Open AccessArticle
trans-Cinnamic and Chlorogenic Acids Affect the Secondary Metabolic Profiles and Ergosterol Biosynthesis by Fusarium culmorum and F. graminearum Sensu Stricto
Toxins 2017, 9(7), 198; doi:10.3390/toxins9070198 -
Abstract
Plant-derived compounds limiting mycotoxin contamination are currently of major interest in food and feed production. However, their potential application requires an evaluation of their effects on fungal secondary metabolism and membrane effects. In this study, different strains of Fusarium culmorum and F. graminearum
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Plant-derived compounds limiting mycotoxin contamination are currently of major interest in food and feed production. However, their potential application requires an evaluation of their effects on fungal secondary metabolism and membrane effects. In this study, different strains of Fusarium culmorum and F. graminearum sensu stricto were exposed to trans-cinnamic and chlorogenic acids on solid YES media. Fusaria produced phenolic acids, whose accumulation was lowered by exogenous phenolic compounds. In addition, fungi reduced exogenous phenolic acids, leading either to their conversion or degradation. trans-Cinnamic acid was converted to caffeic and ferulic acids, while chlorogenic acid was degraded to caffeic acid. The latter underwent further degradation to protocatechuic acid. Fungal-derived trans-cinnamic acid, as the first intermediate of the shikimate pathway, increased after chlorogenic acid treatment, presumably due to the further inhibition of the conversion of trans-cinnamic acid. Exogenous trans-cinnamic and chlorogenic acid displayed the inhibition of mycotoxin production by Fusaria, which appeared to be largely dependent on the phenolic compound and its concentration and the assayed strain. Exogenous phenolic acids showed different effects on ergosterol biosynthesis by fungi. It was found that the production of this membrane sterol was stimulated by trans-cinnamic acid, while chlorogenic acid negatively impacted ergosterol biosynthesis, suggesting that phenolic acids with stronger antifungal activities may upregulate ergosterol biosynthesis by Fusaria. This paper reports on the production of phenolic acids by Fusaria for the first time. Full article
Open AccessArticle
Deoxynivalenol Biomarkers in the Urine of UK Vegetarians
Toxins 2017, 9(7), 196; doi:10.3390/toxins9070196 -
Abstract
Deoxynivalenol (DON) is produced by Fusarium graminearum and is one of the most commonly occurring trichothecenes. Vegetarians are alleged to be a high-risk group for DON exposure due to high intakes of cereals susceptible to the growth of the mycotoxin. This study provides
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Deoxynivalenol (DON) is produced by Fusarium graminearum and is one of the most commonly occurring trichothecenes. Vegetarians are alleged to be a high-risk group for DON exposure due to high intakes of cereals susceptible to the growth of the mycotoxin. This study provides the levels of DON and de-epoxi Deoxynivalenol (DOM-1) in urine analysed by liquid chromatography-mass spectrometry (LC-MS) in UK vegetarians. Over two consecutive days, morning urine samples were collected from 32 vegetarians and 31 UK adult volunteers, and associated food consumption 24 h prior to the sample was recorded. Statistically significant differences between the weight of the UK adults and vegetarians (t = 3.15. df = 61, p ≤ 0.005 two-tailed) were observed. The mean levels of DON in urine for adults on day 1 was 3.05 ng free DON/mg creatinine, and on day 2 was 2.98 ng free DON/mg creatinine. Even though high mean levels were observed, most adults were within the tolerable daily intake. However, for vegetarians, the mean level of urinary DON on day 1 was 6.69 ng free DON/mg creatinine, and on day 2 was 3.42 ng free DON/mg creatinine. These levels equate to up to 32% of vegetarians exceeding recommended tolerable daily intakes (TDI) of exposure (1 µg/kg b.w./day). Full article
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Open AccessArticle
Impact of Mycotoxins Secreted by Aspergillus Molds on the Inflammatory Response of Human Corneal Epithelial Cells
Toxins 2017, 9(7), 197; doi:10.3390/toxins9070197 -
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
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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
Virulence Genes of S. aureus from Dairy Cow Mastitis and Contagiousness Risk
Toxins 2017, 9(6), 195; doi:10.3390/toxins9060195 -
Abstract
