Fructosyl Amino Oxidase as a Therapeutic Enzyme in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is an age-related disorder that is a global public health problem. The non-enzymatic Maillard reaction results in the formation of advanced glycation end products (AGEs). Accumulation of AGEs in drusen plays a key role in AMD. AGE-reducing drugs may contribute to the prevention and treatment of AGE-related disease. Fructosamine oxidase (FAOD) acts on fructosyl lysine and fructosyl valine. Based upon the published results of fructosamine 3-kinase (FN3K) and FAOD obtained in cataract and presbyopia, we studied ex vivo FAOD treatment as a non-invasive AMD therapy. On glycolaldehyde-treated porcine retinas, FAOD significantly reduced AGE autofluorescence (p = 0.001). FAOD treatment results in a breakdown of AGEs, as evidenced using UV fluorescence, near-infrared microspectroscopy on stained tissue sections of human retina, and gel permeation chromatography. Drusen are accumulations of AGEs that build up between Bruch's membrane and the retinal pigment epithelium. On microscopy slides of human retina affected by AMD, a significant reduction in drusen surface to 45 ± 21% was observed following FAOD treatment. Enzymatic digestion followed by mass spectrometry of fructose- and glucose-based AGEs (produced in vitro) revealed a broader spectrum of substrates for FAOD, as compared to FN3K, including the following: fructosyllysine, carboxymethyllysine, carboxyethyllysine, and imidazolone. In contrast to FN3K digestion, agmatine (4-aminobutyl-guanidine) was formed following FAOD treatment in vitro. The present study highlights the therapeutic potential of FAOD in AMD by repairing glycation-induced damage.

Chemical repertoire and biosynthetic machinery of the Aspergillus flavus secondary metabolome: A review.

Filamentous fungi represent a rich source of extrolites, including secondary metabolites (SMs) comprising a great variety of astonishing structures and interesting bioactivities. State-of-the-art techniques in genome mining, genetic manipulation, and secondary metabolomics have enabled the scientific community to better elucidate and more deeply appreciate the genetic and biosynthetic chemical arsenal of these microorganisms. Aspergillus flavus is best known as a contaminant of food and feed commodities and a producer of the carcinogenic family of SMs, aflatoxins. This fungus produces many SMs including polyketides, ribosomal and nonribosomal peptides, terpenoids, and other hybrid molecules. This review will discuss the chemical diversity, biosynthetic pathways, and biological/ecological role of A. flavus SMs, as well as their significance concerning food safety and security.

Secondary Metabolite Dereplication and Phylogenetic Analysis Identify Various Emerging Mycotoxins and Reveal the High Intra-Species Diversity in Aspergillus flavus.

Aspergillus flavus is one of the most important mycotoxigenic species from the genus Aspergillus, due to its ability to synthesize the potent hepatocarcinogen, aflatoxin B1. Moreover, this fungus is capable of producing several other toxic metabolites from the class of indole-tetramates, non-ribosomal peptides, and indole-diterpenoids. Populations of A. flavus are characterized by considerable diversity in terms of morphological, functional and genetic features. Although for many years A. flavus was considered an asexual fungus, researchers have shown evidence that at best these fungi can exhibit a predominantly asexual existence. We now know that A. flavus contains functional genes for mating, uncovering sexuality as potential contributor for its diversification. Based on our results, we reconfirm that A. flavus is a predominant producer of B-type aflatoxins. Moreover, this fungus can decisively produce AFM1 and AFM2. We did not observe any clear relationship between mating-type genes and particular class of metabolites, probably other parameters such as sexual/asexual ratio should be investigated. A dynamic secondary metabolism was found also in strains intended to be used as biocontrol agents. In addition we succeeded to provide mass spectrometry fragmentation spectra for the most important classes of A. flavus metabolites, which will serve as identification cards for future studies. Both, metabolic and phylogenetic analysis proved a high intra-species diversity for A. flavus. These findings contribute to our understanding about the diversity of Aspergillus section Flavi species, raising the necessity for polyphasic approaches (morphological, metabolic, genetic, etc.) when dealing with this type of complex group of species.

Identification and functional analysis of the aspergillic acid gene cluster in Aspergillus flavus.

