted to an electronic optical sorter or blanched, peeled, and manually sorted to visually identify and sort discolored kernels (dark and spotted) from healthy ones. The samples obtained from the two sorting approaches were ground, homogenized, and analysed by HPLC-FLD for their aflatoxin content. A mass balance approach was used to measure the distribution of aflatoxins in the collected fractions. Aflatoxin B1 and B2 were identified and quantitated in all collected fractions at levels ranging from 1.7 to 22,451.5 µg/kg of AFB1 + AFB2, whereas AFG1 and AFG2 were not detected. Excellent results were obtained by manual sorting of peeled kernels since the removal of discolored kernels (2.6%–19.9% of total peeled kernels) removed 97.3%–99.5% of total aflatoxins. The combination of peeling and visual/manual separation of discolored kernels is a feasible strategy to remove 97%–99% of aflatoxins accumulated in naturally-contaminated samples. Electronic optical sorter gave highly variable results since the amount of AFB1 + AFB2 measured in rejected fractions (15%–18% of total kernels) ranged from 13% to 59% of total aflatoxins. An improved immunoaffinity-based HPLC-FLD method having low limits of detection for the four aflatoxins (0.01–0.05 µg/kg) was developed and used to monitor the occurrence of aflatoxins in 47 commercial products containing apricot kernels and/or almonds commercialized in Italy. Low aflatoxin levels were found in 38% of the tested samples and ranged from 0.06 to 1.50 μg/kg for AFB1 and from 0.06 to 1.79 μg/kg for total aflatoxins. Full article
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Open AccessArticle Hepatic Transcriptome Responses of Domesticated and Wild Turkey Embryos to Aflatoxin B1
Received: 30 November 2015 / Revised: 23 December 2015 / Accepted: 30 December 2015 / Published: 6 January 2016
Cited by 6 | PDF Full-text (2254 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
The mycotoxin, aflatoxin B1 (AFB1) is a hepatotoxic, immunotoxic, and mutagenic contaminant of food and animal feeds. In poultry, AFB1 can be maternally transferred to embryonated eggs, affecting development, viability and performance after hatch. Domesticated turkeys (Meleagris gallopavo [...] Read more.
The mycotoxin, aflatoxin B1 (AFB1) is a hepatotoxic, immunotoxic, and mutagenic contaminant of food and animal feeds. In poultry, AFB1 can be maternally transferred to embryonated eggs, affecting development, viability and performance after hatch. Domesticated turkeys (Meleagris gallopavo) are especially sensitive to aflatoxicosis, while Eastern wild turkeys (M. g. silvestris) are likely more resistant. In ovo exposure provided a controlled AFB1 challenge and comparison of domesticated and wild turkeys. Gene expression responses to AFB1 in the embryonic hepatic transcriptome were examined using RNA-sequencing (RNA-seq). Eggs were injected with AFB1 (1 μg) or sham control and dissected for liver tissue after 1 day or 5 days of exposure. Libraries from domesticated turkey (n = 24) and wild turkey (n = 15) produced 89.2 Gb of sequence. Approximately 670 M reads were mapped to a turkey gene set. Differential expression analysis identified 1535 significant genes with |log2 fold change| ≥ 1.0 in at least one pair-wise comparison. AFB1 effects were dependent on exposure time and turkey type, occurred more rapidly in domesticated turkeys, and led to notable up-regulation in cell cycle regulators, NRF2-mediated response genes and coagulation factors. Further investigation of NRF2-response genes may identify targets to improve poultry resistance. Full article
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Open AccessArticle Identification and Quantification of a Toxigenic and Non-Toxigenic Aspergillus flavus Strain in Contaminated Maize Using Quantitative Real-Time PCR
Received: 26 October 2015 / Revised: 17 December 2015 / Accepted: 28 December 2015 / Published: 4 January 2016
Cited by 6 | PDF Full-text (1862 KB) | HTML Full-text | XML Full-text
Abstract
Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective [...] Read more.
Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective strategies for reducing aflatoxin accumulation in maize grain. Distinguishing between the toxin and non-toxin producing strains is important in determining the effectiveness of bio-control strategies and understanding inter-strain interactions. Using polymorphisms found in the fungal rRNA intergenic spacer region (IGS) between a toxigenic strain of A. flavus (NRRL 3357) and the non-toxigenic strain used in the biological control agent Afla-Guard® (NRRL 21882), we developed a set of primers that allows for the identification and quantification of the two strains using quantitative PCR. This primer set has been used to screen maize grain that was inoculated with the two strains individually and co-inoculated with both strains, and it has been shown to be effective in both the identification and quantification of both strains. Screening of co-inoculated ears from multiple resistant and susceptible genotypic crosses revealed no significant differences in fungal biomass accumulation of either strain in the field tests from 2010 and 2011 when compared across the means of all genotypes. Only one genotype/year combination showed significant differences in strain accumulation. Aflatoxin accumulation analysis showed that, as expected, genotypes inoculated with the toxigenic strain accumulated more aflatoxin than when co-inoculated with both strains or inoculated with only the non-toxigenic strain. Furthermore, accumulation of toxigenic fungal mass was significantly correlated with aflatoxin accumulation while non-toxigenic fungal accumulation was not. This primer set will allow researchers to better determine how the two fungal strains compete on the maize ear and investigate the interaction between different maize lines and these A. flavus strains. Full article
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Open AccessReview AFM1 in Milk: Physical, Biological, and Prophylactic Methods to Mitigate Contamination
Toxins 2015, 7(10), 4330-4349; https://doi.org/10.3390/toxins7104330
Received: 16 September 2015 / Revised: 15 October 2015 / Accepted: 19 October 2015 / Published: 23 October 2015
Cited by 21 | PDF Full-text (502 KB) | HTML Full-text | XML Full-text
Abstract
Aflatoxins (AFs) are toxic, carcinogenic, immunosuppressive secondary metabolites produced by some Aspergillus species which colonize crops, including many dietary staple foods and feed components. AFB1 is the prevalent and most toxic among AFs. In the liver, it is biotransformed into AFM1 [...] Read more.
