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Open AccessEditor’s ChoiceArticle Degradation of Aflatoxins by Means of Laccases from Trametes versicolor: An In Silico Insight

by Luca Dellafiora, Gianni Galaverna, Massimo Reverberi and Chiara Dall’Asta

Toxins 2017, 9(1), 17; https://doi.org/10.3390/toxins9010017

Received: 16 November 2016 / Revised: 16 December 2016 / Accepted: 26 December 2016 / Published: 1 January 2017

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Abstract

Mycotoxins are secondary metabolites of fungi that contaminate food and feed, and are involved in a series of foodborne illnesses and disorders in humans and animals. The mitigation of mycotoxin content via enzymatic degradation is a strategy to ensure safer food and feed,

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Mycotoxins are secondary metabolites of fungi that contaminate food and feed, and are involved in a series of foodborne illnesses and disorders in humans and animals. The mitigation of mycotoxin content via enzymatic degradation is a strategy to ensure safer food and feed, and to address the forthcoming issues in view of the global trade and sustainability. Nevertheless, the search for active enzymes is still challenging and time-consuming. The in silico analysis may strongly support the research by providing the evidence-based hierarchization of enzymes for a rational design of more effective experimental trials. The present work dealt with the degradation of aflatoxin B₁ and M₁ by laccase enzymes from Trametes versicolor. The enzymes–substrate interaction for various enzyme isoforms was investigated through 3D molecular modeling techniques. Structural differences among the isoforms have been pinpointed, which may cause different patterns of interaction between aflatoxin B₁ and M₁. The possible formation of different products of degradation can be argued accordingly. Moreover, the laccase gamma isoform was identified as the most suitable for protein engineering aimed at ameliorating the substrate specificity. Overall, 3D modeling proved to be an effective analytical tool to assess the enzyme–substrate interaction and provided a solid foothold for supporting the search of degrading enzyme at the early stage. Full article

(This article belongs to the Special Issue Promising Detoxification Strategies to Mitigate Mycotoxins in Food and Feed) Printed Edition available

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Open AccessEditor’s ChoiceArticle Bioactivity of Natural and Engineered Antimicrobial Peptides from Venom of the Scorpions Urodacus yaschenkoi and U. manicatus

by Karen Luna-Ramirez, Miray Tonk, Mohammad Rahnamaeian and Andreas Vilcinskas

Toxins 2017, 9(1), 22; https://doi.org/10.3390/toxins9010022

Received: 26 November 2016 / Revised: 26 December 2016 / Accepted: 29 December 2016 / Published: 6 January 2017

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Abstract

The spread of multidrug-resistant human pathogens has drawn attention towards antimicrobial peptides (AMPs), which are major players in the innate immune systems of many organisms, including vertebrates, invertebrates, plants and microbes. Scorpion venom is an abundant source of novel and potent AMPs. Here,

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The spread of multidrug-resistant human pathogens has drawn attention towards antimicrobial peptides (AMPs), which are major players in the innate immune systems of many organisms, including vertebrates, invertebrates, plants and microbes. Scorpion venom is an abundant source of novel and potent AMPs. Here, we investigated natural and engineered AMPs from the scorpions Urodacus yaschenkoi and U. manicatus to determine their antimicrobial spectra as well as their hemolytic/cytotoxic activity. None of the AMPs were active against fungi, but many of them were active at low concentrations (0.25–30 µM) against seven different bacteria. Hemolytic and cytotoxic activities were determined using pig erythrocytes and baby hamster kidney cells, respectively. The amino acid substitutions in the engineered AMPs did not inhibit cytotoxicity, but reduced hemolysis and therefore increased the therapeutic indices. The phylogenetic analysis of scorpion AMPs revealed they are closely related and the GXK motif is highly conserved. The engineered scorpion AMPs offer a promising alternative for the treatment of multidrug-resistant bacterial infections and could be modified further to reduce their hemolytic/cytotoxic activity. Full article

(This article belongs to the Section Animal Venoms)

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Open AccessEditor’s ChoiceArticle Novel Aflatoxin-Degrading Enzyme from Bacillus shackletonii L7

by Liang Xu, Mohamed Farah Eisa Ahmed, Lancine Sangare, Yueju Zhao, Jonathan Nimal Selvaraj, Fuguo Xing, Yan Wang, Hongping Yang and Yang Liu

Toxins 2017, 9(1), 36; https://doi.org/10.3390/toxins9010036

Received: 22 September 2016 / Revised: 10 January 2017 / Accepted: 11 January 2017 / Published: 14 January 2017

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Abstract

Food and feed contamination by aflatoxin (AF)B₁ has adverse economic and health consequences. AFB₁ degradation by microorganisms or microbial enzymes provides a promising preventive measure. To this end, the present study tested 43 bacterial isolates collected from maize, rice, and soil

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Food and feed contamination by aflatoxin (AF)B₁ has adverse economic and health consequences. AFB₁ degradation by microorganisms or microbial enzymes provides a promising preventive measure. To this end, the present study tested 43 bacterial isolates collected from maize, rice, and soil samples for AFB₁-reducing activity. The higher activity was detected in isolate L7, which was identified as Bacillus shackletonii. L7 reduced AFB₁, AFB₂, and AFM₁ levels by 92.1%, 84.1%, and 90.4%, respectively, after 72 h at 37 °C. The L7 culture supernatant degraded more AFB₁ than viable cells and cell extracts; and the degradation activity was reduced from 77.9% to 15.3% in the presence of proteinase K and sodium dodecyl sulphate. A thermostable enzyme purified from the boiled supernatant was designated as Bacillus aflatoxin-degrading enzyme (BADE). An overall 9.55-fold purification of BADE with a recovery of 39.92% and an activity of 3.85 × 10³ U·mg⁻¹ was obtained using chromatography on DEAE-Sepharose. BADE had an estimated molecular mass of 22 kDa and exhibited the highest activity at 70 °C and pH 8.0, which was enhanced by Cu²⁺ and inhibited by Zn²⁺, Mn²⁺, Mg²⁺, and Li⁺. BADE is the major protein involved in AFB₁ detoxification. This is the first report of a BADE isolated from B. shackletonii, which has potential applications in the detoxification of aflatoxins during food and feed processing. Full article

(This article belongs to the Special Issue Promising Detoxification Strategies to Mitigate Mycotoxins in Food and Feed) Printed Edition available

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Open AccessEditor’s ChoiceArticle Metabolism of Zearalenone and Its Major Modified Forms in Pigs

by Sabina B. Binder, Heidi E. Schwartz-Zimmermann, Elisabeth Varga, Gerlinde Bichl, Herbert Michlmayr, Gerhard Adam and Franz Berthiller

Toxins 2017, 9(2), 56; https://doi.org/10.3390/toxins9020056

Received: 15 January 2017 / Revised: 2 February 2017 / Accepted: 6 February 2017 / Published: 8 February 2017

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Abstract

The Fusarium mycotoxin zearalenone (ZEN) can be conjugated with polar molecules, like sugars or sulfates, by plants and fungi. To date, the fate of these modified forms of ZEN has not yet been elucidated in animals. In order to investigate whether ZEN conjugates

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The Fusarium mycotoxin zearalenone (ZEN) can be conjugated with polar molecules, like sugars or sulfates, by plants and fungi. To date, the fate of these modified forms of ZEN has not yet been elucidated in animals. In order to investigate whether ZEN conjugates contribute to the total ZEN exposure of an individual, ZEN (10 µg/kg b.w.) and equimolar amounts of two of its plant metabolites (ZEN-14-O-β-glucoside, ZEN-16-O-β-glucoside) and of one fungal metabolite (ZEN-14-sulfate) were orally administered to four pigs as a single bolus using a repeated measures design. The concentrations of ZEN, its modified forms and its mammalian metabolites ZEN-14-glucuronide, α-zearalenol (α-ZEL) and α-ZEL-14-glucuronide in excreta were analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) based methods. The biological recovery of ZEN in urine was 26% ± 10%, the total biological recovery in excreta was 40% ± 8%. Intact ZEN-14-sulfate, ZEN-14-O-β-glucoside and ZEN-16-O-β-glucoside were neither detected in urine nor in feces. After ZEN-14-sulfate application, 19% ± 5% of the administered dose was recovered in urine. In feces, no ZEN metabolites were detected. The total biological recoveries of ZEN-14-O-β-glucoside and ZEN-16-O-β-glucoside in the form of their metabolites in urine were 19% ± 11% and 13% ± 7%, respectively. The total biological recoveries in urine and feces amounted to 48% ± 7% and 34 ± 3%. An explanation for the low biological recoveries could be extensive metabolization by intestinal bacteria to yet unknown metabolites. In summary, ZEN-14-sulfate, ZEN-14-O-β-glucoside, and ZEN-16-O-β-glucoside were completely hydrolyzed in the gastrointestinal tract of swine, thus contributing to the overall toxicity of ZEN. Full article

(This article belongs to the collection Fusarium Toxins – Relevance for Human and Animal Health)

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Open AccessEditor’s ChoiceArticle Patulin Degradation by the Biocontrol Yeast Sporobolomyces sp. Is an Inducible Process

by Giuseppe Ianiri, Cristina Pinedo, Alessandra Fratianni, Gianfranco Panfili and Raffaello Castoria

Toxins 2017, 9(2), 61; https://doi.org/10.3390/toxins9020061

Received: 12 January 2017 / Revised: 2 February 2017 / Accepted: 7 February 2017 / Published: 10 February 2017

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Abstract

Patulin is a mycotoxin produced by Penicillium expansum and a common contaminant of pome fruits and their derived products worldwide. It is considered to be mutagenic, genotoxic, immunotoxic, teratogenic and cytotoxic, and the development of strategies to reduce this contamination is an active

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Patulin is a mycotoxin produced by Penicillium expansum and a common contaminant of pome fruits and their derived products worldwide. It is considered to be mutagenic, genotoxic, immunotoxic, teratogenic and cytotoxic, and the development of strategies to reduce this contamination is an active field of research. We previously reported that Sporobolomyces sp. is able to degrade patulin and convert it into the breakdown products desoxypatulinic acid and ascladiol, both of which were found to be less toxic than patulin. The specific aim of this study was the evaluation of the triggering of the mechanisms involved in patulin resistance and degradation by Sporobolomyces sp. Cells pre-incubated in the presence of a low patulin concentration showed a higher resistance to patulin toxicity and a faster kinetics of degradation. Similarly, patulin degradation was faster when crude intracellular protein extracts of Sporobolomyces sp. were prepared from cells pre-treated with the mycotoxin, indicating the induction of the mechanisms involved in the resistance and degradation of the mycotoxin by Sporobolomyces sp. This study contributes to the understanding of the mechanisms of patulin resistance and degradation by Sporobolomyces sp., which is an essential prerequisite for developing an industrial approach aiming at the production of patulin-free products. Full article

(This article belongs to the Special Issue Promising Detoxification Strategies to Mitigate Mycotoxins in Food and Feed) Printed Edition available

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Open AccessEditor’s ChoiceArticle The Cardiovascular and Neurotoxic Effects of the Venoms of Six Bony and Cartilaginous Fish Species

by Han Han, Kate Baumann, Nicholas R. Casewell, Syed A. Ali, James Dobson, Ivan Koludarov, Jordan Debono, Scott C. Cutmore, Niwanthi W. Rajapakse, Timothy N. W. Jackson, Rob Jones, Wayne C. Hodgson, Bryan G. Fry and Sanjaya Kuruppu

Toxins 2017, 9(2), 67; https://doi.org/10.3390/toxins9020067

Received: 15 September 2016 / Accepted: 3 February 2017 / Published: 16 February 2017

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Abstract

Fish venoms are often poorly studied, in part due to the difficulty in obtaining, extracting, and storing them. In this study, we characterize the cardiovascular and neurotoxic effects of the venoms from the following six species of fish: the cartilaginous stingrays Neotrygon kuhlii

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Fish venoms are often poorly studied, in part due to the difficulty in obtaining, extracting, and storing them. In this study, we characterize the cardiovascular and neurotoxic effects of the venoms from the following six species of fish: the cartilaginous stingrays Neotrygon kuhlii and Himantura toshi, and the bony fish Platycephalus fucus, Girella tricuspidata, Mugil cephalus, and Dentex tumifrons. All venoms (10–100 μg/kg, i.v.), except G. tricuspidata and P. fuscus, induced a biphasic response on mean arterial pressure (MAP) in the anesthetised rat. P. fucus venom exhibited a hypotensive response, while venom from G. tricuspidata displayed a single depressor response. All venoms induced cardiovascular collapse at 200 μg/kg, i.v. The in vitro neurotoxic effects of venom were examined using the chick biventer cervicis nerve‐muscle (CBCNM) preparation. N. kuhlii, H. toshi, and P. fucus venoms caused concentration‐dependent inhibition of indirect twitches in the CBCNM preparation. These three venoms also inhibited responses to exogenous acetylcholine (ACh) and carbachol (CCh), but not potassium chloride (KCl), indicating a post‐synaptic mode of action. Venom from G. tricuspidata, M. cephalus, and D. tumifrons had no significant effect on indirect twitches or agonist responses in the CBCNM. Our results demonstrate that envenoming by these species of fish may result in moderate cardiovascular and/or neurotoxic effects. Future studies aimed at identifying the molecules responsible for these effects could uncover potentially novel lead compounds for future pharmaceuticals, in addition to generating new knowledge about the evolutionary relationships between venomous animals. Full article

(This article belongs to the Section Animal Venoms)

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Open AccessEditor’s ChoiceCommunication Acute Oral Toxicity of Tetrodotoxin in Mice: Determination of Lethal Dose 50 (LD50) and No Observed Adverse Effect Level (NOAEL)

by Paula Abal, M. Carmen Louzao, Alvaro Antelo, Mercedes Alvarez, Eva Cagide, Natalia Vilariño, Mercedes R. Vieytes and Luis M. Botana

Toxins 2017, 9(3), 75; https://doi.org/10.3390/toxins9030075

Received: 9 January 2017 / Revised: 26 January 2017 / Accepted: 22 February 2017 / Published: 24 February 2017

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Abstract

Tetrodotoxin (TTX) is starting to appear in molluscs from the European waters and is a hazard to seafood consumers. This toxin blocks sodium channels resulting in neuromuscular paralysis and even death. As a part of the risk assessment process leading to a safe

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Tetrodotoxin (TTX) is starting to appear in molluscs from the European waters and is a hazard to seafood consumers. This toxin blocks sodium channels resulting in neuromuscular paralysis and even death. As a part of the risk assessment process leading to a safe seafood level for TTX, oral toxicity data are required. In this study, a 4-level Up and Down Procedure was designed in order to determine for the first time the oral lethal dose 50 (LD₅₀) and the No Observed Adverse Effect Level (NOAEL) in mice by using an accurate well-characterized TTX standard. Full article

(This article belongs to the collection Marine and Freshwater Toxins)

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Open AccessEditor’s ChoiceArticle Detection of Cyanotoxins in Algae Dietary Supplements

by Audrey Roy-Lachapelle, Morgan Solliec, Maryse F. Bouchard and Sébastien Sauvé

Toxins 2017, 9(3), 76; https://doi.org/10.3390/toxins9030076

Received: 6 December 2016 / Revised: 17 February 2017 / Accepted: 21 February 2017 / Published: 25 February 2017