Aspergillus flavus can colonize important food staples and produce aflatoxins, a group of toxic and carcinogenic secondary metabolites. Previous in silico analysis of the A. flavus genome revealed 56 gene clusters predicted to be involved in the biosynthesis of secondary metabolites. A. flavus secondary metabolites produced during infection of maize seed are of particular interest, especially with respect to their roles in the biology of the fungus. A predicted nonribosomal peptide synthetase-like (NRPS-like) gene, designated asaC (AFLA_023020), present in the uncharacterized A. flavus secondary metabolite gene cluster 11 was previously shown to be expressed during the earliest stages of maize kernel infection. Cluster 11 is composed of six additional genes encoding a number of putative decorating enzymes as well as a transporter and transcription factor. We generated knock-out mutants of the seven predicted cluster 11 genes. LC-MS analysis of extracts from knockout mutants of these genes showed that they were responsible for the synthesis of the previously characterized antimicrobial mycotoxin aspergillic acid. Extracts of the asaC mutant showed no production of aspergillic acid or its precursors. Knockout of the cluster 11 P450 oxidoreductase afforded a pyrazinone metabolite, the aspergillic acid precursor deoxyaspergillic acid. The formation of hydroxyaspergillic acid was abolished in a desaturase/hydroxylase mutant. The hydroxamic acid functional group in aspergillic acid allows the molecule to bind to iron resulting in the production of a red pigment in A. flavus identified previously as ferriaspergillin. A reduction of aflatoxin B1 and cyclopiazonic acid that correlated with reduced fungal growth was observed in maize kernel infection assays when aspergillic acid biosynthesis in A. flavus is halted.

In-house validation of a rapid and efficient procedure for simultaneous determination of ergot alkaloids and other mycotoxins in wheat and maize.

A fundamental step in addressing the global problem of mycotoxins is the development of highly sensitive, multi-class extraction and detection methods. This constitutes a field of research that has in recent years enjoyed a steady advance. Such methods, generally based on liquid chromatography coupled to mass spectrometry, are widely reported successfully detecting various mycotoxins in different food and feed samples. In this work, an innovative approach to multi-class mycotoxin control is proposed, offering specific advantages: a broader inclusion of more mycotoxin classes, robust and thorough extraction for all target compounds despite their varied chemical properties, and determination of all analytes from a single injection. The method involved the extraction and quantification of the main mycotoxins produced by Aspergillus, Fusarium, and Penicillium fungi, as well as their reported derivatives, together with 12 other compounds most commonly produced by Claviceps purpurea. The popularly reported QuEChERS technique has been reduced to a simple "salting-out liquid-liquid extraction" (SO-LLE) to obtain the most efficient extraction of the aforementioned mycotoxin classes in a very short time. This is in particular extremely important in ensuring correct determination of individual ergot alkaloids, for which short and robust sample preparation as well as short analytical sequences were key for minimizing the epimerization during analysis. The analyses of wheat and maize samples were performed using ultra-high performance liquid chromatography coupled with tandem mass spectrometry. Matrix-matched calibration curves were established and limits of quantification were below the maximum levels established by the EU regulation. The precision (repeatability and intermediate precision) was lower than 13% in all cases and recoveries ranged between 60 and 98% in maize and between 62 and 103% in wheat, fulfilling the current legislation. The method was applied to study the co-occurrence of these mycotoxins in wheat (n = 13) and maize (n = 15) samples from six European countries. A successful quantification of 23 different mycotoxins, from all major classes, in 85% of wheat and 93% of maize samples was achieved.

Genetic and Toxigenic Variability within Aspergillus flavus Population Isolated from Maize in Two Diverse Environments in Kenya.