Aflatoxins (AFs) are toxic, carcinogenic, immunosuppressive secondary metabolites produced by some Aspergillus species which colonize crops, including many dietary staple foods and feed components. AFB1 is the prevalent and most toxic among AFs. In the liver, it is biotransformed into AFM1, which is then excreted into the milk of lactating mammals, including dairy animals. AFM1 has been shown to be cause of both acute and chronic toxicoses. The presence of AFM1 in milk and dairy products represents a worldwide concern since even small amounts of this metabolite may be of importance as long-term exposure is concerned. Contamination of milk may be mitigated either directly, decreasing the AFM1 content in contaminated milk, or indirectly, decreasing AFB1 contamination in the feed of dairy animals. Current strategies for AFM1 mitigation include good agricultural practices in pre-harvest and post-harvest management of feed crops (including storage) and physical or chemical decontamination of feed and milk. However, no single strategy offers a complete solution to the issue. Full article
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Open AccessArticle Detoxification of Aflatoxin-Contaminated Maize by Neutral Electrolyzed Oxidizing Water
Toxins 2015, 7(10), 4294-4314; https://doi.org/10.3390/toxins7104294
Received: 17 September 2015 / Revised: 14 October 2015 / Accepted: 15 October 2015 / Published: 23 October 2015
Cited by 8 | PDF Full-text (267 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Aflatoxins, a group of extremely toxic mycotoxins produced by Aspergillus flavus, A. parasiticus and A. nomius, can occur as natural contaminants of certain agricultural commodities, particularly maize. These toxins have been shown to be hepatotoxic, carcinogenic, mutagenic and cause severe human [...] Read more.
Aflatoxins, a group of extremely toxic mycotoxins produced by Aspergillus flavus, A. parasiticus and A. nomius, can occur as natural contaminants of certain agricultural commodities, particularly maize. These toxins have been shown to be hepatotoxic, carcinogenic, mutagenic and cause severe human and animal diseases. The effectiveness of neutral electrolyzed oxidizing water (NEW) on aflatoxin detoxification was investigated in HepG2 cells using several validation methodologies such as the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay, the induction of lipid peroxidation, the oxidative damage by means of glutathione modulation, the Ames test and the alkaline Comet assay. Our results showed that, after the aflatoxin-contaminated maize containing 360 ng/g was soaked in NEW (60 mg/L available chlorine, pH 7.01) during 15 min at room temperature, the aflatoxin content did not decrease as confirmed by the immunoaffinity column and ultra performance liquid chromatography methods. Aflatoxin fluorescence strength of detoxified samples was similar to untreated samples. However, aflatoxin-associated cytotoxicity and OPEN ACCESS Toxins 2015, 7 4295 genotoxicity effects were markedly reduced upon treatment. According to these results, NEW can be effectively used to detoxify aflatoxin-contaminated maize. Full article
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Open AccessArticle Extracellular Xylanolytic and Pectinolytic Hydrolase Production by Aspergillus flavus Isolates Contributes to Crop Invasion
Toxins 2015, 7(8), 3257-3266; https://doi.org/10.3390/toxins7083257
Received: 6 July 2015 / Revised: 30 July 2015 / Accepted: 3 August 2015 / Published: 19 August 2015
Cited by 3 | PDF Full-text (450 KB) | HTML Full-text | XML Full-text
Abstract
Several atoxigenic Aspergillus flavus isolates, including some being used as biocontrol agents, and one toxigenic isolate were surveyed for the ability to produce extracellular xylanolytic and pectinolytic hydrolases. All of the tested isolates displayed good production of endoxylanases when grown on a medium [...] Read more.
Several atoxigenic Aspergillus flavus isolates, including some being used as biocontrol agents, and one toxigenic isolate were surveyed for the ability to produce extracellular xylanolytic and pectinolytic hydrolases. All of the tested isolates displayed good production of endoxylanases when grown on a medium utilizing larch xylan as a sole carbon substrate. Four of the tested isolates produced reasonably high levels of esterase activity, while the atoxigenic biocontrol agent NRRL 21882 isolate esterase level was significantly lower than the others. Atoxigenic A. flavus isolates 19, 22, K49, AF36 (the latter two are biocontrol agents) and toxigenic AF13 produced copious levels of pectinolytic activity when grown on a pectin medium. The pectinolytic activity levels of the atoxigenic A. flavus 17 and NRRL 21882 isolates were significantly lower than the other tested isolates. In addition, A. flavus isolates that displayed high levels of pectinolytic activity in the plate assay produced high levels of endopolygalacturonase (pectinase) P2c, as ascertained by isoelectric focusing electrophoresis. Isolate NRRL 21882 displayed low levels of both pectinase P2c and pectin methyl esterase. A. flavus appears capable of producing these hydrolytic enzymes irrespective of aflatoxin production. This ability of atoxigenic isolates to produce xylanolytic and pectinolytic hydrolases mimics that of toxigenic isolates and, therefore, contributes to the ability of atoxigenic isolates to occupy the same niche as A. flavus toxigenic isolates. Full article
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