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Abstract

Algae dietary supplements are marketed worldwide as natural health products. Although their proprieties have been claimed as beneficial to improve overall health, there have been several previous reports of contamination by cyanotoxins. These products generally contain non-toxic cyanobacteria, but the methods of cultivation

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Algae dietary supplements are marketed worldwide as natural health products. Although their proprieties have been claimed as beneficial to improve overall health, there have been several previous reports of contamination by cyanotoxins. These products generally contain non-toxic cyanobacteria, but the methods of cultivation in natural waters without appropriate quality controls allow contamination by toxin producer species present in the natural environment. In this study, we investigated the presence of total microcystins, seven individual microcystins (RR, YR, LR, LA, LY, LW, LF), anatoxin-a, dihydroanatoxin-a, epoxyanatoxin-a, cylindrospermopsin, saxitoxin, and β-methylamino-l-alanine in 18 different commercially available products containing Spirulina or Aphanizomenon flos-aquae. Total microcystins analysis was accomplished using a Lemieux oxidation and a chemical derivatization using dansyl chloride was needed for the simultaneous analysis of cylindrospermopsin, saxitoxin, and β-methylamino-l-alanine. Moreover, the use of laser diode thermal desorption (LDTD) and ultra-high performance liquid chromatography (UHPLC) both coupled to high resolution mass spectrometry (HRMS) enabled high performance detection and quantitation. Out of the 18 products analyzed, 8 contained some cyanotoxins at levels exceeding the tolerable daily intake values. The presence of cyanotoxins in these algal dietary supplements reinforces the need for a better quality control as well as consumer’s awareness on the potential risks associated with the consumption of these supplements. Full article

(This article belongs to the Section Marine and Freshwater Toxins)

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Open AccessEditor’s ChoiceArticle Effects of Electron Beam Irradiation on Zearalenone and Ochratoxin A in Naturally Contaminated Corn and Corn Quality Parameters

by Xiaohu Luo, Lijun Qi, Yuntao Liu, Ren Wang, Dan Yang, Ke Li, Li Wang, Yanan Li, Yuwei Zhang and Zhengxing Chen

Toxins 2017, 9(3), 84; https://doi.org/10.3390/toxins9030084

Received: 26 November 2016 / Accepted: 6 February 2017 / Published: 27 February 2017

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Abstract

Zearalenone (ZEN) and ochratoxin A (OTA) are secondary toxic metabolites widely present in grains and grain products. In this study, the effects of electron beam irradiation (EBI) on ZEN and OTA in corn and the quality of irradiated corn were investigated. Results indicated

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Zearalenone (ZEN) and ochratoxin A (OTA) are secondary toxic metabolites widely present in grains and grain products. In this study, the effects of electron beam irradiation (EBI) on ZEN and OTA in corn and the quality of irradiated corn were investigated. Results indicated that EBI significantly affected ZEN and OTA. The degradation rates of ZEN and OTA at 10 kGy in solution were 65.6% and 75.2%, respectively. The initial amounts significantly affected the degradation rate. ZEN and OTA in corn were decreased by the irradiation dose, and their degradation rates at 50 kGy were 71.1% and 67.9%, respectively. ZEN and OTA were more easily degraded in corn kernel than in corn flour. Moisture content (MC) played a vital role in ZEN and OTA degradation. High MC was attributed to high ZEN and OTA degradation. The quality of irradiated corn was evaluated on the basis of irradiation dose. L* value changed, but this change was not significant (p > 0.05). By contrast, a* and b* decreased significantly (p < 0.05) with irradiation dose. The fatty acid value increased significantly. The pasting properties, including peak, trough, breakdown, and final and setback viscosities, were also reduced significantly (p < 0.05) by irradiation. Our study verified that EBI could effectively degrade ZEN and OTA in corn. Irradiation could also affect corn quality. Full article

(This article belongs to the Special Issue Selected papers from 1^st China Mycotoxin Conference: Current trends in mycotoxin prevention and control)

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Open AccessEditor’s ChoiceArticle Silver Nanoparticle-Based Fluorescence-Quenching Lateral Flow Immunoassay for Sensitive Detection of Ochratoxin A in Grape Juice and Wine

by Hu Jiang, Xiangmin Li, Ying Xiong, Ke Pei, Lijuan Nie and Yonghua Xiong

Toxins 2017, 9(3), 83; https://doi.org/10.3390/toxins9030083

Received: 24 November 2016 / Revised: 23 February 2017 / Accepted: 23 February 2017 / Published: 28 February 2017

Cited by 14 | PDF Full-text (2766 KB) | HTML Full-text | XML Full-text | Supplementary Files

Abstract

A silver nanoparticle (AgNP)-based fluorescence-quenching lateral flow immunoassay with competitive format (cLFIA) was developed for sensitive detection of ochratoxin A (OTA) in grape juice and wine samples in the present study. The Ru(phen)

_{3}^{2 +}

-doped silica nanoparticles (RuNPs) were sprayed on

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A silver nanoparticle (AgNP)-based fluorescence-quenching lateral flow immunoassay with competitive format (cLFIA) was developed for sensitive detection of ochratoxin A (OTA) in grape juice and wine samples in the present study. The Ru(phen)

_{3}^{2 +}

-doped silica nanoparticles (RuNPs) were sprayed on the test and control line zones as background fluorescence signals. The AgNPs were designed as the fluorescence quenchers of RuNPs because they can block the exciting light transferring to the RuNP molecules. The proposed method exhibited high sensitivity for OTA detection, with a detection limit of 0.06 µg/L under optimized conditions. The method also exhibited a good linear range for OTA quantitative analysis from 0.08 µg/L to 5.0 µg/L. The reliability of the fluorescence-quenching cLFIA method was evaluated through analysis of the OTA-spiked red grape wine and juice samples. The average recoveries ranged from 88.0% to 110.0% in red grape wine and from 92.0% to 110.0% in grape juice. Meanwhile, less than a 10% coefficient variation indicated an acceptable precision of the cLFIA method. In summary, the new AgNP-based fluorescence-quenching cLFIA is a simple, rapid, sensitive, and accurate method for quantitative detection of OTA in grape juice and wine or other foodstuffs. Full article

(This article belongs to the Special Issue Selected papers from 1^st China Mycotoxin Conference: Current trends in mycotoxin prevention and control)

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Open AccessEditor’s ChoiceArticle Identification of the Anti-Aflatoxinogenic Activity of Micromeria graeca and Elucidation of Its Molecular Mechanism in Aspergillus flavus

by Rhoda El Khoury, Isaura Caceres, Olivier Puel, Sylviane Bailly, Ali Atoui, Isabelle P. Oswald, André El Khoury and Jean-Denis Bailly

Toxins 2017, 9(3), 87; https://doi.org/10.3390/toxins9030087

Received: 18 January 2017 / Revised: 22 February 2017 / Accepted: 24 February 2017 / Published: 1 March 2017

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Abstract

Of all the food-contaminating mycotoxins, aflatoxins, and most notably aflatoxin B₁ (AFB₁), are found to be the most toxic and economically costly. Green farming is striving to replace fungicides and develop natural preventive strategies to minimize crop contamination by these

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Of all the food-contaminating mycotoxins, aflatoxins, and most notably aflatoxin B₁ (AFB₁), are found to be the most toxic and economically costly. Green farming is striving to replace fungicides and develop natural preventive strategies to minimize crop contamination by these toxic fungal metabolites. In this study, we demonstrated that an aqueous extract of the medicinal plant Micromeria graeca—known as hyssop—completely inhibits aflatoxin production by Aspergillus flavus without reducing fungal growth. The molecular inhibitory mechanism was explored by analyzing the expression of 61 genes, including 27 aflatoxin biosynthesis cluster genes and 34 secondary metabolism regulatory genes. This analysis revealed a three-fold down-regulation of aflR and aflS encoding the two internal cluster co-activators, resulting in a drastic repression of all aflatoxin biosynthesis genes. Hyssop also targeted fifteen regulatory genes, including veA and mtfA, two major global-regulating transcription factors. The effect of this extract is also linked to a transcriptomic variation of several genes required for the response to oxidative stress such as msnA, srrA, catA, cat2, sod1, mnsod, and stuA. In conclusion, hyssop inhibits AFB₁ synthesis at the transcriptomic level. This aqueous extract is a promising natural-based solution to control AFB₁ contamination. Full article

(This article belongs to the Special Issue Promising Detoxification Strategies to Mitigate Mycotoxins in Food and Feed) Printed Edition available

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Open AccessEditor’s ChoiceArticle A Greener, Quick and Comprehensive Extraction Approach for LC-MS of Multiple Mycotoxins

by Andreas Breidbach

Toxins 2017, 9(3), 91; https://doi.org/10.3390/toxins9030091

Received: 12 January 2017 / Revised: 18 February 2017 / Accepted: 1 March 2017 / Published: 7 March 2017

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Abstract

In food/feed control, mycotoxin analysis is often still performed “one analyte at a time”. Here a method is presented which aims at making mycotoxin analysis environmentally friendlier through replacing acetonitrile by ethyl acetate and reducing chemical waste production by analyzing four mycotoxins together,

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In food/feed control, mycotoxin analysis is often still performed “one analyte at a time”. Here a method is presented which aims at making mycotoxin analysis environmentally friendlier through replacing acetonitrile by ethyl acetate and reducing chemical waste production by analyzing four mycotoxins together, forgoing sample extract clean-up, and minimizing solvent consumption. For this, 2 g of test material were suspended in 8 mL water and 16 mL ethyl acetate were added. Extraction was accelerated through sonication for 30 min and subsequent addition of 8 g sodium sulfate. After centrifugation, 500 µL supernatant were spiked with isotopologues, dried down, reconstituted in mobile phase, and measured with LC-MS. The method was validated in-house and through a collaborative study and the performance was fit-for-purpose. Repeatability relative standard deviation (RSDs) between 16% at low and 4% at higher contaminations were obtained. The reproducibility RSDs were mostly between 12% and 32%. The trueness of results for T-2 toxin and Zearalenone were not different from 100%, for Deoxynivalenol and HT-2 toxin they were larger than 89%. The extraction was also adapted to a quick screening of Aflatoxin B1 in maize by flow-injection–mass spectrometry. Semi-quantitative results were obtained through standard addition and scan-based ion ratio calculations. The method proved to be a viable greener and quicker alternative to existing methods. Full article

(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis) Printed Edition available

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Open AccessEditor’s ChoiceArticle Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

by Guorui Yao, Kwok-ho Lam, Kay Perry, Jasmin Weisemann, Andreas Rummel and Rongsheng Jin

Toxins 2017, 9(3), 93; https://doi.org/10.3390/toxins9030093

Received: 25 January 2017 / Revised: 3 March 2017 / Accepted: 4 March 2017 / Published: 8 March 2017

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Abstract

Botulinum neurotoxins (BoNTs), which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin

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Botulinum neurotoxins (BoNTs), which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A–G), a new mosaic toxin type termed BoNT/HA (aka type FA or H) was reported recently. Sequence analyses indicate that the receptor-binding domain (H_C) of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2) that target the H_C. Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-H_C at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures. Full article

(This article belongs to the Section Bacterial Toxins)

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Open AccessEditor’s ChoiceArticle Multiple Stressors at the Land-Sea Interface: Cyanotoxins at the Land-Sea Interface in the Southern California Bight

by Avery O. Tatters, Meredith D.A. Howard, Carey Nagoda, Lilian Busse, Alyssa G. Gellene and David A. Caron

Toxins 2017, 9(3), 95; https://doi.org/10.3390/toxins9030095

Received: 11 February 2017 / Revised: 2 March 2017 / Accepted: 3 March 2017 / Published: 9 March 2017

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Abstract

Blooms of toxic cyanobacteria in freshwater ecosystems have received considerable attention in recent years, but their occurrence and potential importance at the land-sea interface has not been widely recognized. Here we present the results of a survey of discrete samples conducted in more

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Blooms of toxic cyanobacteria in freshwater ecosystems have received considerable attention in recent years, but their occurrence and potential importance at the land-sea interface has not been widely recognized. Here we present the results of a survey of discrete samples conducted in more than fifty brackish water sites along the coastline of southern California. Our objectives were to characterize cyanobacterial community composition and determine if specific groups of cyanotoxins (anatoxins, cylindrospermopsins, microcystins, nodularins, and saxitoxins) were present. We report the identification of numerous potentially harmful taxa and the co-occurrence of multiple toxins, previously undocumented, at several locations. Our findings reveal a potential health concern based on the range of organisms present and the widespread prevalence of recognized toxic compounds. Our results raise concerns for recreation, harvesting of finfish and shellfish, and wildlife and desalination operations, highlighting the need for assessments and implementation of monitoring programs. Such programs appear to be particularly necessary in regions susceptible to urban influence. Full article

(This article belongs to the collection Marine and Freshwater Toxins)

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Open AccessEditor’s ChoiceArticle Plasma-Based Degradation of Mycotoxins Produced by Fusarium, Aspergillus and Alternaria Species

by Lars ten Bosch, Katharina Pfohl, Georg Avramidis, Stephan Wieneke, Wolfgang Viöl and Petr Karlovsky

Toxins 2017, 9(3), 97; https://doi.org/10.3390/toxins9030097

Received: 13 January 2017 / Revised: 28 February 2017 / Accepted: 7 March 2017 / Published: 10 March 2017

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Abstract

The efficacy of cold atmospheric pressure plasma (CAPP) with ambient air as working gas for the degradation of selected mycotoxins was studied. Deoxynivalenol, zearalenone, enniatins, fumonisin B1, and T2 toxin produced by Fusarium spp., sterigmatocystin produced by Aspergillus spp. and AAL toxin produced

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The efficacy of cold atmospheric pressure plasma (CAPP) with ambient air as working gas for the degradation of selected mycotoxins was studied. Deoxynivalenol, zearalenone, enniatins, fumonisin B1, and T2 toxin produced by Fusarium spp., sterigmatocystin produced by Aspergillus spp. and AAL toxin produced by Alternaria alternata were used. The kinetics of the decay of mycotoxins exposed to plasma discharge was monitored. All pure mycotoxins exposed to CAPP were degraded almost completely within 60 s. Degradation rates varied with mycotoxin structure: fumonisin B1 and structurally related AAL toxin were degraded most rapidly while sterigmatocystin exhibited the highest resistance to degradation. As compared to pure compounds, the degradation rates of mycotoxins embedded in extracts of fungal cultures on rice were reduced to a varying extent. Our results show that CAPP efficiently degrades pure mycotoxins, the degradation rates vary with mycotoxin structure, and the presence of matrix slows down yet does not prevent the degradation. CAPP appears promising for the decontamination of food commodities with mycotoxins confined to or enriched on surfaces such as cereal grains. Full article

(This article belongs to the Special Issue Promising Detoxification Strategies to Mitigate Mycotoxins in Food and Feed) Printed Edition available

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Open AccessEditor’s ChoiceArticle How the Cobra Got Its Flesh-Eating Venom: Cytotoxicity as a Defensive Innovation and Its Co-Evolution with Hooding, Aposematic Marking, and Spitting

Toxins 2017, 9(3), 103; https://doi.org/10.3390/toxins9030103

Received: 23 January 2017 / Revised: 19 February 2017 / Accepted: 5 March 2017 / Published: 13 March 2017