Aspergillus flavus is the main producer of carcinogenic aflatoxins in agricultural commodities such as maize. This fungus occurs naturally on crops, and produces aflatoxins when environmental conditions are favorable. The aim of this study is to analyse the genetic variability among 109 A. flavus isolates previously recovered from maize sampled from a known aflatoxin-hotspot (Eastern region, Kenya) and the major maize-growing area in the Rift Valley (Kenya), and to determine their toxigenic potential. DNA analyses of internal transcribed spacer (ITS) regions of ribosomal DNA, partial ß-tubulin gene (benA) and calmodulin gene (CaM) sequences were used. The strains were further analyzed for the presence of four aflatoxin-biosynthesis genes in relation to their capability to produce aflatoxins and other metabolites, targeting the regulatory gene aflR and the structural genes aflP, aflD, and aflQ. In addition, the metabolic profile of the fungal strains was unraveled using state-of-the-art LC-MS/MS instrumentation. The three gene-sequence data grouped the isolates into two major clades, A. minisclerotigenes and A. flavus. A. minisclerotigenes was most prevalent in Eastern Kenya, while A. flavus was common in both regions. A. parasiticus was represented by a single isolate collected from Rift Valley. Diversity existed within the A. flavus population, which formed several subclades. An inconsistency in identification of some isolates using the three markers was observed. The calmodulin gene sequences showed wider variation of polymorphisms. The aflatoxin production pattern was not consistent with the presence of aflatoxigenic genes, suggesting an inability of the primers to always detect the genes or presence of genetic mutations. Significant variation was observed in toxin profiles of the isolates. This is the first time that a profound metabolic profiling of A. flavus isolates was done in Kenya. Positive associations were evident for some metabolites, while for others no associations were found and for a few metabolite-pairs negative associations were seen. Additionally, the growth medium influenced the mycotoxin metabolite production. These results confirm the wide variation that exists among the group A. flavus and the need for more insight in clustering the group.

Aspergillus flavus aswA, a gene homolog of Aspergillus nidulans oefC, regulates sclerotial development and biosynthesis of sclerotium-associated secondary metabolites.

Aspergillus flavus aswA (AFLA_085170) is a gene encoding a Zn(II)2Cys6 DNA-binding domain and a transcriptional activation domain, DUF3468. Disruption of aswA yielded strains that made a truncated gene transcript and generated a fungus that produced a greatly increased number of sclerotia. These sclerotia were odd-shaped and non-pigmented (white) and different from oval and pigmented (dark brown to black) mature sclerotia. Transcriptomic analysis of the ΔaswA strain grown on potato dextrose agar plates and Wickerham agar plates showed that expression of clustering genes involved in the biosynthesis of three sclerotium-associated secondary metabolites was down-regulated. These included gene clusters of asparasone, aflatrem, and aflavarin. In contrast, those of aflatoxin, cyclopiazonic acid and kojic acid were not affected. Metabolite analyses confirmed that the non-pigmented sclerotia contained aflatoxin and cyclopiazonic acid but not other aforementioned metabolites, three asparasone analogs and dihydroxyaflavinine commonly present in mature sclerotia. Impairment in aswA gene function stalls normal sclerotial development, which in turn prevents biosynthesis and accumulation of sclerotium-specific metabolites.

Analytical strategy for determination of known and unknown destruxins using hybrid quadrupole-Orbitrap high-resolution mass spectrometry.

An analytical strategy based on a hybrid quadrupole-Orbitrap mass spectrometry was proposed for the simultaneous screening of known destruxins and characterization of potential members of this class of secondary metabolites, in order to evaluate the metabolite production of entomopathogenic fungi used as biocontrol agents. Initially, the fragmentation pathway of the known and commercially available destruxin A was established combining high resolution mass spectrometry (HRMS) and multiple stage MS data in order to obtain the strategy for the characterization of other destruxins for which reference standards were not available. Nineteen known destruxins including A, B, C, D, Ed, F, A1, B1, Ed1, A2, B2, D2, A3, DesmA, DesmB, DesmC, DesmB2, and two chloro-derivatives (Cl and E2 chlorohydrin) were unequivocally identified in Metarhizium brunneum using the proposed strategy. In addition, four unknown destruxins, namely C1, Ed2, G, and G1, were structurally elucidated and characterized for the first time in this fungal strain.

Unravelling the Diversity of the Cyclopiazonic Acid Family of Mycotoxins in Aspergillus flavus by UHPLC Triple-TOF HRMS.