Staphylococcus aureus (S. aureus) is a major agent of dairy cow intramammary infections: the different prevalences of mastitis reported might be related to a combination of S. aureus virulence factors beyond host factors. The present study considered 169 isolates from different
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Staphylococcus aureus (S. aureus) is a major agent of dairy cow intramammary infections: the different prevalences of mastitis reported might be related to a combination of S. aureus virulence factors beyond host factors. The present study considered 169 isolates from different Italian dairy herds that were classified into four groups based on the prevalence of S. aureus infection at the first testing: low prevalence (LP), medium–low (MLP), medium–high (MHP) and high (HP). We aimed to correlate the presence of virulence genes with the prevalence of intramammary infections in order to develop new strategies for the control of S. aureus mastitis. Microarray data were statistically evaluated using binary logistic regression and correspondence analysis to screen the risk factors and the relationship between prevalence group and gene. The analysis showed: (1) 24 genes at significant risk of being detected in all the herds with infection prevalence >5%, including genes belonging to microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), immune evasion and serine proteases; and (2) a significant correlation coefficient between the genes interacting with the host immune response and HP isolates against LP ones. These results support the hypothesis that virulence factors, in addition to cow management, could be related to strain contagiousness, offering new insights into vaccine development. Full article
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Open AccessArticle
Characterization of Hemagglutinin Negative Botulinum Progenitor Toxins
Toxins 2017, 9(6), 193; doi:10.3390/toxins9060193 -
Abstract
Botulism is a disease involving intoxication with botulinum neurotoxins (BoNTs), toxic proteins produced by Clostridium botulinum and other clostridia. The 150 kDa neurotoxin is produced in conjunction with other proteins to form the botulinum progenitor toxin complex (PTC), alternating in size from 300
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Botulism is a disease involving intoxication with botulinum neurotoxins (BoNTs), toxic proteins produced by Clostridium botulinum and other clostridia. The 150 kDa neurotoxin is produced in conjunction with other proteins to form the botulinum progenitor toxin complex (PTC), alternating in size from 300 kDa to 500 kDa. These progenitor complexes can be classified into hemagglutinin positive or hemagglutinin negative, depending on the ability of some of the neurotoxin-associated proteins (NAPs) to cause hemagglutination. The hemagglutinin positive progenitor toxin complex consists of BoNT, nontoxic non-hemagglutinin (NTNH), and three hemagglutinin proteins; HA-70, HA-33, and HA-17. Hemagglutinin negative progenitor toxin complexes contain BoNT and NTNH as the minimally functional PTC (M-PTC), but not the three hemagglutinin proteins. Interestingly, the genome of hemagglutinin negative progenitor toxin complexes comprises open reading frames (orfs) which encode for three proteins, but the existence of these proteins has not yet been extensively demonstrated. In this work, we demonstrate that these three proteins exist and form part of the PTC for hemagglutinin negative complexes. Several hemagglutinin negative strains producing BoNT/A, /E, and /F were found to contain the three open reading frame proteins. Additionally, several BoNT/A-containing bivalent strains were examined, and NAPs from both genes, including the open reading frame proteins, were associated with BoNT/A. The open reading frame encoded proteins are more easily removed from the botulinum complex than the hemagglutinin proteins, but are present in several BoNT/A and /F toxin preparations. These are not easily removed from the BoNT/E complex, however, and are present even in commercially-available purified BoNT/E complex. Full article
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Open AccessArticle
Occurrence of Penicillium brocae and Penicillium citreonigrum, which Produce a Mutagenic Metabolite and a Mycotoxin Citreoviridin, Respectively, in Selected Commercially Available Rice Grains in Thailand
Toxins 2017, 9(6), 194; doi:10.3390/toxins9060194 -
Abstract
Commercially available rice grains in Thailand were examined to isolate the monoverticillate Penicillium species responsible for toxic yellowed rice. Penicillium species were obtained from seven out of 10 rice samples tested. Among them, one Penicillium citreonigrum isolate and six Penicillium brocae isolates were