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Abstract

The cytotoxicity of the venom of 25 species of Old World elapid snake was tested and compared with the morphological and behavioural adaptations of hooding and spitting. We determined that, contrary to previous assumptions, the venoms of spitting species are not consistently more

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The cytotoxicity of the venom of 25 species of Old World elapid snake was tested and compared with the morphological and behavioural adaptations of hooding and spitting. We determined that, contrary to previous assumptions, the venoms of spitting species are not consistently more cytotoxic than those of closely related non-spitting species. While this correlation between spitting and non-spitting was found among African cobras, it was not present among Asian cobras. On the other hand, a consistent positive correlation was observed between cytotoxicity and utilisation of the defensive hooding display that cobras are famous for. Hooding and spitting are widely regarded as defensive adaptations, but it has hitherto been uncertain whether cytotoxicity serves a defensive purpose or is somehow useful in prey subjugation. The results of this study suggest that cytotoxicity evolved primarily as a defensive innovation and that it has co-evolved twice alongside hooding behavior: once in the Hemachatus + Naja and again independently in the king cobras (Ophiophagus). There was a significant increase of cytotoxicity in the Asian Naja linked to the evolution of bold aposematic hood markings, reinforcing the link between hooding and the evolution of defensive cytotoxic venoms. In parallel, lineages with increased cytotoxicity but lacking bold hood patterns evolved aposematic markers in the form of high contrast body banding. The results also indicate that, secondary to the evolution of venom rich in cytotoxins, spitting has evolved three times independently: once within the African Naja, once within the Asian Naja, and once in the Hemachatus genus. The evolution of cytotoxic venom thus appears to facilitate the evolution of defensive spitting behaviour. In contrast, a secondary loss of cytotoxicity and reduction of the hood occurred in the water cobra Naja annulata, which possesses streamlined neurotoxic venom similar to that of other aquatic elapid snakes (e.g., hydrophiine sea snakes). The results of this study make an important contribution to our growing understanding of the selection pressures shaping the evolution of snake venom and its constituent toxins. The data also aid in elucidating the relationship between these selection pressures and the medical impact of human snakebite in the developing world, as cytotoxic cobras cause considerable morbidity including loss-of-function injuries that result in economic and social burdens in the tropics of Asia and sub-Saharan Africa. Full article

(This article belongs to the Special Issue Animal Venoms and Pain)

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Open AccessEditor’s ChoiceArticle Cubozoan Sting-Site Seawater Rinse, Scraping, and Ice Can Increase Venom Load: Upending Current First Aid Recommendations

by Angel Anne Yanagihara and Christie L. Wilcox

Toxins 2017, 9(3), 105; https://doi.org/10.3390/toxins9030105

Received: 3 February 2017 / Revised: 9 March 2017 / Accepted: 13 March 2017 / Published: 15 March 2017

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Abstract

Cnidarian envenomations are the leading cause of severe and lethal human sting injuries from marine life. The total amount of venom discharged into sting-site tissues, sometimes referred to as “venom load”, has been previously shown to correlate with tentacle contact length and sequelae

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Cnidarian envenomations are the leading cause of severe and lethal human sting injuries from marine life. The total amount of venom discharged into sting-site tissues, sometimes referred to as “venom load”, has been previously shown to correlate with tentacle contact length and sequelae severity. Since <1% of cnidae discharge upon initial tentacle contact, effective and safe removal of adherent tentacles is of paramount importance in the management of life-threatening cubozoan stings. We evaluated whether common rinse solutions or scraping increased venom load as measured in a direct functional assay of venom activity (hemolysis). Scraping significantly increased hemolysis by increasing cnidae discharge. For Alatina alata, increases did not occur if the tentacles were first doused with vinegar or if heat was applied. However, in Chironex fleckeri, vinegar dousing and heat treatment were less effective, and the best outcomes occurred with the use of venom-inhibiting technologies (Sting No More^® products). Seawater rinsing, considered a “no-harm” alternative, significantly increased venom load. The application of ice severely exacerbated A. alata stings, but had a less pronounced effect on C. fleckeri stings, while heat application markedly reduced hemolysis for both species. Our results do not support scraping or seawater rinsing to remove adherent tentacles. Full article

(This article belongs to the Section Animal Venoms)

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Open AccessEditor’s ChoiceArticle Venom Profiling of a Population of the Theraphosid Spider Phlogius crassipes Reveals Continuous Ontogenetic Changes from Juveniles through Adulthood

by Renan C. Santana, David Perez, James Dobson, Nadya Panagides, Robert J. Raven, Amanda Nouwens, Alun Jones, Glenn F. King and Bryan G. Fry

Toxins 2017, 9(4), 116; https://doi.org/10.3390/toxins9040116

Received: 3 February 2017 / Revised: 27 February 2017 / Accepted: 5 March 2017 / Published: 25 March 2017

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Abstract

Theraphosid spiders (tarantulas) are venomous arthropods found in most tropical and subtropical regions of the world. Tarantula venoms are a complex cocktail of toxins with potential use as pharmacological tools, drugs and bioinsecticides. Although numerous toxins have been isolated from tarantula venoms, little

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Theraphosid spiders (tarantulas) are venomous arthropods found in most tropical and subtropical regions of the world. Tarantula venoms are a complex cocktail of toxins with potential use as pharmacological tools, drugs and bioinsecticides. Although numerous toxins have been isolated from tarantula venoms, little research has been carried out on the venom of Australian tarantulas. We therefore investigated the venom profile of the Australian theraphosid spider Phlogius crassipes and examined whether there are ontogenetic changes in venom composition. Spiders were divided into four ontogenic groups according to cephalothorax length, then the venom composition of each group was examined using gel electrophoresis and mass spectrometry. We found that the venom of P. crassipes changes continuously during development and throughout adulthood. Our data highlight the need to investigate the venom of organisms over the course of their lives to uncover and understand the changing functions of venom and the full range of toxins expressed. This in turn should lead to a deeper understanding of the organism’s ecology and enhance the potential for biodiscovery. Full article

(This article belongs to the collection Evolution of Venom Systems)

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Open AccessEditor’s ChoiceArticle Changes in the Fusarium Head Blight Complex of Malting Barley in a Three-Year Field Experiment in Italy

by Giovanni Beccari, Antonio Prodi, Francesco Tini, Umberto Bonciarelli, Andrea Onofri, Souheib Oueslati, Marwa Limayma and Lorenzo Covarelli

Toxins 2017, 9(4), 120; https://doi.org/10.3390/toxins9040120

Received: 6 February 2017 / Revised: 23 March 2017 / Accepted: 24 March 2017 / Published: 29 March 2017

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Abstract

In this study, conducted for three years on eleven malting barley varieties cultivated in central Italy, the incidence of different mycotoxigenic fungal genera, the identification of the Fusarium species associated with the Fusarium Head Blight (FHB) complex, and kernels contamination with deoxynivalenol (DON)

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In this study, conducted for three years on eleven malting barley varieties cultivated in central Italy, the incidence of different mycotoxigenic fungal genera, the identification of the Fusarium species associated with the Fusarium Head Blight (FHB) complex, and kernels contamination with deoxynivalenol (DON) and T-2 mycotoxins were determined. The influence of climatic conditions on Fusarium infections and FHB complex composition was also investigated. Fusarium species were always present in the three years and the high average and maximum temperatures during anthesis mainly favored their occurrence. The FHB complex was subject to changes during the three years and the main causal agents were F. poae, F. avenaceum, F. tricinctum and F. graminearum, which, even if constantly present, never represented the principal FHB agent. The relative incidence of Fusarium species changed because of climatic conditions occurring during the seasons. The FHB complex was composed of many different Fusarium species and some of them were associated with a specific variety and/or with specific weather parameters, indicating that the interaction between a certain plant genotype and climatic conditions may influence the presence of Fusarium spp. causing infections. With regard to mycotoxin contamination, T-2 toxin, in some cases, was found in kernels at levels that exceeded EU recommended values. Full article

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Open AccessFeature PaperEditor’s ChoiceArticle Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis

by Yolanda Bel, Núria Banyuls, Maissa Chakroun, Baltasar Escriche and Juan Ferré

Toxins 2017, 9(4), 131; https://doi.org/10.3390/toxins9040131

Received: 14 March 2017 / Revised: 28 March 2017 / Accepted: 4 April 2017 / Published: 7 April 2017

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Abstract

Vip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its

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Vip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its structure. The Vip3Aa protoxin (of 89 kDa) was treated with trypsin at concentrations from 1:100 to 120:100 (trypsin:Vip3A, w:w). If the action of trypsin was not properly neutralized, the results of SDS-PAGE analysis (as well as those with Agrotis ipsilon midgut juice) equivocally indicated that the protoxin could be completely processed. However, when the proteolytic reaction was efficiently stopped, it was revealed that the protoxin was only cleaved at a primary cleavage site, regardless of the amount of trypsin used. The 66 kDa and the 19 kDa peptides generated by the proteases co-eluted after gel filtration chromatography, indicating that they remain together after cleavage. The 66 kDa fragment was found to be extremely resistant to proteases. The trypsin treatment of the protoxin in the presence of SDS revealed the presence of secondary cleavage sites at S-509, and presumably at T-466 and V-372, rendering C-terminal fragments of approximately 29, 32, and 42 kDa, respectively. The fact that the predicted secondary structure of the Vip3Aa protein shows a cluster of beta sheets in the C-terminal region of the protein might be the reason behind the higher stability to proteases compared to the rest of the protein, which is mainly composed of alpha helices. Full article

(This article belongs to the Special Issue The Insecticidal Bacterial Toxins in Modern Agriculture) Printed Edition available

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Open AccessEditor’s ChoiceArticle Modification of the Mycotoxin Deoxynivalenol Using Microorganisms Isolated from Environmental Samples

by Nina M. Wilson, Nicole McMaster, Dash Gantulga, Cara Soyars, Susan P. McCormick, Ken Knott, Ryan S. Senger and David G. Schmale

Toxins 2017, 9(4), 141; https://doi.org/10.3390/toxins9040141

Received: 2 March 2017 / Revised: 1 April 2017 / Accepted: 11 April 2017 / Published: 15 April 2017

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Abstract

The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of wheat, barley, and maize. New strategies are needed to reduce or eliminate DON in feed and food products. Microorganisms from plant and soil samples collected in Blacksburg, VA, USA, were screened by incubation

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The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of wheat, barley, and maize. New strategies are needed to reduce or eliminate DON in feed and food products. Microorganisms from plant and soil samples collected in Blacksburg, VA, USA, were screened by incubation in a mineral salt media containing 100 μg/mL DON and analysis by gas chromatography mass spectrometry (GC/MS). Two mixed cultures derived from soil samples consistently decreased DON levels in assays using DON as the sole carbon source. Nuclear magnetic resonance (NMR) analysis indicated that 3-keto-4-deoxynivalenol was the major by-product of DON. Via 16S rRNA sequencing, these mixed cultures, including mostly members of the genera Acinetobacter, Leadbetterella, and Gemmata, were revealed. Incubation of one of these mixed cultures with wheat samples naturally contaminated with 7.1 μg/mL DON indicated nearly complete conversion of DON to the less toxic 3-epimer-DON (3-epi-DON). Our work extends previous studies that have demonstrated the potential for bioprospecting for microorganisms from the environment to remediate or modify mycotoxins for commercial applications, such as the reduction of mycotoxins in fuel ethanol co-products. Full article

(This article belongs to the Special Issue Promising Detoxification Strategies to Mitigate Mycotoxins in Food and Feed) Printed Edition available

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Open AccessEditor’s ChoiceArticle Assessing the Efficacy of First-Aid Measures in Physalia sp. Envenomation, Using Solution- and Blood Agarose-Based Models

by Christie L. Wilcox, Jasmine L. Headlam, Thomas K. Doyle and Angel A. Yanagihara

Toxins 2017, 9(5), 149; https://doi.org/10.3390/toxins9050149

Received: 20 March 2017 / Revised: 18 April 2017 / Accepted: 21 April 2017 / Published: 26 April 2017

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Abstract

Stings from the hydrozoan species in the genus Physalia cause intense, immediate skin pain and elicit serious systemic effects. There has been much scientific debate about the most appropriate first aid for these stings, particularly with regard to whether vinegar use is appropriate

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Stings from the hydrozoan species in the genus Physalia cause intense, immediate skin pain and elicit serious systemic effects. There has been much scientific debate about the most appropriate first aid for these stings, particularly with regard to whether vinegar use is appropriate (most current recommendations recommend against vinegar). We found that only a small percentage (≤1.0%) of tentacle cnidae discharge during a sting event using an ex vivo tissue model which elicits spontaneous stinging from live cnidarian tentacles. We then tested a variety of rinse solutions on both Atlantic and Pacific Physalia species to determine if they elicit cnidae discharge, further investigating any that did not cause immediate significant discharge to determine if they are able to inhibit cnidae discharge in response to chemical and physical stimuli. We found commercially available vinegars, as well as the recently developed Sting No More^® Spray, were the most effective rinse solutions, as they irreversibly inhibited cnidae discharge. However, even slight dilution of vinegar reduced its protective effects. Alcohols and folk remedies, such as urine, baking soda and shaving cream, caused varying amounts of immediate cnidae discharge and failed to inhibit further discharge, and thus likely worsen stings. Full article

(This article belongs to the Section Animal Venoms)

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Open AccessEditor’s ChoiceArticle Cytotoxic Indole Alkaloid 3α-Acetonyltabersonine Induces Glioblastoma Apoptosis via Inhibition of DNA Damage Repair

by Yuan Li, Yunli Zhao, Xia Zhou, Wei Ni, Zhi Dai, Dong Yang, Junjun Hao, Lin Luo, Yaping Liu, Xiaodong Luo and Xudong Zhao

Toxins 2017, 9(5), 150; https://doi.org/10.3390/toxins9050150

Received: 5 March 2017 / Revised: 16 April 2017 / Accepted: 19 April 2017 / Published: 28 April 2017

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Abstract

Cytotoxic indole alkaloids from Melodinus suaveolens, which belongs to the toxic plant family Apocynaceae, demonstrated impressive antitumor activities in many tumor types, but less application in glioblastoma, which is the lethal brain tumor. In the present study, we reported the anti-glioblastoma activity

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Cytotoxic indole alkaloids from Melodinus suaveolens, which belongs to the toxic plant family Apocynaceae, demonstrated impressive antitumor activities in many tumor types, but less application in glioblastoma, which is the lethal brain tumor. In the present study, we reported the anti-glioblastoma activity of an indole alkaloid, 3α-acetonyltabersonine, which was isolated from Melodinus suaveolens. 3α-acetonyltabersonine was cytotoxic to glioblastoma cell lines (U87 and T98G) and stem cells at low concentrations. We verified 3α-acetonyltabersonine could suppress tumor cell proliferation and cause apoptosis in glioblastoma stem cells (GSCs). Moreover, detailed investigation of transcriptome study and Western blotting analysis indicated the mitogen activated protein kinase (MAPK) pathway was activated by phosphorylation upon 3α-acetonyltabersonine treatment. Additionally, we found 3α-acetonyltabersonine inhibited DNA damage repair procedures, the accumulated DNA damage stimulated activation of MAPK pathway and, finally, induced apoptosis. Further evidence was consistently obtained from vivo experiments on glioblastoma mouse model: treatment of 3α-acetonyltabersonine could exert pro-apoptotic function and prolong the life span of tumor-bearing mice. These results in vitro and in vivo suggested that 3α-acetonyltabersonine could be a potential candidate antitumor agent. Full article