Cyclopiazonic acid (α-cyclopiazonic acid, α-CPA) is an indole-hydrindane-tetramic acid neurotoxin produced by various fungal species, including the notorious food and feed contaminant Aspergillus flavus. Despite its discovery in A. flavus cultures approximately 40 years ago, its contribution to the A. flavus mycotoxin burden is consistently minimized by our focus on the more potent carcinogenic aflatoxins also produced by this fungus. Here, we report the screening and identification of several CPA-type alkaloids not previously found in A. flavus cultures. Our identifications of these CPA-type alkaloids are based on a dereplication strategy involving accurate mass high resolution mass spectrometry data and a careful study of the α-CPA fragmentation pattern. In total, 22 CPA-type alkaloids were identified in extracts from the A. flavus strains examined. Of these metabolites, 13 have been previously reported in other fungi, though this is the first report of their existence in A. flavus. Two of our metabolite discoveries, 11,12-dehydro α-CPA and 3-hydroxy-2-oxo CPA, have never been reported for any organism. The conspicuous presence of CPA and its numerous derivatives in A. flavus cultures raises concerns about the long-term and cumulative toxicological effects of these fungal secondary metabolites and their contributions to the entire A. flavus mycotoxin problem.

The Aspergillus flavus fluP-associated metabolite promotes sclerotial production.

Aspergillus flavus is able to synthesize a variety of polyketide-derived secondary metabolites including the hepatocarcinogen, aflatoxin B1. The fungus reproduces and disseminates predominantly by production of conidia. It also produces hardened mycelial aggregates called sclerotia that are used to cope with unfavourable growth environments. In the present study, we examined the role of A. flavus fluP, the backbone polyketide synthase gene of secondary metabolite gene cluster 41, on fungal development. The A. flavus CA14 fluP deletion mutant (AfΔfluP) grew and accumulated aflatoxin normally but produced a lower amount of sclerotia than the parental strain. This was also true for the Aspergillus parasiticus BN9 fluP deletion mutant (ApΔfluP). The A. flavus fluP gene was positively regulated by developmental regulators of VeA and VelB but not by the global regulator of secondary metabolism, LaeA. Overexpression of fluP in AfΔfluP (OEfluP) elevated its ability to produce sclerotia compared to that of the parental strain. Coculture of OEfluP with CA14, AfΔfluP, ApΔfluP, or an A. flavus pptA deletion mutant incapable of producing functional polyketide synthases also allowed increased sclerotial production of the respective strains at edges where colonies made contact. Acetone extracts of OEfluP but not of AfΔfluP exhibited the same effect in promoting sclerotial production of AfΔfluP. These results suggest that FluP polyketide synthase is involved in the synthesis of a diffusible metabolite that could serve as a signal molecule to regulate sclerotiogenesis.

Validated UPLC-MS/MS Methods To Quantitate Free and Conjugated Alternaria Toxins in Commercially Available Tomato Products and Fruit and Vegetable Juices in Belgium.

Ultraperformance liquid chromatography tandem mass spectrometry and Quick, Easy, Cheap, Effective, Rugged, and Safe based analytical methodologies to quantitate both free (alternariol (1), alternariol monomethyl ether (2), tenuazonic acid (3), tentoxin (4), altenuene (5), altertoxin-I (6)) and conjugated (sulfates and glucosides of 1 and 2) Alternaria toxins in fruit and vegetable juices and tomato products were developed and validated. Acceptable limits of quantitation (0.7-5.7 µg/kg), repeatability (RSDr < 15.7%), reproducibility (RSDR < 17.9%), and apparent recovery (87.0-110.6%) were obtained for all analytes in all matrices investigated. 129 commercial foodstuffs were analyzed, and 3 was detected in 100% of tomato product samples (

Holistic approach based on high resolution and multiple stage mass spectrometry to investigate ergot alkaloids in cereals.

A holistic approach based on high resolution and multiple stage mass spectrometry was developed for identification of less studied or novel ergot alkaloid derivatives. Initially, the fragmentation of nine known ergot alkaloids was studied to establish a strategy for the identification of novel ergot alkaloids. Ions with m/z 223 and m/z 251 were found to be common for all ergopeptines, ergoamides and ergopeptams. Subsequently, parent scan experiments using these ions were performed to screen grain samples for the presence of possible ergot alkaloid derivatives. Besides the six most common ergot alkaloids and their corresponding epimers (for which reference standards were available), eleven other ergot alkaloid derivatives were identified following the proposed strategy.