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Commercially available rice grains in Thailand were examined to isolate the monoverticillate Penicillium species responsible for toxic yellowed rice. Penicillium species were obtained from seven out of 10 rice samples tested. Among them, one Penicillium citreonigrum isolate and six Penicillium brocae isolates were morphologically identified. The P. citreonigrum isolate produced the mycotoxin citreoviridin on a yeast extract sucrose broth medium. Mycotoxin surveys showed that citreoviridin was not detected in any samples, but one out of 10 rice samples tested was positive for aflatoxin B1 at a level of 5.9 μg/kg. An Ames test revealed that methanol extracts from rice grains inoculated with selected P. brocae isolates were positive for strains TA100 and YG7108 of Salmonella typhimurium, suggesting the presence of base-pair substitution and DNA alkylation mutagens. Our data obtained here demonstrated that aflatoxin B1 and toxic P. citreonigrum were present on domestic rice grains in Thailand, although limited samples were tested. Penicillium brocae, which may produce mutagenic metabolites, was isolated for the first time from the surface of Thai rice grains. Full article
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Open AccessReview
Talaromyces marneffei Genomic, Transcriptomic, Proteomic and Metabolomic Studies Reveal Mechanisms for Environmental Adaptations and Virulence
Toxins 2017, 9(6), 192; doi:10.3390/toxins9060192 -
Abstract
Talaromycesmarneffei is a thermally dimorphic fungus causing systemic infections in patients positive for HIV or other immunocompromised statuses. Analysis of its ~28.9 Mb draft genome and additional transcriptomic, proteomic and metabolomic studies revealed mechanisms for environmental adaptations and virulence. Meiotic genes and
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Talaromycesmarneffei is a thermally dimorphic fungus causing systemic infections in patients positive for HIV or other immunocompromised statuses. Analysis of its ~28.9 Mb draft genome and additional transcriptomic, proteomic and metabolomic studies revealed mechanisms for environmental adaptations and virulence. Meiotic genes and genes for pheromone receptors, enzymes which process pheromones, and proteins involved in pheromone response pathway are present, indicating its possibility as a heterothallic fungus. Among the 14 Mp1p homologs, only Mp1p is a virulence factor binding a variety of host proteins, fatty acids and lipids. There are 23 polyketide synthase genes, one for melanin and two for mitorubrinic acid/mitorubrinol biosynthesis, which are virulence factors. Another polyketide synthase is for biogenesis of the diffusible red pigment, which consists of amino acid conjugates of monascorubin and rubropunctatin. Novel microRNA-like RNAs (milRNAs) and processing proteins are present. The dicer protein, dcl-2, is required for biogenesis of two milRNAs, PM-milR-M1 and PM-milR-M2, which are more highly expressed in hyphal cells. Comparative transcriptomics showed that tandem repeat-containing genes were overexpressed in yeast phase, generating protein polymorphism among cells, evading host’s immunity. Comparative proteomics between yeast and hyphal cells revealed that glyceraldehyde-3-phosphate dehydrogenase, up-regulated in hyphal cells, is an adhesion factor for conidial attachment. Full article
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Open AccessArticle
Recombinant Phospholipase D from Loxosceles gaucho Binds to Platelets and Promotes Phosphatidylserine Exposure
Toxins 2017, 9(6), 191; doi:10.3390/toxins9060191 -
Abstract
Spider envenomation, from the genus Loxosceles, is frequently reported as a cause of necrotic lesions in humans around the world. Among the many components found in the venom of Loxosceles genus, phospholipases D (PLDs) are the most investigated, since they can cause