(This article belongs to the Section Plant Toxins)

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Open AccessFeature PaperEditor’s ChoiceArticle Insect-Active Toxins with Promiscuous Pharmacology from the African Theraphosid Spider Monocentropus balfouri

by Jennifer J. Smith, Volker Herzig, Maria P. Ikonomopoulou, Sławomir Dziemborowicz, Frank Bosmans, Graham M. Nicholson and Glenn F. King

Toxins 2017, 9(5), 155; https://doi.org/10.3390/toxins9050155

Received: 28 March 2017 / Revised: 24 April 2017 / Accepted: 28 April 2017 / Published: 5 May 2017

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Abstract

Many chemical insecticides are becoming less efficacious due to rising resistance in pest species, which has created much interest in the development of new, eco-friendly bioinsecticides. Since insects are the primary prey of most spiders, their venoms are a rich source of insect-active

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Many chemical insecticides are becoming less efficacious due to rising resistance in pest species, which has created much interest in the development of new, eco-friendly bioinsecticides. Since insects are the primary prey of most spiders, their venoms are a rich source of insect-active peptides that can be used as leads for new bioinsecticides or as tools to study molecular receptors that are insecticidal targets. In the present study, we isolated two insecticidal peptides, µ/ω-TRTX-Mb1a and -Mb1b, from venom of the African tarantula Monocentropus balfouri. Recombinant µ/ω-TRTX-Mb1a and -Mb1b paralyzed both Lucilia cuprina (Australian sheep blowfly) and Musca domestica (housefly), but neither peptide affected larvae of Helicoverpa armigera (cotton bollworms). Both peptides inhibited currents mediated by voltage-gated sodium (Na_V) and calcium channels in Periplaneta americana (American cockroach) dorsal unpaired median neurons, and they also inhibited the cloned Blattella germanica (German cockroach) Na_V channel (BgNa_V1). An additional effect seen only with Mb1a on BgNa_V1 was a delay in fast inactivation. Comparison of the Na_V channel sequences of the tested insect species revealed that variations in the S1–S2 loops in the voltage sensor domains might underlie the differences in activity between different phyla. Full article

(This article belongs to the Section Animal Venoms)

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Open AccessFeature PaperEditor’s ChoiceArticle Third Generation Antivenomics: Pushing the Limits of the In Vitro Preclinical Assessment of Antivenoms

by Davinia Pla, Yania Rodríguez and Juan J. Calvete

Toxins 2017, 9(5), 158; https://doi.org/10.3390/toxins9050158

Received: 20 April 2017 / Revised: 5 May 2017 / Accepted: 5 May 2017 / Published: 10 May 2017

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Abstract

Second generation antivenomics is a translational venomics approach designed to complement in vivo preclinical neutralization assays. It provides qualitative and quantitative information on the set of homologous and heterologous venom proteins presenting antivenom-recognized epitopes and those exhibiting impaired immunoreactivity. In a situation of

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Second generation antivenomics is a translational venomics approach designed to complement in vivo preclinical neutralization assays. It provides qualitative and quantitative information on the set of homologous and heterologous venom proteins presenting antivenom-recognized epitopes and those exhibiting impaired immunoreactivity. In a situation of worrying antivenom shortage in many tropical and sub-tropical regions with high snakebite mortality and morbidity rates, such knowledge has the potential to facilitate the optimal deployment of currently existing antivenoms and to aid in the rational design of novel broad specificity antidotes. The aim of the present work was to expand the analytical capability of the immunoaffinity second-generation antivenomics platform, endowing it with the ability to determine the maximal binding capacity of an antivenom toward the different toxins present in a venom, and to quantify the fraction of venom-specific antibodies present in a given antivenom. The application of this new platform, termed third generation (3G) antivenomics, in the preclinical evaluation of antivenoms is illustrated in this paper for the case of antivenom EchiTAb-Plus-ICP^® reactivity towards the toxins of homologous (B. arietans) and heterologous (N. melanoleuca) venoms. Full article

(This article belongs to the Special Issue Use of Antibodies/Antivenom Against Envenoming)

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Open AccessEditor’s ChoiceArticle The Vip3Ag4 Insecticidal Protoxin from Bacillus thuringiensis Adopts A Tetrameric Configuration That Is Maintained on Proteolysis

by Leopoldo Palma, David J. Scott, Gemma Harris, Salah-Ud Din, Thomas L. Williams, Oliver J. Roberts, Mark T. Young, Primitivo Caballero and Colin Berry

Toxins 2017, 9(5), 165; https://doi.org/10.3390/toxins9050165

Received: 17 January 2017 / Revised: 11 May 2017 / Accepted: 12 May 2017 / Published: 14 May 2017

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Abstract

The Vip3 proteins produced during vegetative growth by strains of the bacterium Bacillus thuringiensis show insecticidal activity against lepidopteran insects with a mechanism of action that may involve pore formation and apoptosis. These proteins are promising supplements to our arsenal of insecticidal proteins,

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The Vip3 proteins produced during vegetative growth by strains of the bacterium Bacillus thuringiensis show insecticidal activity against lepidopteran insects with a mechanism of action that may involve pore formation and apoptosis. These proteins are promising supplements to our arsenal of insecticidal proteins, but the molecular details of their activity are not understood. As a first step in the structural characterisation of these proteins, we have analysed their secondary structure and resolved the surface topology of a tetrameric complex of the Vip3Ag4 protein by transmission electron microscopy. Sites sensitive to proteolysis by trypsin are identified and the trypsin-cleaved protein appears to retain a similar structure as an octomeric complex comprising four copies each of the ~65 kDa and ~21 kDa products of proteolysis. This processed form of the toxin may represent the active toxin. The quality and monodispersity of the protein produced in this study make Vip3Ag4 a candidate for more detailed structural analysis using cryo-electron microscopy. Full article

(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins) Printed Edition available

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Open AccessEditor’s ChoiceArticle Coagulating Colubrids: Evolutionary, Pathophysiological and Biodiscovery Implications of Venom Variations between Boomslang (Dispholidus typus) and Twig Snake (Thelotornis mossambicanus)

by Jordan Debono, James Dobson, Nicholas R. Casewell, Anthony Romilio, Bin Li, Nyoman Kurniawan, Karine Mardon, Vera Weisbecker, Amanda Nouwens, Hang Fai Kwok and Bryan G. Fry

Toxins 2017, 9(5), 171; https://doi.org/10.3390/toxins9050171

Received: 16 January 2017 / Revised: 15 May 2017 / Accepted: 15 May 2017 / Published: 19 May 2017

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Abstract

Venoms can deleteriously affect any physiological system reachable by the bloodstream, including directly interfering with the coagulation cascade. Such coagulopathic toxins may be anticoagulants or procoagulants. Snake venoms are unique in their use of procoagulant toxins for predatory purposes. The boomslang (Dispholidus

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Venoms can deleteriously affect any physiological system reachable by the bloodstream, including directly interfering with the coagulation cascade. Such coagulopathic toxins may be anticoagulants or procoagulants. Snake venoms are unique in their use of procoagulant toxins for predatory purposes. The boomslang (Dispholidus typus) and the twig snakes (Thelotornis species) are iconic African snakes belonging to the family Colubridae. Both species produce strikingly similar lethal procoagulant pathologies. Despite these similarities, antivenom is only produced for treating bites by D. typus, and the mechanisms of action of both venoms have been understudied. In this study, we investigated the venom of D. typus and T. mossambicanus utilising a range of proteomic and bioactivity approaches, including determining the procoagulant properties of both venoms in relation to the human coagulation pathways. In doing so, we developed a novel procoagulant assay, utilising a Stago STA-R Max analyser, to accurately detect real time clotting in plasma at varying concentrations of venom. This approach was used to assess the clotting capabilities of the two venoms both with and without calcium and phospholipid co-factors. We found that T. mossambicanus produced a significantly stronger coagulation response compared to D. typus. Functional enzyme assays showed that T. mossambicanus also exhibited a higher metalloprotease and phospholipase activity but had a much lower serine protease activity relative to D. typus venom. The neutralising capability of the available boomslang antivenom was also investigated on both species, with it being 11.3 times more effective upon D. typus venom than T. mossambicanus. In addition to being a faster clotting venom, T. mossambicanus was revealed to be a much more complex venom composition than D. typus. This is consistent with patterns seen for other snakes with venom complexity linked to dietary complexity. Consistent with the external morphological differences in head shape between the two species, CT and MRI analyses revealed significant internal structural differences in skull architecture and venom gland anatomy. This study increases our understanding of not only the biodiscovery potential of these medically important species but also increases our knowledge of the pathological relationship between venom and the human coagulation cascade. Full article

(This article belongs to the Section Animal Venoms)

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Open AccessEditor’s ChoiceArticle Venom On-a-Chip: A Fast and Efficient Method for Comparative Venomics

by Giulia Zancolli, Libia Sanz, Juan J. Calvete and Wolfgang Wüster

Toxins 2017, 9(6), 179; https://doi.org/10.3390/toxins9060179

Received: 20 April 2017 / Revised: 23 May 2017 / Accepted: 24 May 2017 / Published: 28 May 2017

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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

(This article belongs to the Section Animal Venoms)

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Open AccessEditor’s ChoiceArticle Presence of Enniatins and Beauvericin in Romanian Wheat Samples: From Raw Material to Products for Direct Human Consumption

by Oana Stanciu, Cristina Juan, Doina Miere, Felicia Loghin and Jordi Mañes

Toxins 2017, 9(6), 189; https://doi.org/10.3390/toxins9060189

Received: 3 May 2017 / Revised: 1 June 2017 / Accepted: 6 June 2017 / Published: 12 June 2017

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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

(This article belongs to the collection Understanding Mycotoxin Occurrence in Food and Feed Chains)

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Open AccessEditor’s ChoiceArticle Prevalence, Variability and Bioconcentration of Saxitoxin-Group in Different Marine Species Present in the Food Chain

by Javiera Oyaneder Terrazas, Héctor R. Contreras and Carlos García

Toxins 2017, 9(6), 190; https://doi.org/10.3390/toxins9060190

Received: 17 May 2017 / Revised: 7 June 2017 / Accepted: 8 June 2017 / Published: 12 June 2017

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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

(This article belongs to the Special Issue Public Health Outreach to Prevention of Aquatic Toxin Exposure)

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Open AccessEditor’s ChoiceArticle Evaluation of Cyanea capillata Sting Management Protocols Using Ex Vivo and In Vitro Envenomation Models

by Thomas K. Doyle, Jasmine L. Headlam, Christie L. Wilcox, Eoin MacLoughlin and Angel A. Yanagihara

Toxins 2017, 9(7), 215; https://doi.org/10.3390/toxins9070215

Received: 2 June 2017 / Revised: 27 June 2017 / Accepted: 3 July 2017 / Published: 7 July 2017

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Abstract

Lion’s mane jellyfish (Cyanea capillata) stings cause severe pain and can lead to dangerous systemic effects, including Irukandji-like syndrome. As is the case for most cnidarian stings, recommended medical protocols in response to such stings lack rigorous scientific support. In this

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Lion’s mane jellyfish (Cyanea capillata) stings cause severe pain and can lead to dangerous systemic effects, including Irukandji-like syndrome. As is the case for most cnidarian stings, recommended medical protocols in response to such stings lack rigorous scientific support. In this study, we sought to evaluate potential first aid care protocols using previously described envenomation models that allow for direct measurements of venom activity. We found that seawater rinsing, the most commonly recommended method of tentacle removal for this species, induced significant increases in venom delivery, while rinsing with vinegar or Sting No More^® Spray did not. Post-sting temperature treatments affected sting severity, with 40 min of hot-pack treatment reducing lysis of sheep’s blood (in agar plates), a direct representation of venom load, by over 90%. Ice pack treatment had no effect on sting severity. These results indicate that sting management protocols for Cyanea need to be revised immediately to discontinue rinsing with seawater and include the use of heat treatment. Full article

(This article belongs to the Section Animal Venoms)

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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

by Kurin Iimura, Tomohiro Furukawa, Toshiyoshi Yamamoto, Lumi Negishi, Michio Suzuki and Shohei Sakuda

Toxins 2017, 9(7), 219; https://doi.org/10.3390/toxins9070219

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

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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

(This article belongs to the collection Aflatoxins)

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Open AccessEditor’s ChoiceArticle Venomics of Remipede Crustaceans Reveals Novel Peptide Diversity and Illuminates the Venom’s Biological Role

by Björn M. von Reumont, Eivind A. B. Undheim, Robin-Tobias Jauss and Ronald A. Jenner

Toxins 2017, 9(8), 234; https://doi.org/10.3390/toxins9080234

Received: 27 June 2017 / Accepted: 24 July 2017 / Published: 26 July 2017

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Abstract

We report the first integrated proteomic and transcriptomic investigation of a crustacean venom. Remipede crustaceans are the venomous sister group of hexapods, and the venom glands of the remipede Xibalbanus tulumensis express a considerably more complex cocktail of proteins and peptides than previously

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We report the first integrated proteomic and transcriptomic investigation of a crustacean venom. Remipede crustaceans are the venomous sister group of hexapods, and the venom glands of the remipede Xibalbanus tulumensis express a considerably more complex cocktail of proteins and peptides than previously thought. We identified 32 venom protein families, including 13 novel peptide families that we name xibalbins, four of which lack similarities to any known structural class. Our proteomic data confirm the presence in the venom of 19 of the 32 families. The most highly expressed venom components are serine peptidases, chitinase and six of the xibalbins. The xibalbins represent Inhibitory Cystine Knot peptides (ICK), a double ICK peptide, peptides with a putative Cystine-stabilized α-helix/β-sheet motif, a peptide similar to hairpin-like β-sheet forming antimicrobial peptides, two peptides related to different hormone families, and four peptides with unique structural motifs. Remipede venom components represent the full range of evolutionary recruitment frequencies, from families that have been recruited into many animal venoms (serine peptidases, ICKs), to those having a very narrow taxonomic range (double ICKs), to those unique for remipedes. We discuss the most highly expressed venom components to shed light on their possible functional significance in the predatory and defensive use of remipede venom, and to provide testable ideas for any future bioactivity studies. Full article

(This article belongs to the Section Animal Venoms)

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Open AccessEditor’s ChoiceArticle Analysis of the Masked Metabolite of Deoxynivalenol and Fusarium Resistance in CIMMYT Wheat Germplasm

by Hiroyuki Nakagawa, Xinyao He, Yosuke Matsuo, Pawan K. Singh and Masayo Kushiro

Toxins 2017, 9(8), 238; https://doi.org/10.3390/toxins9080238

Received: 15 June 2017 / Revised: 26 July 2017 / Accepted: 27 July 2017 / Published: 29 July 2017

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Abstract

Fusarium head blight (FHB) causes significant grain loss and contamination of grains with harmful mycotoxins, especially deoxynivalenol (DON). Fusarium resistance and DON accumulation have been extensively investigated in various cultivars; however, the level of DON-3-O-glucoside (D3G) has not been as carefully