Multimycotoxin analysis in urines to assess infant exposure: a case study in Cameroon.

This study was conducted to investigate mycotoxin exposure in children (n=220, aged 1.5-4.5years) from high mycotoxin contamination regions of Cameroon and to examine the association between the mycotoxin levels (in total 18 analytes) and several socio-demographic factors and anthropometric characteristics. A cross-sectional study was conducted in six villages in Cameroon with 220 children. Mycotoxins and their metabolites were detected in 160/220 (73%) urine samples. There were significant differences in the mean contamination levels of ochratoxin A (p=0.01) and ß-zearalenol (p=0.017) between the two agro-ecological zones investigated. Likewise significant differences were observed in the mean levels of aflatoxin M1 (p=0.001) across the weaning categories of these children. The mean concentration of aflatoxin M1 detected in the urine of the partially breastfed children (1.43ng/mL) was significantly higher (p=0.001) than those of the fully weaned children (0.282ng/mL). Meanwhile, the mean concentrations of deoxynivalenol (3.0ng/mL) and fumonisin B1 (0.59ng/mL) detected in the urine of the male children was significantly (p value 0.021 for deoxynivalenol and 0.004 for fumonisin B1) different from the levels detected in the urine of female children; 0.71ng/mL and 0.01ng/mL for deoxynivalenol and fumonisin B1 respectively. In this study, there was no association between the different malnutrition categories (stunted, wasting and underweight) and the mycotoxin concentrations detected in the urine of these children. However, there is sufficient evidence to suggest that children in Cameroon under the age 5 are exposed to high levels of carcinogenic substances such as fumonisin B1, aflatoxin M1 and ochratoxin A through breastfeeding. To the best of our knowledge, this is the first report of its kind carried out in West Africa to determine multi-mycotoxin exposure in infants.

A systematic assessment of the variability of matrix effects in LC-MS/MS analysis of ergot alkaloids in cereals and evaluation of method robustness.

The study presents for the first time a systematic investigation of matrix effects in the LC-MS/MS analysis of ergot alkaloids in cereals. In order to assure the accuracy of the results, several approaches to minimize/eliminate matrix effects were investigated including variation of ionization techniques, chromatography and sample preparation on different grain types and grain varieties. It was revealed that the use of UPLC and careful choice of sample preparation might reduce signal suppression/enhancement. In general, ergometrine was found to be the most susceptible among the ergot alkaloids studied, but none of the used approaches suggested a total elimination of matrix effects; only less than half of its MS signal could be recovered. The late-eluting compounds were less affected by matrix components in all conditions tested. Further, the robustness of the applied LC-MS method was checked by means of a fractional factorial design. The results indicate that small changes to the sample preparation parameters, namely pH and concentration of extraction buffer, shaking time, drying temperature and extraction volumes, did not significantly (α = 0.05) affect the recoveries of ergot alkaloids.

Development of suspension polymerized molecularly imprinted beads with metergoline as template and application in a solid-phase extraction procedure toward ergot alkaloids.

The first successfully developed molecularly imprinted polymer toward six ergot alkaloids and their respective epimers is described. A new imprinting molecule, metergoline, was used as template analogue in the production of suspension polymerized beads. These spherical particles functioned as selective sorbent in a solid-phase extraction column. The application of this column in the cleanup of barley samples prior to liquid chromatography coupled with tandem mass spectrometry allowed simple and cost-efficient sample preparation. The performance of the imprinted polymer and a non-imprinted control polymer was evaluated. This includes determination of the recovery values and the matrix effect of each of the 12 tested ergot alkaloids as well as a cross-reactivity study with 25 common mycotoxins. The binding isotherms were obtained for metergoline, thus allowing comparison with other (imprinted) sorbents. A comparison between bulk and suspension polymerization is provided to determine the appropriate production technique.

Improved positive electrospray ionization of patulin by adduct formation: usefulness in liquid chromatography-tandem mass spectrometry multi-mycotoxin analysis.