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Spider envenomation, from the genus Loxosceles, is frequently reported as a cause of necrotic lesions in humans around the world. Among the many components found in the venom of Loxosceles genus, phospholipases D (PLDs) are the most investigated, since they can cause a massive inflammatory response, dermonecrosis, hemolysis and platelet aggregation, among other effects. Even though the PLDs induce strong platelet aggregation, there are no studies showing how the PLDs interact with platelets to promote this effect. Since many agonists must interact with specific receptors on the platelet membrane to induce aggregation, it is reasonable to expect that the PLDs may, in some way, also interact with platelets, to induce this activity. Therefore, to address this possibility, in this work, a recombinant PLD, called LgRec1, from L. gaucho was fused to enhanced green fluorescent protein (EGFP) and used as a probe to detect the interaction of LgRec1 to platelets, by fluorescence-activated cell sorter (FACS) and confocal microscopy. The preservation of biological activities of this chimera toxin was also analyzed. As a first, the results show that LgRec1 does not require plasma components to bind to platelets, although these components are necessary to LgRec1 to induce platelet aggregation. Also, the attachment of LgRec1 to human platelets’ cell membranes suggests that the exposure of phosphatidylserine (PS) may act as a scaffold for coagulation factors. Therefore, the results add new information about the binding of Loxosceles PLDs to platelets, which may help unravel how these toxins promote platelet aggregation. Full article
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Open AccessArticle
Presence of Enniatins and Beauvericin in Romanian Wheat Samples: From Raw Material to Products for Direct Human Consumption
Toxins 2017, 9(6), 189; doi:10.3390/toxins9060189 -
Abstract
In this study, a total of 244 wheat and wheat-based products collected from Romania were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) in order to evaluate the presence of four enniatins (ENs; i.e., ENA, ENA1, ENB, and ENB1) and beauvericin (BEA). For
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In this study, a total of 244 wheat and wheat-based products collected from Romania were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) in order to evaluate the presence of four enniatins (ENs; i.e., ENA, ENA1, ENB, and ENB1) and beauvericin (BEA). For the wheat samples, the influence of agricultural practices was assessed, whereas the results for the wheat-based products were used to calculate the estimated daily intake of emerging mycotoxins through wheat consumption for the Romanian population. ENB presented the highest incidence (41% in wheat and 32% in wheat-based products), with its maximum levels of 815 μg kg−1 and 170 μg kg−1 in wheat and wheat-based products, respectively. The correlation between the concentrations of ENB and ENB1 in wheat grain samples and farm practices (organic or conventional) was confirmed statistically (p < 0.05). This is the first study that provides comprehensive information about the influence of agricultural practice on emerging Fusarium mycotoxin presence in Romanian wheat samples and the estimated daily intake of ENs and BEA present in wheat-based products for human consumption commercialized in Romania. Full article
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Open AccessArticle
Prevalence, Variability and Bioconcentration of Saxitoxin-Group in Different Marine Species Present in the Food Chain
Toxins 2017, 9(6), 190; doi:10.3390/toxins9060190 -
Abstract
The saxitoxin-group (STX-group) corresponds to toxic metabolites produced by cyanobacteria and dinoflagellates of the genera Alexandrium, Gymnodinium, and Pyrodinium. Over the last decade, it has been possible to extrapolate the areas contaminated with the STX-group worldwide, including Chile, a phenomenon that
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The saxitoxin-group (STX-group) corresponds to toxic metabolites produced by cyanobacteria and dinoflagellates of the genera Alexandrium, Gymnodinium, and Pyrodinium. Over the last decade, it has been possible to extrapolate the areas contaminated with the STX-group worldwide, including Chile, a phenomenon that has affected ≈35% of the Southern Pacific coast territory, generating a high economic impact. The objective of this research was to study the toxicity of the STX-group in all aquatic organisms (bivalves, algae, echinoderms, crustaceans, tunicates, cephalopods, gastropods, and fish) present in areas with a variable presence of harmful algal blooms (HABs). Then, the toxic profiles of each species and dose of STX equivalents ingested by a 60 kg person from 400 g of shellfish were determined to establish the health risk assessment. The toxins with the highest prevalence detected were gonyautoxin-4/1 (GTX4/GTX1), gonyautoxin-3/2 (GTX3/GTX2), neosaxitoxin (neoSTX), decarbamoylsaxitoxin (dcSTX), and saxitoxin (STX), with average concentrations of 400, 2800, 280, 200, and 2000 µg kg−1 respectively, a species-specific variability, dependent on the evaluated tissue, which demonstrates the biotransformation of the analogues in the trophic transfer with a predominance of α-epimers in all toxic profiles. The identification in multiple vectors, as well as in unregulated species, suggests that a risk assessment and risk management update are required; also, chemical and specific analyses for the detection of all analogues associated with the STX-group need to be established. Full article