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Fusarium head blight (FHB) causes significant grain loss and contamination of grains with harmful mycotoxins, especially deoxynivalenol (DON). Fusarium resistance and DON accumulation have been extensively investigated in various cultivars; however, the level of DON-3-O-glucoside (D3G) has not been as carefully studied. In this study, we measured accumulated DON and D3G levels in CIMMYT wheat elite germplasm using an analytical method validated in-house. Co-occurring nivalenol (NIV) and ergostrerol (ERG) were also analyzed. LC-MS/MS and LC-UV analyses were applied to the 50 CIMMYT elite wheat lines. D3G showed rather high correlation with DON (r = 0.82), while FHB symptoms showed slight correlation with DON and D3G (r = 0.36 and 0.32, respectively). D3G/DON ratio varied widely from 8.1 to 37.7%, and the ratio was not related with FHB resistance in this dataset. Full article

(This article belongs to the collection Understanding Mycotoxin Occurrence in Food and Feed Chains)

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Open AccessEditor’s ChoiceArticle Insights into the Mechanisms Involved in Strong Hemorrhage and Dermonecrosis Induced by Atroxlysin-Ia, a PI-Class Snake Venom Metalloproteinase

by Luciana Aparecida Freitas-de-Sousa, Mônica Colombini, Mônica Lopes-Ferreira, Solange M. T. Serrano and Ana Maria Moura-da-Silva

Toxins 2017, 9(8), 239; https://doi.org/10.3390/toxins9080239

Received: 30 June 2017 / Revised: 28 July 2017 / Accepted: 29 July 2017 / Published: 2 August 2017

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Abstract

Hemorrhage is the most prominent effect of snake venom metalloproteinases (SVMPs) in human envenomation. The capillary injury is a multifactorial effect caused by hydrolysis of the components of the basement membrane (BM). The PI and PIII classes of SVMPs are abundant in viperid

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Hemorrhage is the most prominent effect of snake venom metalloproteinases (SVMPs) in human envenomation. The capillary injury is a multifactorial effect caused by hydrolysis of the components of the basement membrane (BM). The PI and PIII classes of SVMPs are abundant in viperid venoms and hydrolyze BM components. However, hemorrhage is associated mostly with PIII-class SVMPs that contain non-catalytic domains responsible for the binding of SVMPs to BM proteins, facilitating enzyme accumulation in the tissue and enhancing its catalytic efficiency. Here we report on Atroxlysin-Ia, a PI-class SVMP that induces hemorrhagic lesions in levels comparable to those induced by Batroxrhagin (PIII-class), and a unique SVMP effect characterized by the rapid onset of dermonecrotic lesions. Atroxlysin-Ia was purified from B. atrox venom, and sequence analyses indicated that it is devoid of non-catalytic domains and unable to bind to BM proteins as collagen IV and laminin in vitro or in vivo. The presence of Atroxlysin-Ia was diffuse in mice skin, and localized mainly in the epidermis with no co-localization with BM components. Nevertheless, the skin lesions induced by Atroxlysin-Ia were comparable to those induced by Batroxrhagin, with induction of leukocyte infiltrates and hemorrhagic areas soon after toxin injection. Detachment of the epidermis was more intense in skin injected with Atroxlysin-Ia. Comparing the catalytic activity of both toxins, Batroxrhagin was more active in the hydrolysis of a peptide substrate while Atroxlysin-Ia hydrolyzed more efficiently fibrin, laminin, collagen IV and nidogen. Thus, the results suggest that Atroxlysin-Ia bypasses the binding step to BM proteins, essential for hemorrhagic lesions induced by PII- and P-III class SVMPs, causing a significantly fast onset of hemorrhage and dermonecrosis, due to its higher proteolytic capacity on BM components. Full article

(This article belongs to the Section Animal Venoms)

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Open AccessEditor’s ChoiceArticle Enter the Dragon: The Dynamic and Multifunctional Evolution of Anguimorpha Lizard Venoms

Toxins 2017, 9(8), 242; https://doi.org/10.3390/toxins9080242

Received: 5 June 2017 / Revised: 4 August 2017 / Accepted: 4 August 2017 / Published: 6 August 2017

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Abstract

While snake venoms have been the subject of intense study, comparatively little work has been done on lizard venoms. In this study, we have examined the structural and functional diversification of anguimorph lizard venoms and associated toxins, and related these results to dentition

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While snake venoms have been the subject of intense study, comparatively little work has been done on lizard venoms. In this study, we have examined the structural and functional diversification of anguimorph lizard venoms and associated toxins, and related these results to dentition and predatory ecology. Venom composition was shown to be highly variable across the 20 species of Heloderma, Lanthanotus, and Varanus included in our study. While kallikrein enzymes were ubiquitous, they were also a particularly multifunctional toxin type, with differential activities on enzyme substrates and also ability to degrade alpha or beta chains of fibrinogen that reflects structural variability. Examination of other toxin types also revealed similar variability in their presence and activity levels. The high level of venom chemistry variation in varanid lizards compared to that of helodermatid lizards suggests that venom may be subject to different selection pressures in these two families. These results not only contribute to our understanding of venom evolution but also reveal anguimorph lizard venoms to be rich sources of novel bioactive molecules with potential as drug design and development lead compounds. Full article

(This article belongs to the collection Evolution of Venom Systems)

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Open AccessEditor’s ChoiceArticle Enzymatic and Pro-Inflammatory Activities of Bothrops lanceolatus Venom: Relevance for Envenomation

by Marie Delafontaine, Isadora Maria Villas-Boas, Laurence Mathieu, Patrice Josset, Joël Blomet and Denise V. Tambourgi

Toxins 2017, 9(8), 244; https://doi.org/10.3390/toxins9080244

Received: 2 July 2017 / Revised: 27 July 2017 / Accepted: 31 July 2017 / Published: 7 August 2017

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Abstract

Bothrops lanceolatus, commonly named ‘Fer-de-Lance’, is an endemic snake of the French Caribbean Island of Martinique. Envenomations by B. lanceolatus present clinical aspects characterized by systemic thrombotic syndrome and important local inflammation, involving edema and pain but limited hemorrhage. To investigate mechanisms

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Bothrops lanceolatus, commonly named ‘Fer-de-Lance’, is an endemic snake of the French Caribbean Island of Martinique. Envenomations by B. lanceolatus present clinical aspects characterized by systemic thrombotic syndrome and important local inflammation, involving edema and pain but limited hemorrhage. To investigate mechanisms of venom-induced inflammation, B. lanceolatus venom was characterized, its cross-reactivity with bothropic antivenom explored, its cytotoxicity on human keratinocytes and vascular cells, and the production of cytokines and chemokines were analyzed. We used electrophoretic separation, zymography, colorimetric or fluorimetric enzymatic assays, and immunochemical assays. Therapeutic South American bothropic antivenom cross-reacted with B. lanceolatus venom and completely or partially abolished its PLA2, hyaluronidase, and proteolytic activities, as well as its cytotoxicity for keratinocytes. The substrate specificity of B. lanceolatus venom proteases was emphasized. B. lanceolatus venom cytotoxicity was compared to the B. jararaca venom. Both venoms were highly cytotoxic for keratinocytes (HaCaT), whereas B. lanceolatus venom showed particularly low toxicity for endothelial cells (EAhy926). Patterns of cytokine and chemokine production by cells exposed to the venoms were highly pro-inflammatory. Thus, the results presented here show that B. lanceolatus venom toxins share important antigenic similarities with South American Bothrops species toxins, although their proteases have acquired particular substrate specificity. Moreover, the venom displays important cytotoxic and pro-inflammatory action on human cell types such as keratinocytes and endothelial cells, which are important players in the local and systemic compartments affected by the envenomation. Full article

(This article belongs to the Section Animal Venoms)

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Open AccessEditor’s ChoiceArticle Chemical Identity of Interaction of Protein with Reactive Metabolite of Diosbulbin B In Vitro and In Vivo

by Kai Wang, Dongju Lin, Xiucai Guo, Wenlin Huang, Jiang Zheng and Ying Peng

Toxins 2017, 9(8), 249; https://doi.org/10.3390/toxins9080249

Received: 10 July 2017 / Revised: 24 July 2017 / Accepted: 28 July 2017 / Published: 14 August 2017

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Abstract

Diosbulbin B (DIOB), a hepatotoxic furan-containing compound, is a primary ingredient in Dioscorea bulbifera L., a common herbal medicine. Metabolic activation is required for DIOB-induced liver injury. Protein covalent binding of an electrophilic reactive intermediate of DIOB is considered to be one of

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Diosbulbin B (DIOB), a hepatotoxic furan-containing compound, is a primary ingredient in Dioscorea bulbifera L., a common herbal medicine. Metabolic activation is required for DIOB-induced liver injury. Protein covalent binding of an electrophilic reactive intermediate of DIOB is considered to be one of the key mechanisms of cytotoxicity. A bromine-based analytical technique was developed to characterize the chemical identity of interaction of protein with reactive intermediate of DIOB. Cysteine (Cys) and lysine (Lys) residues were found to react with the reactive intermediate to form three types of protein modification, including Cys adduction, Schiff’s base, and Cys/Lys crosslink. The crosslink showed time- and dose-dependence in animals given DIOB. Ketoconazole pretreatment decreased the formation of the crosslink derived from DIOB, whereas pretreatment with dexamethasone or buthionine sulfoximine increased such protein modification. These data revealed that the levels of hepatic protein adductions were proportional to the severity of hepatotoxicity of DIOB. Full article

(This article belongs to the Section Plant Toxins)

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Open AccessEditor’s ChoiceArticle Orally Delivered Scorpion Antimicrobial Peptides Exhibit Activity against Pea Aphid (Acyrthosiphon pisum) and Its Bacterial Symbionts

by Karen Luna-Ramirez, Marisa Skaljac, Jens Grotmann, Phillipp Kirfel and Andreas Vilcinskas

Toxins 2017, 9(9), 261; https://doi.org/10.3390/toxins9090261

Received: 4 July 2017 / Revised: 3 August 2017 / Accepted: 22 August 2017 / Published: 24 August 2017

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Abstract

Aphids are severe agricultural pests that damage crops by feeding on phloem sap and vectoring plant pathogens. Chemical insecticides provide an important aphid control strategy, but alternative and sustainable control measures are required to avoid rapidly emerging resistance, environmental contamination, and the risk

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Aphids are severe agricultural pests that damage crops by feeding on phloem sap and vectoring plant pathogens. Chemical insecticides provide an important aphid control strategy, but alternative and sustainable control measures are required to avoid rapidly emerging resistance, environmental contamination, and the risk to humans and beneficial organisms. Aphids are dependent on bacterial symbionts, which enable them to survive on phloem sap lacking essential nutrients, as well as conferring environmental stress tolerance and resistance to parasites. The evolution of aphids has been accompanied by the loss of many immunity-related genes, such as those encoding antibacterial peptides, which are prevalent in other insects, probably because any harm to the bacterial symbionts would inevitably affect the aphids themselves. This suggests that antimicrobial peptides (AMPs) could replace or at least complement conventional insecticides for aphid control. We fed the pea aphids (Acyrthosiphon pisum) with AMPs from the venom glands of scorpions. The AMPs reduced aphid survival, delayed their reproduction, displayed in vitro activity against aphid bacterial symbionts, and reduced the number of symbionts in vivo. Remarkably, we found that some of the scorpion AMPs compromised the aphid bacteriome, a specialized organ that harbours bacterial symbionts. Our data suggest that scorpion AMPs holds the potential to be developed as bio-insecticides, and are promising candidates for the engineering of aphid-resistant crops. Full article

(This article belongs to the Section Animal Venoms)

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Open AccessEditor’s ChoiceArticle Listeriolysin O Regulates the Expression of Optineurin, an Autophagy Adaptor That Inhibits the Growth of Listeria monocytogenes

by Madhu Puri, Luigi La Pietra, Mobarak Abu Mraheil, Rudolf Lucas, Trinad Chakraborty and Helena Pillich

Toxins 2017, 9(9), 273; https://doi.org/10.3390/toxins9090273

Received: 31 July 2017 / Revised: 31 August 2017 / Accepted: 2 September 2017 / Published: 5 September 2017

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Abstract

Autophagy, a well-established defense mechanism, enables the elimination of intracellular pathogens including Listeria monocytogenes. Host cell recognition results in ubiquitination of L. monocytogenes and interaction with autophagy adaptors p62/SQSTM1 and NDP52, which target bacteria to autophagosomes by binding to microtubule-associated protein

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Autophagy, a well-established defense mechanism, enables the elimination of intracellular pathogens including Listeria monocytogenes. Host cell recognition results in ubiquitination of L. monocytogenes and interaction with autophagy adaptors p62/SQSTM1 and NDP52, which target bacteria to autophagosomes by binding to microtubule-associated protein 1 light chain 3 (LC3). Although studies have indicated that L. monocytogenes induces autophagy, the significance of this process in the infectious cycle and the mechanisms involved remain poorly understood. Here, we examined the role of the autophagy adaptor optineurin (OPTN), the phosphorylation of which by the TANK binding kinase 1 (TBK1) enhances its affinity for LC3 and promotes autophagosomal degradation, during L. monocytogenes infection. In LC3- and OPTN-depleted host cells, intracellular replicating L. monocytogenes increased, an effect not seen with a mutant lacking the pore-forming toxin listeriolysin O (LLO). LLO induced the production of OPTN. In host cells expressing an inactive TBK1, bacterial replication was also inhibited. Our studies have uncovered an OPTN-dependent pathway in which L. monocytogenes uses LLO to restrict bacterial growth. Hence, manipulation of autophagy by L. monocytogenes, either through induction or evasion, represents a key event in its intracellular life style and could lead to either cytosolic growth or persistence in intracellular vacuolar structures. Full article

(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins) Printed Edition available

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Open AccessEditor’s ChoiceArticle Typhonium giganteum Lectin Exerts A Pro-Inflammatory Effect on RAW 264.7 via ROS and The NF-κB Signaling Pathway

by Wei Wang, Hao Wu, Hongli Yu, Xingde Zhang, Guojing Cui, Kuilong Wang, Shanhu Mao and Yaozong Pan

Toxins 2017, 9(9), 275; https://doi.org/10.3390/toxins9090275

Received: 8 August 2017 / Revised: 4 September 2017 / Accepted: 5 September 2017 / Published: 7 September 2017

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Abstract

Typhonii rhizoma, a widely used herb in traditional Chinese medicine, has acute irritating toxicity related to Typhonium giganteum lectin (TGL). TGL exhibits acute inflammatory effects, but the underlying molecular mechanisms are largely unknown. This paper is designed to assess the pro-inflammatory response

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Typhonii rhizoma, a widely used herb in traditional Chinese medicine, has acute irritating toxicity related to Typhonium giganteum lectin (TGL). TGL exhibits acute inflammatory effects, but the underlying molecular mechanisms are largely unknown. This paper is designed to assess the pro-inflammatory response of TGL on RAW 264.7 cells. RAW 264.7 treated with 6.25, 12.5, 25, and 50 µg/mL TGL showed elevated levels of inflammatory factors (TNF-α, IL-1β) and of p-IκB and p-p65, all dose-dependent, indicating that TGL had a substantial inflammatory effect and mobilized the nuclear factor-κB (NF-κB) pathway. All four TGL treatments also induced the up-regulation of reactive oxygen species (ROS) and cytosolic free Ca²⁺ and down-regulation of mitochondrial membrane potential (MMP). The production of cytokines and p-IκB, p-p65 were reduced by N-acetylcysteine (NAC), an ROS scavenger, which somewhat abrogated ROS production. The results showed the TGL-activated inflammatory signaling pathway NF-κB to be associated with the overproduction of ROS. Moreover, 50 μg/mL treatment with TGL led to cell apoptosis after 1 h and increased necrosis over time. These results provided potential molecular mechanisms for the observed inflammatory response to TGL including up-regulation of ROS and cytosolic free Ca²⁺, down-regulation of MMP, the mobilization of the NF-κB pathway, and the subsequent overproduction of pro-inflammatory factors resulting in apoptosis. Long-term stimulation with TGL resulted in strong toxic effects related to inflammation that induced necrosis in macrophages. Full article