Several sensitive methods have been developed for patulin determination; however, mass spectrometric (MS) detection of this toxin in the positive electrospray ionization (ESI(+)) mode is not straightforward. Furthermore, the combined determination of patulin with other mycotoxins in one single run has not been reported yet. The present paper demonstrates the formation and use of a methanol adduct of patulin in ESI(+). A study of the fragmentation pathway confirmed the authenticity of the patulin adduct, while the use of ion trap and high resolution Orbitrap mass spectrometry allowed reliable assignment of the patulin fragment ions. Exploiting the formation of the methanol adduct, patulin has been successfully included in a single run multi-mycotoxin liquid chromatography tandem mass spectrometric (LC-MS/MS) method in support of ex vivo-in vitro biomedical studies.

Human skin penetration of selected model mycotoxins.

Dermal exposure data for mycotoxins are very scarce and fragmentary, despite their widespread skin contact and hazard toxicity. In this study, the transdermal kinetics of aflatoxin B1 (AFB1), ochratoxin A (OTA), fumonisin B1 (FB1), citrinin (CIT), zearalenone (ZEA) and T-2 toxin (T-2) were quantitatively evaluated, using human skin in an in vitro Franz diffusion cell set-up. All mycotoxins penetrated through the skin, except for FB1, which showed concentrations in the receptor fluid below the LoD, resulting in a K(p)<3.24×10(-6)cm/h. OTA showed the highest permeation (K(p)=8.20×10(-4)cm/h), followed by CIT (K(p)=4.67×10(-4)cm/h). AFB1 and ZEA showed lower permeability rates (K(p)=2.11 and 2.33×10(-4)cm/h, respectively). T-2 was found to have the lowest permeability (K(p)=6.07×10(-5)cm/h). From literature-based mycotoxin-concentrations, dermal contact surface, exposure time and apparent K(p)'s obtained in this study, the daily dermal exposure (DDE) in two industrial and one residential scenario was estimated. Dermal exposure to the DNA-reactive genotoxic carcinogenic AFB1 can lead to a health risk for agricultural workers which are exposed to a mycotoxin contaminated solution in a worst case situation. For all the other investigated mycotoxins, no significant health risk is calculated after dermal contact in neither agricultural nor residential environments.

LC-MS/MS multi-analyte method for mycotoxin determination in food supplements.

A multi-analyte method for the liquid chromatography-tandem mass spectrometric determination of mycotoxins in food supplements is presented. The analytes included A and B trichothecenes (nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin and T-2 toxin), aflatoxins (aflatoxin-B(1), aflatoxin-B(2), aflatoxin-G(1) and aflatoxin-G(2)), Alternaria toxins (alternariol, alternariol methyl ether and altenuene), fumonisins (fumonisin-B(1), fumonisin-B(2) and fumonisin-B(3)), ochratoxin A, zearalenone, beauvericin and sterigmatocystin. Optimization of the simultaneous extraction of these toxins and the sample pretreatment procedure, as well as method validation were performed on maca (Lepidium meyenii) food supplements. The results indicated that the solvent mixture ethyl acetate/formic acid (95:5, v/v) was the best compromise for the extraction of the analytes from food supplements. Liquid-liquid partition with n-hexane was applied as partial clean-up step to remove excess of co-extracted non-polar components. Further clean-up was performed on Oasis HLB cartridges. Samples were analysed using an Acquity UPLC system coupled to a Micromass Quattro Micro triple quadrupole mass spectrometer equipped with an electrospray interface operated in the positive-ion mode. Limits of detection and quantification were in the range of 0.3-30 ng g(-1) and 1-100 ng g(-1), respectively. Recovery yields were above 60% for most of the analytes, except for nivalenol, sterigmatocystine and the fumonisins. The method showed good precision and trueness. Analysis of different food supplements such as soy (Glycine max) isoflavones, St John's wort (Hypericum perforatum), garlic (Allium sativum), Ginkgo biloba, and black radish (Raphanus niger) demonstrated the general applicability of the method. Due to different matrix effects observed in different food supplement samples, the standard addition approach was applied to perform correct quantitative analysis. In 56 out of 62 samples analysed, none of the 23 mycotoxins investigated was detected. Positive samples contained at least one of the toxins fumonisin-B(1), fumonisin-B(2), fumonisin-B(3) and ochratoxin A.