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Open AccessArticle
Embryotoxicity Caused by DON-Induced Oxidative Stress Mediated by Nrf2/HO-1 Pathway
Toxins 2017, 9(6), 188; doi:10.3390/toxins9060188 -
Abstract
Deoxynivalenol (DON) belongs to the type B group of trichothecenes family, which is composed of sesquiterpenoid metabolites produced by Fusarium and other fungi in grain. DON may cause various toxicities, such as cytotoxicity, immunotoxicity, genotoxicity as well as teratogenicity and carcinogenicity. In the
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Deoxynivalenol (DON) belongs to the type B group of trichothecenes family, which is composed of sesquiterpenoid metabolites produced by Fusarium and other fungi in grain. DON may cause various toxicities, such as cytotoxicity, immunotoxicity, genotoxicity as well as teratogenicity and carcinogenicity. In the present study, we focus on a hypothesis that DON alters the expressions of Nrf2/HO-1 pathway by inducing embryotoxicity in C57BL/6 mouse (5.0, 2.5, 1.0, and 0 mg/kg/day) and BeWo cell lines (0 and 50 nM; 3 h, 12 h and 24 h). Our results indicate that DON treatment in mice during pregnancy leads to ROS accumulation in the placenta, which results in embryotoxicity. At the same time Nrf2/HO-1 pathway is up-regulated by ROS to protect placenta cells from oxidative damage. In DON-treated BeWo cells, the level of ROS has time–effect and dose–effect relationships with HO-1 expression. Moderate increase in HO-1 protects the cell from oxidative damage, while excessive increase in HO-1 aggravates the oxidative damage, which is called in some studies the “threshold effect”. Therefore, oxidative stress may be the critical molecular mechanism for DON-induced embryotoxicity. Besides, Nrf2/HO-1 pathway accompanied by the “threshold effect” also plays an important role against DON-induced oxidative damage in this process. Full article
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Open AccessArticle
Coralsnake Venomics: Analyses of Venom Gland Transcriptomes and Proteomes of Six Brazilian Taxa
Toxins 2017, 9(6), 187; doi:10.3390/toxins9060187 -
Abstract
Venom gland transcriptomes and proteomes of six Micrurus taxa (M. corallinus, M. lemniscatus carvalhoi, M. lemniscatus lemniscatus, M. paraensis, M. spixii spixii, and M. surinamensis) were investigated, providing the most comprehensive, quantitative data on Micrurus venom
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Venom gland transcriptomes and proteomes of six Micrurus taxa (M. corallinus, M. lemniscatus carvalhoi, M. lemniscatus lemniscatus, M. paraensis, M. spixii spixii, and M. surinamensis) were investigated, providing the most comprehensive, quantitative data on Micrurus venom composition to date, and more than tripling the number of Micrurus venom protein sequences previously available. The six venomes differ dramatically. All are dominated by 2–6 toxin classes that account for 91–99% of the toxin transcripts. The M. s. spixii venome is compositionally the simplest. In it, three-finger toxins (3FTxs) and phospholipases A2 (PLA2s) comprise >99% of the toxin transcripts, which include only four additional toxin families at levels ≥0.1%. Micrurus l. lemniscatus venom is the most complex, with at least 17 toxin families. However, in each venome, multiple structural subclasses of 3FTXs and PLA2s are present. These almost certainly differ in pharmacology as well. All venoms also contain phospholipase B and vascular endothelial growth factors. Minor components (0.1–2.0%) are found in all venoms except that of M. s. spixii. Other toxin families are present in all six venoms at trace levels (<0.005%). Minor and trace venom components differ in each venom. Numerous novel toxin chemistries include 3FTxs with previously unknown 8- and 10-cysteine arrangements, resulting in new 3D structures and target specificities. 