(This article belongs to the Section Plant Toxins)

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Open AccessEditor’s ChoiceArticle α-Conotoxin Decontamination Protocol Evaluation: What Works and What Doesn’t

by Matthew W. Turner, John R. Cort and Owen M. McDougal

Toxins 2017, 9(9), 281; https://doi.org/10.3390/toxins9090281

Received: 11 August 2017 / Revised: 4 September 2017 / Accepted: 9 September 2017 / Published: 14 September 2017

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Abstract

Nine publically available biosafety protocols for safely handling conotoxin peptides were tested to evaluate their decontamination efficacy. Circular dichroism (CD) spectroscopy and mass spectrometry (MS) were used to assess the effect of each chemical treatment on the secondary and primary structure of α-CTx

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Nine publically available biosafety protocols for safely handling conotoxin peptides were tested to evaluate their decontamination efficacy. Circular dichroism (CD) spectroscopy and mass spectrometry (MS) were used to assess the effect of each chemical treatment on the secondary and primary structure of α-CTx MII (L10V, E11A). Of the nine decontamination methods tested, treatment with 1% (m/v) solution of the enzymatic detergent Contrex™ EZ resulted in a 76.8% decrease in α-helical content as assessed by the mean residue ellipticity at 222 nm, and partial peptide digestion was demonstrated using high performance liquid chromatography mass spectrometry (HPLC-MS). Additionally, treatment with 6% sodium hypochlorite (m/v) resulted in 80.5% decrease in α-helical content and complete digestion of the peptide. The Contrex™ EZ treatment was repeated with three additional α-conotoxins (α-CTxs), α-CTxs LvIA, ImI and PeIA, which verified the decontamination method was reasonably robust. These results support the use of either 1% Contrex™ EZ solution or 6% sodium hypochlorite in biosafety protocols for the decontamination of α-CTxs in research laboratories. Full article

(This article belongs to the collection Marine and Freshwater Toxins)

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Open AccessEditor’s ChoiceArticle Antiallodynic Effects of Bee Venom in an Animal Model of Complex Regional Pain Syndrome Type 1 (CRPS-I)

by Sung Hyun Lee, Jae Min Lee, Yun Hong Kim, Jung Hyun Choi, Seung Hwan Jeon, Dong Kyu Kim, Hyeon Do Jeong, You Jung Lee and Hue Jung Park

Toxins 2017, 9(9), 285; https://doi.org/10.3390/toxins9090285

Received: 25 August 2017 / Revised: 11 September 2017 / Accepted: 13 September 2017 / Published: 15 September 2017

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Abstract

Neuropathic pain in a chronic post-ischaemic pain (CPIP) model mimics the symptoms of complex regional pain syndrome type I (CRPS I). The administration of bee venom (BV) has been utilized in Eastern medicine to treat chronic inflammatory diseases accompanying pain. However, the analgesic

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Neuropathic pain in a chronic post-ischaemic pain (CPIP) model mimics the symptoms of complex regional pain syndrome type I (CRPS I). The administration of bee venom (BV) has been utilized in Eastern medicine to treat chronic inflammatory diseases accompanying pain. However, the analgesic effect of BV in a CPIP model remains unknown. The application of a tight-fitting O-ring around the left ankle for a period of 3 h generated CPIP in C57/Bl6 male adult mice. BV (1 mg/kg ; 1, 2, and 3 times) was administered into the SC layer of the hind paw, and the antiallodynic effects were investigated using the von Frey test and by measuring the expression of neurokinin type 1 (NK-1) receptors in dorsal root ganglia (DRG). The administration of BV dose-dependently reduced the pain withdrawal threshold to mechanical stimuli compared with the pre-administration value and with that of the control group. After the development of the CPIP model, the expression of NK-1 receptors in DRG increased and then decreased following the administration of BV. SC administration of BV results in the attenuation of allodynia in a mouse model of CPIP. The antiallodynic effect was objectively proven through a reduction in the increased expression of NK-1 receptors in DRG. Full article

(This article belongs to the Special Issue Toxins in Drug Discovery and Pharmacology) Printed Edition available

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Open AccessEditor’s ChoiceArticle Evidence for Complex Formation of the Bacillus cereus Haemolysin BL Components in Solution

by Franziska Tausch, Richard Dietrich, Kristina Schauer, Robert Janowski, Dierk Niessing, Erwin Märtlbauer and Nadja Jessberger

Toxins 2017, 9(9), 288; https://doi.org/10.3390/toxins9090288

Received: 16 August 2017 / Revised: 11 September 2017 / Accepted: 12 September 2017 / Published: 16 September 2017

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Abstract

Haemolysin BL is an important virulence factor regarding the diarrheal type of food poisoning caused by Bacillus cereus. However, the pathogenic importance of this three-component enterotoxin is difficult to access, as nearly all natural B. cereus culture supernatants additionally contain the highly

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Haemolysin BL is an important virulence factor regarding the diarrheal type of food poisoning caused by Bacillus cereus. However, the pathogenic importance of this three-component enterotoxin is difficult to access, as nearly all natural B. cereus culture supernatants additionally contain the highly cytotoxic Nhe, the second three-component toxin involved in the aetiology of B. cereus-induced food-borne diseases. To better address the toxic properties of the Hbl complex, a system for overexpression and purification of functional, cytotoxic, recombinant (r)Hbl components L₂, L₁ and B from E. coli was established and an nheABC deletion mutant was constructed from B. cereus reference strain F837/76. Furthermore, 35 hybridoma cell lines producing monoclonal antibodies (mAbs) against Hbl L₂, L₁ and B were generated. While mAbs 1H9 and 1D8 neutralized Hbl toxicity and thus, represent important tools for future investigations of the mode-of-action of Hbl on the target cell surface, mAb 1D7, in contrast, even enhanced Hbl toxicity by supporting the binding of Hbl B to the cell surface. By using the specific mAbs in Dot blots, indirect and hybrid sandwich enzyme immuno assays (EIAs), complex formation between Hbl L₁ and B, as well as L₁ and L₂ in solution could be shown for the first time. Surface plasmon resonance experiments with the rHbl components confirmed these results with K_D values of 4.7 × 10⁻⁷ M and 1.5 × 10⁻⁷ M, respectively. These findings together with the newly created tools lay the foundation for the detailed elucidation of the molecular mode-of-action of the highly complex three-component Hbl toxin. Full article

(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins) Printed Edition available

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Open AccessEditor’s ChoiceArticle Gas Chromatography-Mass Spectrometry for Metabolite Profiling of Japanese Black Cattle Naturally Contaminated with Zearalenone and Sterigmatocystin

by Katsuki Toda, Emiko Kokushi, Seiichi Uno, Ayaka Shiiba, Hiroshi Hasunuma, Yasuo Fushimi, Missaka P. B. Wijayagunawardane, Chunhua Zhang, Osamu Yamato, Masayasu Taniguchi, Johanna Fink-Gremmels and Mitsuhiro Takagi

Toxins 2017, 9(10), 294; https://doi.org/10.3390/toxins9100294

Received: 1 May 2017 / Revised: 14 September 2017 / Accepted: 18 September 2017 / Published: 21 September 2017

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Abstract

The objective of this study was to evaluate the metabolic profile of cattle fed with or without zearalenone (ZEN) and sterigmatocystin (STC)-contaminated diets using a gas chromatography-mass spectrometry metabolomics approach. Urinary samples were collected from individual animals (n = 6 per herd)

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The objective of this study was to evaluate the metabolic profile of cattle fed with or without zearalenone (ZEN) and sterigmatocystin (STC)-contaminated diets using a gas chromatography-mass spectrometry metabolomics approach. Urinary samples were collected from individual animals (n = 6 per herd) from fattening female Japanese Black (JB) cattle herds (23 months old, 550–600 kg). Herd 1 had persistently high urinary ZEN and STC concentrations due to the presence of contaminated rice straw. Herd 2, the second female JB fattening herd (23 months old, 550–600 kg), received the same dietary feed as Herd 1, with non-contaminated rice straw. Urine samples were collected from Herd 1, two weeks after the contaminated rice straw was replaced with uncontaminated rice straw (Herd 1N). Identified metabolites were subjected to principal component analysis (PCA) and ANOVA. The PCA revealed that the effects on cattle metabolites depended on ZEN and STC concentrations. The contamination of cattle feed with multiple mycotoxins may alter systemic metabolic processes, including metabolites associated with ATP generation, amino acids, glycine-conjugates, organic acids, and purine bases. The results obtained from Herd 1N indicate that a two-week remedy period was not sufficient to improve the levels of urinary metabolites, suggesting that chronic contamination with mycotoxins may have long-term harmful effects on the systemic metabolism of cattle. Full article

(This article belongs to the collection Fusarium Toxins – Relevance for Human and Animal Health)

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Open AccessEditor’s ChoiceArticle Asymmetric Cryo-EM Structure of Anthrax Toxin Protective Antigen Pore with Lethal Factor N-Terminal Domain

by Alexandra J. Machen, Narahari Akkaladevi, Caleb Trecazzi, Pierce T. O’Neil, Srayanta Mukherjee, Yifei Qi, Rebecca Dillard, Wonpil Im, Edward P. Gogol, Tommi A. White and Mark T. Fisher

Toxins 2017, 9(10), 298; https://doi.org/10.3390/toxins9100298

Received: 17 August 2017 / Revised: 18 September 2017 / Accepted: 19 September 2017 / Published: 22 September 2017

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Abstract

The anthrax lethal toxin consists of protective antigen (PA) and lethal factor (LF). Understanding both the PA pore formation and LF translocation through the PA pore is crucial to mitigating and perhaps preventing anthrax disease. To better understand the interactions of the LF-PA

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The anthrax lethal toxin consists of protective antigen (PA) and lethal factor (LF). Understanding both the PA pore formation and LF translocation through the PA pore is crucial to mitigating and perhaps preventing anthrax disease. To better understand the interactions of the LF-PA engagement complex, the structure of the LF_N-bound PA pore solubilized by a lipid nanodisc was examined using cryo-EM. CryoSPARC was used to rapidly sort particle populations of a heterogeneous sample preparation without imposing symmetry, resulting in a refined 17 Å PA pore structure with 3 LF_N bound. At pH 7.5, the contributions from the three unstructured LF_N lysine-rich tail regions do not occlude the Phe clamp opening. The open Phe clamp suggests that, in this translocation-compromised pH environment, the lysine-rich tails remain flexible and do not interact with the pore lumen region. Full article

(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins) Printed Edition available

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Open AccessFeature PaperEditor’s ChoiceArticle Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain

by Alexey S. Ladokhin, Mauricio Vargas-Uribe, Mykola V. Rodnin, Chiranjib Ghatak and Onkar Sharma

Toxins 2017, 9(10), 299; https://doi.org/10.3390/toxins9100299

Received: 31 August 2017 / Revised: 20 September 2017 / Accepted: 20 September 2017 / Published: 22 September 2017

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Abstract

Cellular entry of diphtheria toxin is a multistage process involving receptor targeting, endocytosis, and translocation of the catalytic domain across the endosomal membrane into the cytosol. The latter is ensured by the translocation (T) domain of the toxin, capable of undergoing conformational refolding

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Cellular entry of diphtheria toxin is a multistage process involving receptor targeting, endocytosis, and translocation of the catalytic domain across the endosomal membrane into the cytosol. The latter is ensured by the translocation (T) domain of the toxin, capable of undergoing conformational refolding and membrane insertion in response to the acidification of the endosomal environment. While numerous now classical studies have demonstrated the formation of an ion-conducting conformation—the Open-Channel State (OCS)—as the final step of the refolding pathway, it remains unclear whether this channel constitutes an in vivo translocation pathway or is a byproduct of the translocation. To address this question, we measure functional activity of known OCS-blocking mutants with H-to-Q replacements of C-terminal histidines of the T-domain. We also test the ability of these mutants to translocate their own N-terminus across lipid bilayers of model vesicles. The results of both experiments indicate that translocation activity does not correlate with previously published OCS activity. Finally, we determined the topology of TH5 helix in membrane-inserted T-domain using W281 fluorescence and its depth-dependent quenching by brominated lipids. Our results indicate that while TH5 becomes a transbilayer helix in a wild-type protein, it fails to insert in the case of the OCS-blocking mutant H322Q. We conclude that the formation of the OCS is not necessary for the functional translocation by the T-domain, at least in the histidine-replacement mutants, suggesting that the OCS is unlikely to constitute a translocation pathway for the cellular entry of diphtheria toxin in vivo. Full article

(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins) Printed Edition available

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Open AccessEditor’s ChoiceArticle Characterization of Post-Translational Modifications and Cytotoxic Properties of the Adenylate-Cyclase Hemolysin Produced by Various Bordetella pertussis and Bordetella parapertussis Isolates

by Valérie Bouchez, Thibaut Douché, Mélody Dazas, Sophie Delaplane, Mariette Matondo, Julia Chamot-Rooke and Nicole Guiso

Toxins 2017, 9(10), 304; https://doi.org/10.3390/toxins9100304

Received: 29 August 2017 / Revised: 19 September 2017 / Accepted: 20 September 2017 / Published: 26 September 2017

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Abstract

Bordetella pertussis and Bordetella parapertussis are the causal agents of whooping cough in humans. They produce diverse virulence factors, including adenylate cyclase-hemolysin (AC-Hly), a secreted toxin of the repeat in toxins (RTX) family with cyclase, pore-forming, and hemolytic activities. Post-translational modifications (PTMs) are

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Bordetella pertussis and Bordetella parapertussis are the causal agents of whooping cough in humans. They produce diverse virulence factors, including adenylate cyclase-hemolysin (AC-Hly), a secreted toxin of the repeat in toxins (RTX) family with cyclase, pore-forming, and hemolytic activities. Post-translational modifications (PTMs) are essential for the biological activities of the toxin produced by B. pertussis. In this study, we compared AC-Hly toxins from various clinical isolates of B. pertussis and B. parapertussis, focusing on (i) the genomic sequences of cyaA genes, (ii) the PTMs of partially purified AC-Hly, and (iii) the cytotoxic activity of the various AC-Hly toxins. The genes encoding the AC-Hly toxins of B. pertussis and B. parapertussis displayed very limited polymorphism in each species. Most of the sequence differences between the two species were found in the C-terminal part of the protein. Both toxins harbored PTMs, mostly corresponding to palmitoylations of the lysine 860 residue and palmoylations and myristoylations of lysine 983 for B. pertussis and AC-Hly and palmitoylations of lysine 894 and myristoylations of lysine 1017 for B. parapertussis AC-Hly. Purified AC-Hly from B. pertussis was cytotoxic to macrophages, whereas that from B. parapertussis was not. Full article

(This article belongs to the Special Issue Adenylate Cyclase (CyaA) Toxin)

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Open AccessFeature PaperEditor’s ChoiceArticle Determination of Ochratoxin A in Rye and Rye-Based Products by Fluorescence Polarization Immunoassay

by Vincenzo Lippolis, Anna C. R. Porricelli, Marina Cortese, Michele Suman, Sandro Zanardi and Michelangelo Pascale

Toxins 2017, 9(10), 305; https://doi.org/10.3390/toxins9100305

Received: 14 September 2017 / Revised: 21 September 2017 / Accepted: 22 September 2017 / Published: 26 September 2017

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Abstract

A rapid fluorescence polarization immunoassay (FPIA) was optimized and validated for the determination of ochratoxin A (OTA) in rye and rye crispbread. Samples were extracted with a mixture of acetonitrile/water (60:40, v/v) and purified by SPE-aminopropyl column clean-up before performing

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A rapid fluorescence polarization immunoassay (FPIA) was optimized and validated for the determination of ochratoxin A (OTA) in rye and rye crispbread. Samples were extracted with a mixture of acetonitrile/water (60:40, v/v) and purified by SPE-aminopropyl column clean-up before performing the FPIA. Overall mean recoveries were 86 and 95% for spiked rye and rye crispbread with relative standard deviations lower than 6%. Limits of detection (LOD) of the optimized FPIA was 0.6 μg/kg for rye and rye crispbread, respectively. Good correlations (r > 0.977) were observed between OTA contents in contaminated samples obtained by FPIA and high-performance liquid chromatography (HPLC) with immunoaffinity cleanup used as reference method. Furthermore, single laboratory validation and small-scale collaborative trials were carried out for the determination of OTA in rye according to Regulation 519/2014/EU laying down procedures for the validation of screening methods. The precision profile of the method, cut-off level and rate of false suspect results confirm the satisfactory analytical performances of assay as a screening method. These findings show that the optimized FPIA is suitable for high-throughput screening, and permits reliable quantitative determination of OTA in rye and rye crispbread at levels that fall below the EU regulatory limits. Full article

(This article belongs to the collection Biorecognition Assays for Mycotoxins)

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Open AccessEditor’s ChoiceArticle Profiling of Extracellular Toxins Associated with Diarrhetic Shellfish Poison in Prorocentrum lima Culture Medium by High-Performance Liquid Chromatography Coupled with Mass Spectrometry

by Lei Pan, Junhui Chen, Huihui Shen, Xiuping He, Guangjiu Li, Xincheng Song, Deshan Zhou and Chengjun Sun

Toxins 2017, 9(10), 308; https://doi.org/10.3390/toxins9100308

Received: 1 September 2017 / Revised: 22 September 2017 / Accepted: 26 September 2017 / Published: 30 September 2017

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Abstract

Extracellular toxins released by marine toxigenic algae into the marine environment have attracted increasing attention in recent years. In this study, profiling, characterization and quantification of extracellular toxin compounds associated with diarrhetic shellfish poison (DSP) in the culture medium of toxin-producing dinoflagellates were

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Extracellular toxins released by marine toxigenic algae into the marine environment have attracted increasing attention in recent years. In this study, profiling, characterization and quantification of extracellular toxin compounds associated with diarrhetic shellfish poison (DSP) in the culture medium of toxin-producing dinoflagellates were performed using high-performance liquid chromatography–high-resolution mass spectrometry/tandem mass spectrometry for the first time. Results showed that solid-phase extraction can effectively enrich and clean the DSP compounds in the culture medium of Prorocentrum lima (P. lima), and the proposed method achieved satisfactory recoveries (94.80%–100.58%) and repeatability (relative standard deviation ≤9.27%). Commercial software associated with the accurate mass information of known DSP toxins and their derivatives was used to screen and identify DSP compounds. Nine extracellular DSP compounds were identified, of which seven toxins (including OA-D7b, OA-D9b, OA-D10a/b, and so on) were found in the culture medium of P. lima for the first time. The results of quantitative analysis showed that the contents of extracellular DSP compounds in P. lima culture medium were relatively high, and the types and contents of intracellular and extracellular toxins apparently varied in the different growth stages of P. lima. The concentrations of extracellular okadaic acid and dinophysistoxin-1 were within 19.9–34.0 and 15.2–27.9 μg/L, respectively. The total concentration of the DSP compounds was within the range of 57.70–79.63 μg/L. The results showed that the proposed method is an effective tool for profiling the extracellular DSP compounds in the culture medium of marine toxigenic algae. Full article

(This article belongs to the collection Marine and Freshwater Toxins)

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Open AccessEditor’s ChoiceArticle Target-Specificity in Scorpions; Comparing Lethality of Scorpion Venoms across Arthropods and Vertebrates

by Arie van der Meijden, Bjørn Koch, Tom van der Valk, Leidy J. Vargas-Muñoz and Sebastian Estrada-Gómez

Toxins 2017, 9(10), 312; https://doi.org/10.3390/toxins9100312

Received: 6 September 2017 / Revised: 22 September 2017 / Accepted: 27 September 2017 / Published: 4 October 2017

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Abstract

Scorpions use their venom in defensive situations as well as for subduing prey. Since some species of scorpion use their venom more in defensive situations than others, this may have led to selection for differences in effectiveness in defensive situations. Here, we compared

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Scorpions use their venom in defensive situations as well as for subduing prey. Since some species of scorpion use their venom more in defensive situations than others, this may have led to selection for differences in effectiveness in defensive situations. Here, we compared the LD₅₀ of the venom of 10 species of scorpions on five different species of target organisms; two insects and three vertebrates. We found little correlation between the target species in the efficacy of the different scorpion venoms. Only the two insects showed a positive correlation, indicating that they responded similarly to the panel of scorpion venoms. We discuss the lack of positive correlation between the vertebrate target species in the light of their evolution and development. When comparing the responses of the target systems to individual scorpion venoms pairwise, we found that closely related scorpion species tend to elicit a similar response pattern across the target species. This was further reflected in a significant phylogenetic signal across the scorpion phylogeny for the LD₅₀ in mice and in zebrafish. We also provide the first mouse LD₅₀ value for Grosphus grandidieri. Full article

(This article belongs to the Special Issue Scorpion Toxins)

Open AccessFeature PaperEditor’s ChoiceArticle The Aspergillus flavus Homeobox Gene, hbx1, Is Required for Development and Aflatoxin Production

by Jeffrey W. Cary, Pamela Harris-Coward, Leslie Scharfenstein, Brian M. Mack, Perng-Kuang Chang, Qijian Wei, Matthew Lebar, Carol Carter-Wientjes, Rajtilak Majumdar, Chandrani Mitra, Sourav Banerjee and Anindya Chanda

Toxins 2017, 9(10), 315; https://doi.org/10.3390/toxins9100315

Received: 20 September 2017 / Revised: 6 October 2017 / Accepted: 9 October 2017 / Published: 12 October 2017

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Abstract

Homeobox proteins, a class of well conserved transcription factors, regulate the expression of targeted genes, especially those involved in development. In filamentous fungi, homeobox genes are required for normal conidiogenesis and fruiting body formation. In the present study, we identified eight homeobox (

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Homeobox proteins, a class of well conserved transcription factors, regulate the expression of targeted genes, especially those involved in development. In filamentous fungi, homeobox genes are required for normal conidiogenesis and fruiting body formation. In the present study, we identified eight homeobox (hbx) genes in the aflatoxin-producing ascomycete, Aspergillus flavus, and determined their respective role in growth, conidiation and sclerotial production. Disruption of seven of the eight genes had little to no effect on fungal growth and development. However, disruption of the homeobox gene AFLA_069100, designated as hbx1, in two morphologically different A. flavus strains, CA14 and AF70, resulted in complete loss of production of conidia and sclerotia as well as aflatoxins B₁ and B₂, cyclopiazonic acid and aflatrem. Microscopic examination showed that the Δhbx1 mutants did not produce conidiophores. The inability of Δhbx1 mutants to produce conidia was related to downregulation of brlA (bristle) and abaA (abacus), regulatory genes for conidiophore development. These mutants also had significant downregulation of the aflatoxin pathway biosynthetic genes aflC, aflD, aflM and the cluster-specific regulatory gene, aflR. Our results demonstrate that hbx1 not only plays a significant role in controlling A. flavus development but is also critical for the production of secondary metabolites, such as aflatoxins. Full article

(This article belongs to the Special Issue Functional Genomics of Toxigenic Fungi and Regulatory Mechanism in the Biosynthesis of Mycotoxins)

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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

by Hui Li, Daibin Yang, Peiwu Li, Qi Zhang, Wen Zhang, Xiaoxia Ding, Jin Mao and Jing Wu

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

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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

(This article belongs to the collection Aflatoxins)

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Open AccessEditor’s ChoiceArticle Combined Venom Gland Transcriptomic and Venom Peptidomic Analysis of the Predatory Ant Odontomachus monticola

by Kohei Kazuma, Keiichi Masuko, Katsuhiro Konno and Hidetoshi Inagaki

Toxins 2017, 9(10), 323; https://doi.org/10.3390/toxins9100323

Received: 18 September 2017 / Revised: 10 October 2017 / Accepted: 11 October 2017 / Published: 13 October 2017

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Abstract

Ants (hymenoptera: Formicidae) have adapted to many different environments and have become some of the most prolific and successful insects. To date, 13,258 ant species have been reported. They have been classified into 333 genera and 17 subfamilies. Except for a few Formicinae,

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Ants (hymenoptera: Formicidae) have adapted to many different environments and have become some of the most prolific and successful insects. To date, 13,258 ant species have been reported. They have been classified into 333 genera and 17 subfamilies. Except for a few Formicinae, Dolichoderinae, and members of other subfamilies, most ant species have a sting with venom. The venoms are composed of formic acid, alkaloids, hydrocarbons, amines, peptides, and proteins. Unlike the venoms of other animals such as snakes and spiders, ant venoms have seldom been analyzed comprehensively, and their compositions are not yet completely known. In this study, we used both transcriptomic and peptidomic analyses to study the composition of the venom produced by the predatory ant species Odontomachus monticola. The transcriptome analysis yielded 49,639 contigs, of which 92 encoded toxin-like peptides and proteins with 18,106,338 mapped reads. We identified six pilosulin-like peptides by transcriptomic analysis in the venom gland. Further, we found intact pilosulin-like peptide 1 and truncated pilosulin-like peptides 2 and 3 by peptidomic analysis in the venom. Our findings related to ant venom peptides and proteins may lead the way towards development and application of novel pharmaceutical and biopesticidal resources. Full article

(This article belongs to the Section Animal Venoms)

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Open AccessEditor’s ChoiceArticle Mycotoxin Analysis of Human Urine by LC-MS/MS: A Comparative Extraction Study

by Laura Escrivá, Lara Manyes, Guillermina Font and Houda Berrada

Toxins 2017, 9(10), 330; https://doi.org/10.3390/toxins9100330

Received: 29 September 2017 / Revised: 13 October 2017 / Accepted: 15 October 2017 / Published: 19 October 2017

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Abstract

The lower mycotoxin levels detected in urine make the development of sensitive and accurate analytical methods essential. Three extraction methods, namely salting-out liquid–liquid extraction (SALLE), miniQuEChERS (quick, easy, cheap, effective, rugged, and safe), and dispersive liquid–liquid microextraction (DLLME), were evaluated and compared based

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The lower mycotoxin levels detected in urine make the development of sensitive and accurate analytical methods essential. Three extraction methods, namely salting-out liquid–liquid extraction (SALLE), miniQuEChERS (quick, easy, cheap, effective, rugged, and safe), and dispersive liquid–liquid microextraction (DLLME), were evaluated and compared based on analytical parameters for the quantitative LC-MS/MS measurement of 11 mycotoxins (AFB1, AFB2, AFG1, AFG2, OTA, ZEA, BEA, EN A, EN B, EN A1 and EN B1) in human urine. DLLME was selected as the most appropriate methodology, as it produced better validation results for recovery (79–113%), reproducibility (RSDs < 12%), and repeatability (RSDs < 15%) than miniQuEChERS (71–109%, RSDs <14% and <24%, respectively) and SALLE (70–108%, RSDs < 14% and < 24%, respectively). Moreover, the lowest detection (LODS) and quantitation limits (LOQS) were achieved with DLLME (LODs: 0.005–2 μg L⁻¹, LOQs: 0.1–4 μg L⁻¹). DLLME methodology was used for the analysis of 10 real urine samples from healthy volunteers showing the presence of ENs B, B1 and A1 at low concentrations. Full article

(This article belongs to the Section Mycotoxins)

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Open AccessEditor’s ChoiceArticle Effect of Clostridium perfringens β-Toxin on Platelets

by Anne Thiel, Helga Mogel, Julia Bruggisser, Arnaud Baumann, Marianne Wyder, Michael H. Stoffel, Artur Summerfield and Horst Posthaus

Toxins 2017, 9(10), 336; https://doi.org/10.3390/toxins9100336

Received: 2 October 2017 / Revised: 19 October 2017 / Accepted: 20 October 2017 / Published: 24 October 2017

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Abstract

Clostridium perfringens β-toxin (CPB) is the major virulence factor of C. perfringens type C causing a hemorrhagic enteritis in animals and humans. In experimentally infected pigs, endothelial binding of CPB was shown to be associated with early vascular lesions and hemorrhage but

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Clostridium perfringens β-toxin (CPB) is the major virulence factor of C. perfringens type C causing a hemorrhagic enteritis in animals and humans. In experimentally infected pigs, endothelial binding of CPB was shown to be associated with early vascular lesions and hemorrhage but without obvious thrombosis of affected vessels, suggesting altered hemostasis in the early phase of the disease. The objective of the present study was to investigate the effect of CPB on platelets, with respect to primary hemostasis. Our results demonstrate that CPB binds to porcine and human platelets and forms oligomers resulting in a time- and dose-dependent cell death. Platelets showed rapid ultrastructural changes, significantly decreased aggregation and could no longer be activated by thrombin. This indicates that CPB affects the physiological function of platelets and counteracts primary hemostasis. Our results add platelets to the list of target cells of CPB and extend the current hypothesis of its role in the pathogenesis of C. perfringens type C enteritis. Full article

(This article belongs to the collection Clostridium difficile/Clostridium sordellii and Clostridium perfringens Toxins)

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Open AccessEditor’s ChoiceArticle Shiga Toxin Glycosphingolipid Receptors in Human Caco-2 and HCT-8 Colon Epithelial Cell Lines

by Ivan U. Kouzel, Gottfried Pohlentz, Julia S. Schmitz, Daniel Steil, Hans-Ulrich Humpf, Helge Karch and Johannes Müthing

Toxins 2017, 9(11), 338; https://doi.org/10.3390/toxins9110338

Received: 26 September 2017 / Revised: 11 October 2017 / Accepted: 19 October 2017 / Published: 25 October 2017

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Abstract

Shiga toxins (Stxs) released by enterohemorrhagic Escherichia coli (EHEC) into the human colon are the causative agents for fatal outcome of EHEC infections. Colon epithelial Caco-2 and HCT-8 cells are widely used for investigating Stx-mediated intestinal cytotoxicity. Only limited data are available regarding