9-cysteine toxins raise the possibility of covalent, homodimeric 3FTxs or heterodimeric toxins with unknown pharmacologies. Probable muscarinic sequences may be reptile-specific homologs that promote hypotension via vascular mAChRs. The first complete sequences are presented for 3FTxs putatively responsible for liberating glutamate from rat brain synaptosomes. Micrurus C-type lectin-like proteins may have 6–9 cysteine residues and may be monomers, or homo- or heterodimers of unknown pharmacology. Novel KSPIs, 3× longer than any seen previously, appear to have arisen in three species by gene duplication and fusion. Four species have transcripts homologous to the nociceptive toxin, (MitTx) α-subunit, but all six species had homologs to the β-subunit. The first non-neurotoxic, non-catalytic elapid phospholipase A2s are reported. All are probably myonecrotic. Phylogenetic analysis indicates that the six taxa diverged 15–35 million years ago and that they split from their last common ancestor with Old World elapines nearly 55 million years ago. Given their early diversification, many cryptic micrurine taxa are anticipated. Full article
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Open AccessArticle
Characterization of Asian Corn Borer Resistance to Bt Toxin Cry1Ie
Toxins 2017, 9(6), 186; doi:10.3390/toxins9060186 -
Abstract
A strain of the Asian corn borer (ACB), Ostrinia furnacalis (Guenée), has evolved >800-fold resistance to Cry1Ie (ACB-IeR) after 49 generations of selection. The inheritance pattern of resistance to Cry1Ie in ACB-IeR strain and its cross-resistance to other Bt toxins were determined through
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A strain of the Asian corn borer (ACB), Ostrinia furnacalis (Guenée), has evolved >800-fold resistance to Cry1Ie (ACB-IeR) after 49 generations of selection. The inheritance pattern of resistance to Cry1Ie in ACB-IeR strain and its cross-resistance to other Bt toxins were determined through bioassay by exposing neonates from genetic-crosses to toxins incorporated into the diet. The response of progenies from reciprocal F1 crosses were similar (LC50s: 76.07 vs. 74.32 μg/g), which suggested the resistance was autosomal. The effective dominance (h) decreased as concentration of Cry1Ie increased. h was nearly recessive or incompletely recessive on Cry1Ie maize leaf tissue (h = 0.02), but nearly dominant or incompletely dominant (h = 0.98) on Cry1Ie maize silk. Bioassay of the backcross suggested that the resistance was controlled by more than one locus. In addition, the resistant strain did not perform cross-resistance to Cry1Ab (0.8-fold), Cry1Ac (0.8-fold), Cry1F (0.9-fold), and Cry1Ah (1.0-fold). The present study not only offers the manifestation for resistance management, but also recommends that Cry1Ie will be an appropriate candidate for expression with Cry1Ab, Cry1Ac, Cry1F, or Cry1Ah for the development of Bt maize. 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)
Toxins 2017, 9(6), 185; doi:10.3390/toxins9060185 -
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
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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
Screening of Deoxynivalenol Producing Strains and Elucidation of Possible Toxigenic Molecular Mechanism
Toxins 2017, 9(6), 184; doi:10.3390/toxins9060184 -
Abstract
In this study, seven strains of Fusarium graminearum were isolated from wheat, of which six were identified to produce deoxynivalenol and the production of deoxynivalenol was assessed. F. graminearum strain Fg1 was noted to produce 1.0 μg/g deoxynivalenol during the incubation period in
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In this study, seven strains of Fusarium graminearum were isolated from wheat, of which six were identified to produce deoxynivalenol and the production of deoxynivalenol was assessed. F. graminearum strain Fg1 was noted to produce 1.0 μg/g deoxynivalenol during the incubation period in the Czapek yeast broth, while none was detected in F. graminearum strain Fg2. Hence, the differences in proteomes and transcriptomes of Fg1 and Fg2 were compared to analyze the mechanism underlying deoxynivalenol production. Among the 66 significantly differentially expressed proteins in Fg1, 39 and 27 were more or less abundant expressed. Functional analysis suggested that the enzymes involved in the methylerythritol 4-phosphate and mevalonate pathways, which provide a substrate for biosynthesis of farnesyl pyrophosphate, a precursor of DON, were activated in Fg1. The transcriptomics data demonstrated that the expression level of a majority of genes, including trichothecene biosynthetic genes, protein kinases, and transcription factors, involved in trichothecene biosynthesis was higher in Fg1 than in Fg2. The results also revealed differential expression profiles of deoxynivalenol biosynthesis genes in strains Fg1 and Fg2, which emphasized their deoxynivalenol producing ability and the underlying mechanism. Full article