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Shiga toxins (Stxs) released by enterohemorrhagic Escherichia coli (EHEC) into the human colon are the causative agents for fatal outcome of EHEC infections. Colon epithelial Caco-2 and HCT-8 cells are widely used for investigating Stx-mediated intestinal cytotoxicity. Only limited data are available regarding precise structures of their Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), and lipid raft association. In this study we identified Gb3Cer and Gb4Cer lipoforms of serum-free cultivated Caco-2 and HCT-8 cells, chiefly harboring ceramide moieties composed of sphingosine (d18:1) and C16:0, C22:0 or C24:0/C24:1 fatty acid. The most significant difference between the two cell lines was the prevalence of Gb3Cer with C16 fatty acid in HCT-8 and Gb4Cer with C22–C24 fatty acids in Caco-2 cells. Lipid compositional analysis of detergent-resistant membranes (DRMs), which were used as lipid raft-equivalents, indicated slightly higher relative content of Stx receptor Gb3Cer in DRMs of HCT-8 cells when compared to Caco-2 cells. Cytotoxicity assays revealed substantial sensitivity towards Stx2a for both cell lines, evidencing little higher susceptibility of Caco-2 cells versus HCT-8 cells. Collectively, Caco-2 and HCT-8 cells express a plethora of different receptor lipoforms and are susceptible towards Stx2a exhibiting somewhat lower sensitivity when compared to Vero cells. Full article

(This article belongs to the collection Shiga Toxins)

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Open AccessEditor’s ChoiceArticle Investigation of Non-Covalent Interactions of Aflatoxins (B1, B2, G1, G2, and M1) with Serum Albumin

by Miklós Poór, Mónika Bálint, Csaba Hetényi, Beatrix Gődér, Sándor Kunsági-Máté, Tamás Kőszegi and Beáta Lemli

Toxins 2017, 9(11), 339; https://doi.org/10.3390/toxins9110339

Received: 27 September 2017 / Revised: 15 October 2017 / Accepted: 20 October 2017 / Published: 25 October 2017

Cited by 7 | PDF Full-text (2897 KB) | HTML Full-text | XML Full-text | Supplementary Files

Abstract

Aflatoxins are widely spread mycotoxins produced mainly by Aspergillus species. Consumption of aflatoxin-contaminated foods and drinks causes serious health risks for people worldwide. It is well-known that the reactive epoxide metabolite of aflatoxin B1 (AFB1) forms covalent adducts with serum albumin. However, non-covalent

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Aflatoxins are widely spread mycotoxins produced mainly by Aspergillus species. Consumption of aflatoxin-contaminated foods and drinks causes serious health risks for people worldwide. It is well-known that the reactive epoxide metabolite of aflatoxin B1 (AFB1) forms covalent adducts with serum albumin. However, non-covalent interactions of aflatoxins with human serum albumin (HSA) are poorly characterized. Thus, in this study the complex formation of aflatoxins was examined with HSA applying spectroscopic and molecular modelling studies. Our results demonstrate that aflatoxins form stable complexes with HSA as reflected by binding constants between 2.1 × 10⁴ and 4.5 × 10⁴ dm³/mol. A binding free energy value of −26.90 kJ mol⁻¹ suggests a spontaneous binding process between AFB1 and HSA at room-temperature, while the positive entropy change of 55.1 JK⁻¹ mol⁻¹ indicates a partial decomposition of the solvation shells of the interacting molecules. Modeling studies and investigations with site markers suggest that Sudlow’s Site I of subdomain IIA is the high affinity binding site of aflatoxins on HSA. Interaction of AFB1 with bovine, porcine, and rat serum albumins was also investigated. Similar stabilities of the examined AFB1-albumin complexes were observed suggesting the low species differences of the albumin-binding of aflatoxins. Full article

(This article belongs to the Section Mycotoxins)

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Open AccessEditor’s ChoiceArticle The Aromatic Head Group of Spider Toxin Polyamines Influences Toxicity to Cancer Cells

by David Wilson, Glen M. Boyle, Lachlan McIntyre, Matthew J. Nolan, Peter G. Parsons, Jennifer J. Smith, Leon Tribolet, Alex Loukas, Michael J. Liddell, Lachlan D. Rash and Norelle L. Daly

Toxins 2017, 9(11), 346; https://doi.org/10.3390/toxins9110346

Received: 5 September 2017 / Revised: 22 October 2017 / Accepted: 23 October 2017 / Published: 27 October 2017

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Abstract

Spider venoms constitute incredibly diverse libraries of compounds, many of which are involved in prey capture and defence. Polyamines are often prevalent in the venom and target ionotropic glutamate receptors. Here we show that a novel spider polyamine, PA₃₆₆, containing a

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Spider venoms constitute incredibly diverse libraries of compounds, many of which are involved in prey capture and defence. Polyamines are often prevalent in the venom and target ionotropic glutamate receptors. Here we show that a novel spider polyamine, PA₃₆₆, containing a hydroxyphenyl-based structure is present in the venom of several species of tarantula, and has selective toxicity against MCF-7 breast cancer cells. By contrast, a polyamine from an Australian funnel-web spider venom, which contains an identical polyamine tail to PA₃₆₆ but an indole-based head-group, is only cytotoxic at high concentrations. Our results suggest that the ring structure plays a role in the cytotoxicity and that modification to the polyamine head group might lead to more potent and selective compounds with potential as novel cancer treatments. Full article

(This article belongs to the Section Animal Venoms)

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Open AccessEditor’s ChoiceArticle Aspergillus korhogoensis, a Novel Aflatoxin Producing Species from the Côte d’Ivoire

by Amaranta Carvajal-Campos, Ama Lethicia Manizan, Souria Tadrist, David Koffi Akaki, Rose Koffi-Nevry, Geromy G. Moore, Stephen O. Fapohunda, Sylviane Bailly, Didier Montet, Isabelle P. Oswald, Sophie Lorber, Catherine Brabet and Olivier Puel

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

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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

(This article belongs to the collection Aflatoxins)

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Open AccessEditor’s ChoiceArticle Efficacy of Bee Venom Acupuncture for Chronic Low Back Pain: A Randomized, Double-Blinded, Sham-Controlled Trial

by Byung-Kwan Seo, Kyungsun Han, Ojin Kwon, Dae-Jean Jo and Jun-Hwan Lee

Toxins 2017, 9(11), 361; https://doi.org/10.3390/toxins9110361

Received: 14 October 2017 / Revised: 27 October 2017 / Accepted: 3 November 2017 / Published: 7 November 2017

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Abstract

Bee venom acupuncture (BVA) is an effective treatment for chronic low back pain (CLBP) through the pharmacological effects of bee venom and the simultaneous stimulation of acupoints. However, evidence of its efficacy and safety in humans remains unclear. Using a double-blind, randomized study,

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Bee venom acupuncture (BVA) is an effective treatment for chronic low back pain (CLBP) through the pharmacological effects of bee venom and the simultaneous stimulation of acupoints. However, evidence of its efficacy and safety in humans remains unclear. Using a double-blind, randomized study, 54 patients with non-specific CLBP were assigned to the BVA and sham groups. All participants underwent six sessions of real or sham BVA for 3 weeks, in addition to administration of 180 mg of loxonin per day. The primary outcome, that is, “bothersomeness” derived from back pain, was assessed using the visual analog scale. Secondary outcomes included pain intensity, dysfunction related to back pain (Oswestry Disability Index), quality of life (EuroQol 5-Dimension), and depressive mood (Beck’s depression inventory). Outcomes were evaluated every week during the treatment period and followed up at weeks 4, 8, and 12. After 3 weeks of the treatment, significant improvements were observed in the bothersomeness, pain intensity, and functional status in the BVA group compared with the sham group. Although minimal adverse events were observed in both groups, subsequent recovery was achieved without treatment. Consequently, our results suggest that it can be used along with conventional pharmacological therapies for the treatment of CLBP. Full article

(This article belongs to the Special Issue Animal Venoms and Pain)

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Open AccessEditor’s ChoiceArticle Awareness and Prevalence of Mycotoxin Contamination in Selected Nigerian Fermented Foods

by Ifeoluwa Adekoya, Patrick Njobeh, Adewale Obadina, Cynthia Chilaka, Sheila Okoth, Marthe De Boevre and Sarah De Saeger

Toxins 2017, 9(11), 363; https://doi.org/10.3390/toxins9110363

Received: 20 October 2017 / Revised: 3 November 2017 / Accepted: 4 November 2017 / Published: 8 November 2017

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Abstract

Fermented food samples (n = 191) including maize gruel (ogi), sorghum gruel (ogi-baba), melon seed (ogiri), locust bean (iru) and African oil bean seed (ugba) from Southwest Nigeria were quantified for 23

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Fermented food samples (n = 191) including maize gruel (ogi), sorghum gruel (ogi-baba), melon seed (ogiri), locust bean (iru) and African oil bean seed (ugba) from Southwest Nigeria were quantified for 23 mycotoxins, including aflatoxin B₁ (AFB₁), fumonisin B₁ (FB₁), and sterigmatocystin (STE) using liquid chromatography-tandem mass spectrometry. The practices, perceived understanding and health risks related to fungal and mycotoxin contamination amongst fermented food sellers was also established. Data obtained revealed that 82% of the samples had mycotoxins occurring singly or in combination. FB₁ was present in 83% of ogi-baba samples, whereas 20% of ugba samples contained AFB₁ (range: 3 to 36 µg/kg) and STE was present in 29% of the ogi samples. In terms of multi-mycotoxin contamination, FB₁ + FB₂ + FB₃ + STE + AFB₁ + alternariol + HT-2 co-occurred within one sample. The awareness study revealed that 98% of respondents were unaware of mycotoxin contamination, and their education level slightly correlated with their level of awareness (p < 0.01, r = 0.308). The extent to which the analyzed mycotoxins contaminated these food commodities, coupled with the poor perception of the population under study on fungi and mycotoxins, justifies the need to enact fungal and mycotoxin mitigation strategies along the food chain. Full article

(This article belongs to the Special Issue 1^st International MYCOKEY Conference: Advances on Mycotoxin Reduction in the Food and Feed Chain)

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Open AccessEditor’s ChoiceArticle Differential Gene Expression Analysis of Bovine Macrophages after Exposure to the Penicillium Mycotoxins Citrinin and/or Ochratoxin A

by Kristen M. Brennan, Se-Young Oh, Alexandros Yiannikouris, Daniel E. Graugnard and Niel A. Karrow

Toxins 2017, 9(11), 366; https://doi.org/10.3390/toxins9110366

Received: 2 October 2017 / Revised: 8 November 2017 / Accepted: 9 November 2017 / Published: 13 November 2017

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Abstract

Mycotoxins produced by fungal species commonly contaminate livestock feedstuffs, jeopardizing their health and diminishing production. Citrinin (CIT) and ochratoxin A (OTA) are mycotoxins produced by Penicillium spp. and commonly co-occur. Both CIT and OTA can modulate immune response by inhibiting cell proliferation and

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Mycotoxins produced by fungal species commonly contaminate livestock feedstuffs, jeopardizing their health and diminishing production. Citrinin (CIT) and ochratoxin A (OTA) are mycotoxins produced by Penicillium spp. and commonly co-occur. Both CIT and OTA can modulate immune response by inhibiting cell proliferation and differentiation, altering cell metabolism, and triggering programmed cell death. The objective of this study was to determine the effects of sublethal exposure (i.e., the concentration that inhibited cell proliferation by 25% (IC₂₅)) to CIT, OTA or CIT + OTA on the bovine macrophage transcriptome. Gene expression was determined using the Affymetrix Bovine Genome Array. After 6 h of exposure to CIT, OTA or CIT + OTA, the number of differentially expressed genes (DEG), respectively, was as follows: 1471 genes (822 up-regulated, 649 down-regulated), 5094 genes (2611 up-regulated, 2483 down-regulated) and 7624 genes (3984 up-regulated, 3640 down-regulated). Of these, 179 genes (88 up-regulated, 91 down-regulated) were commonly expressed between treatments. After 24 h of exposure to CIT, OTA or CIT + OTA the number of DEG, respectively, was as follows: 3230 genes (1631 up-regulated, 1599 down-regulated), 8558 genes (4167 up-regulated, 4391 down-regulated), and 10,927 genes (6284 up-regulated, 4643 down-regulated). Of these, 770 genes (247 up-regulated, 523 down-regulated) were commonly expressed between treatments. The categorization of common biological functions and pathway analysis suggests that the IC₂₅ of both CIT and OTA, or their combination, induces cellular oxidative stress, a slowing of cell cycle progression, and apoptosis. Collectively, these effects contribute to inhibiting bovine macrophage proliferation. Full article

(This article belongs to the collection Ochratoxins-Collection)

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Open AccessFeature PaperEditor’s ChoiceArticle Membrane-Active Properties of an Amphitropic Peptide from the CyaA Toxin Translocation Region

by Alexis Voegele, Orso Subrini, Nicolas Sapay, Daniel Ladant and Alexandre Chenal

Toxins 2017, 9(11), 369; https://doi.org/10.3390/toxins9110369

Received: 18 October 2017 / Revised: 9 November 2017 / Accepted: 10 November 2017 / Published: 14 November 2017

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Abstract

The adenylate cyclase toxin CyaA is involved in the early stages of infection by Bordetella pertussis, the causative agent of whooping cough. CyaA intoxicates target cells by a direct translocation of its catalytic domain (AC) across the plasma membrane and produces supraphysiological

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The adenylate cyclase toxin CyaA is involved in the early stages of infection by Bordetella pertussis, the causative agent of whooping cough. CyaA intoxicates target cells by a direct translocation of its catalytic domain (AC) across the plasma membrane and produces supraphysiological levels of cAMP, leading to cell death. The molecular process of AC translocation remains largely unknown, however. We have previously shown that deletion of residues 375–485 of CyaA selectively abrogates AC translocation into eukaryotic cells. We further identified within this “translocation region” (TR), P454 (residues 454–484), a peptide that exhibits membrane-active properties, i.e., is able to bind and permeabilize lipid vesicles. Here, we analyze various sequences from CyaA predicted to be amphipatic and show that although several of these peptides can bind membranes and adopt a helical conformation, only the P454 peptide is able to permeabilize membranes. We further characterize the contributions of the two arginine residues of P454 to membrane partitioning and permeabilization by analyzing the peptide variants in which these residues are substituted by different amino acids (e.g., A, K, Q, and E). Our data shows that both arginine residues significantly contribute, although diversely, to the membrane-active properties of P454, i.e., interactions with both neutral and anionic lipids, helix formation in membranes, and disruption of lipid bilayer integrity. These results are discussed in the context of the translocation process of the full-length CyaA toxin. Full article

(This article belongs to the Special Issue Adenylate Cyclase (CyaA) Toxin)

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Open AccessEditor’s ChoiceArticle Ameliorative Effects of Grape Seed Proanthocyanidin Extract on Growth Performance, Immune Function, Antioxidant Capacity, Biochemical Constituents, Liver Histopathology and Aflatoxin Residues in Broilers Exposed to Aflatoxin B₁

by Shahid Ali Rajput, Lvhui Sun, Niya Zhang, Mahmoud Mohamed Khalil, Xin Gao, Zhao Ling, Luoyi Zhu, Farhan Anwar Khan, Jiacai Zhang and Desheng Qi

Toxins 2017, 9(11), 371; https://doi.org/10.3390/toxins9110371

Received: 26 September 2017

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