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Open AccessArticle
Careful with That Axe, Gene, Genome Perturbation after a PEG-Mediated Protoplast Transformation in Fusarium verticillioides
Toxins 2017, 9(6), 183; doi:10.3390/toxins9060183 -
Abstract
Fusarium verticillioides causes ear rot disease in maize and its contamination with fumonisins, mycotoxins harmful for humans and livestock. Lipids, and their oxidized forms, may drive the fate of this disease. In a previous study, we have explored the role of oxylipins in
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Fusarium verticillioides causes ear rot disease in maize and its contamination with fumonisins, mycotoxins harmful for humans and livestock. Lipids, and their oxidized forms, may drive the fate of this disease. In a previous study, we have explored the role of oxylipins in this interaction by deleting by standard transformation procedures a linoleate diol synthase-coding gene, lds1, in F. verticillioides. A profound phenotypic diversity in the mutants generated has prompted us to investigate more deeply the whole genome of two lds1-deleted strains. Bioinformatics analyses pinpoint significant differences in the genome sequences emerged between the wild type and the lds1-mutants further than those trivially attributable to the deletion of the lds1 locus, such as single nucleotide polymorphisms, small deletion/insertion polymorphisms and structural variations. Results suggest that the effect of a (theoretically) punctual transformation event might have enhanced the natural mechanisms of genomic variability and that transformation practices, commonly used in the reverse genetics of fungi, may potentially be responsible for unexpected, stochastic and henceforth off-target rearrangements throughout the genome. Full article
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Open AccessReview
Small and Smaller—sRNAs and MicroRNAs in the Regulation of Toxin Gene Expression in Prokaryotic Cells: A Mini-Review
Toxins 2017, 9(6), 181; doi:10.3390/toxins9060181 -
Abstract
Non-coding small RNAs (sRNAs) have been identified in the wide range of bacteria (also pathogenic species) and found to play an important role in the regulation of many processes, including toxin gene expression. The best characterized prokaryotic sRNAs regulate gene expression by base
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Non-coding small RNAs (sRNAs) have been identified in the wide range of bacteria (also pathogenic species) and found to play an important role in the regulation of many processes, including toxin gene expression. The best characterized prokaryotic sRNAs regulate gene expression by base pairing with mRNA targets and fall into two broad classes: cis-encoded sRNAs (also called antisense RNA) and trans-acting sRNAs. Molecules from the second class are frequently considered as the most related to eukaryotic microRNAs. Interestingly, typical microRNA-size RNA molecules have also been reported in prokaryotic cells, although they have received little attention up to now. In this work we have collected information about all three types of small prokaryotic RNAs in the context of the regulation of toxin gene expression. Full article
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Open AccessArticle
Two Saporin-Containing Immunotoxins Specific for CD20 and CD22 Show Different Behavior in Killing Lymphoma Cells
Toxins 2017, 9(6), 182; doi:10.3390/toxins9060182 -
Abstract
Immunotoxins (ITs) are hybrid proteins combining the binding specificity of antibodies with the cytocidal properties of toxins. They represent a promising approach to lymphoma therapy. The cytotoxicity of two immunotoxins obtained by chemical conjugation of the plant toxin saporin-S6 with the anti-CD20 chimeric
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Immunotoxins (ITs) are hybrid proteins combining the binding specificity of antibodies with the cytocidal properties of toxins. They represent a promising approach to lymphoma therapy. The cytotoxicity of two immunotoxins obtained by chemical conjugation of the plant toxin saporin-S6 with the anti-CD20 chimeric antibody rituximab and the anti-CD22 murine antibody OM124 were evaluated on the CD20-/CD22-positive cell line Raji. Both ITs showed strong cytotoxicity for Raji cells, but the anti-CD22 IT was two logs more efficient in killing, probably because of its faster internalization. The anti-CD22 IT gave slower but greater caspase activation than the anti-CD20 IT. The cytotoxic effect of both immunotoxins can be partially prevented by either the pan-caspase inhibitor Z-VAD or the necroptosis inhibitor necrostatin-1. Oxidative stress seems to be involved in the cell killing activity of anti-CD20 IT, as demonstrated by the protective role of the H2O2 scavenger catalase, but not in that of anti-CD22 IT. Moreover, the IT toxicity can be augmented by the contemporary administration of other chemotherapeutic drugs, such as PS-341, MG-132, and fludarabine. These results contribute to the understanding of the immunotoxin mechanism of action that is required for their clinical use, either alone or in combination with other drugs. Full article
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Open AccessArticle
Role of Homologous Fc Fragment in the Potency and Efficacy of Anti‐Botulinum Antibody Preparations
Toxins 2017, 9(6), 180; doi:10.3390/toxins9060180 -
Abstract
The only approved treatment for botulism relies on passive immunity which is mostly based on antibody preparations collected from hyper‐immune horses. The IgG Fc fragment is commonly removed from these heterologous preparations to reduce the incidence of hyper‐sensitivity reactions. New‐generation therapies entering the
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The only approved treatment for botulism relies on passive immunity which is mostly based on antibody preparations collected from hyper‐immune horses. The IgG Fc fragment is commonly removed from these heterologous preparations to reduce the incidence of hyper‐sensitivity reactions. New‐generation therapies entering the pipeline are based on a combination of humanized monoclonal antibodies (MAbs), which exhibit improved safety and pharmacokinetics. In the current study, a systematic and quantitative approach was applied to measure the direct contribution of homologous Fc to the potency of monoclonal and polyclonal antitoxin preparations in mice. Homologous Fc increased the potency of three individual anti‐botulinum toxin MAbs by up to one order of magnitude. Moreover, Fc fragment removal almost completely abolished the synergistic potency obtained from a combined preparation of these three MAbs. The MAb mixture neutralized a 400‐mouse median lethal dose (MsLD50) of botulinum toxin, whereas the F(ab′)2 combination failed to neutralize 10 MsLD50 of botulinum toxin. Notably, increased avidity did not compensate for this phenomenon, as a polyclonal, hyper‐immune, homologous preparation lost 90% of its potency as well upon Fc removal. Finally, the addition of homologous Fc arms to a heterologous pharmaceutical anti‐botulinum toxin polyclonal horse F(ab′)2 preparation improved its efficacy when administered to intoxicated symptomatic mice. Our study extends the aspects by which switching from animal‐based to human‐based antitoxins will improve not only the safety but also the potency and efficacy of passive immunity against toxins. Full article
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Open AccessArticle
Venom On-a-Chip: A Fast and Efficient Method for Comparative Venomics
Toxins 2017, 9(6), 179; doi:10.3390/toxins9060179 -
Abstract
Venom research has attracted an increasing interest in disparate fields, from drug development and pharmacology, to evolutionary biology and ecology, and rational antivenom production. Advances in “-omics” technologies have allowed the characterization of an increasing number of animal venoms, but the methodology currently
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Venom research has attracted an increasing interest in disparate fields, from drug development and pharmacology, to evolutionary biology and ecology, and rational antivenom production. Advances in “-omics” technologies have allowed the characterization of an increasing number of animal venoms, but the methodology currently available is suboptimal for large-scale comparisons of venom profiles. Here, we describe a fast, reproducible and semi-automated protocol for investigating snake venom variability, especially at the intraspecific level, using the Agilent Bioanalyzer on-chip technology. Our protocol generated a phenotype matrix which can be used for robust statistical analysis and correlations of venom variation with ecological correlates, or other extrinsic factors. We also demonstrate the ease and utility of combining on-chip technology with previously fractionated venoms for detection of specific individual toxin proteins. Our study describes a novel strategy for rapid venom discrimination and analysis of compositional variation at multiple taxonomic levels, allowing researchers to tackle evolutionary questions and unveiling the drivers of the incredible biodiversity of venoms. Full article
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