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Toxins, Volume 9, Issue 10 (October 2017)

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Editorial

Jump to: Research, Review

Open AccessEditorial Introduction to the Toxins Special Issue on LC-MS/MS Methods for Mycotoxin Analysis
Toxins 2017, 9(10), 325; doi:10.3390/toxins9100325
Received: 30 August 2017 / Revised: 27 September 2017 / Accepted: 10 October 2017 / Published: 16 October 2017
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Abstract
Various filamentous fungi can produce secondary metabolites, whose biochemical significance in fungal growth and development has not always been fully clarified; however, some of these metabolites can cause deleterious effects on other organisms and are classified as mycotoxins [...]
Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)

Research

Jump to: Editorial, Review

Open AccessArticle Gas Chromatography-Mass Spectrometry for Metabolite Profiling of Japanese Black Cattle Naturally Contaminated with Zearalenone and Sterigmatocystin
Toxins 2017, 9(10), 294; doi: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 AccessArticle Monitoring the Temporal Expression of Genes Involved in Ochratoxin A Production of Aspergillus carbonarius under the Influence of Temperature and Water Activity
Toxins 2017, 9(10), 296; doi:10.3390/toxins9100296
Received: 25 July 2017 / Revised: 11 September 2017 / Accepted: 19 September 2017 / Published: 22 September 2017
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Abstract
The objective of this study was to investigate the effect of environmental factors, namely temperature and water activity, on genes involved in the regulation of ochratoxin A (OTA) production over time. For this purpose, the previously characterized toxigenic Aspergillus carbonarius Ac29 isolate from
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The objective of this study was to investigate the effect of environmental factors, namely temperature and water activity, on genes involved in the regulation of ochratoxin A (OTA) production over time. For this purpose, the previously characterized toxigenic Aspergillus carbonarius Ac29 isolate from Greek vineyards and the A. carbonarius ITEM 5010 reference strain were subjected to combined temperature and water activity (aw) treatments to study OTA production and relative gene expression. The fungal isolates were grown on a synthetic grape juice liquid medium (SGM) under different temperature (20 °C, 25 °C and 30 °C) and aw (0.94 and 0.98) regimes. The expression of the AcOTApks, AcOTAnrps, and laeA OTA related genes was investigated using real time PCR. Gene expression was monitored at the same time points, along with fungal biomass and OTA accumulation at three, six and nine days of incubation. In gene expression analysis, stimulation of the biosynthetic genes was observed a few days before any toxin could be detected. This fact may underline a possible early indicator of potential toxin contamination of grapes. However, the transcript levels varied with respect to the different combinations of ecophysiological conditions and time, highlighting a complex regulation of OTA related gene expression of A. carbonarius in the specific medium. Full article
(This article belongs to the collection Understanding Mycotoxin Occurrence in Food and Feed Chains)
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Open AccessArticle Humoral Response of Buffaloes to a Recombinant Vaccine against Botulism Serotypes C and D
Toxins 2017, 9(10), 297; doi:10.3390/toxins9100297
Received: 31 August 2017 / Revised: 15 September 2017 / Accepted: 16 September 2017 / Published: 22 September 2017
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Abstract
Botulism is a fatal intoxication caused by botulinum neurotoxins (BoNTs), which are mainly produced by Clostridium botulinum and characterized by flaccid paralysis. The BoNTs C and D are the main serotypes responsible for botulism in animals, including buffaloes. Botulism is one of the
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Botulism is a fatal intoxication caused by botulinum neurotoxins (BoNTs), which are mainly produced by Clostridium botulinum and characterized by flaccid paralysis. The BoNTs C and D are the main serotypes responsible for botulism in animals, including buffaloes. Botulism is one of the leading causes of death in adult ruminants in Brazil due to the high mortality rates, even though botulism in buffaloes is poorly reported and does not reflect the real economic impact of this disease in Brazilian herds. Vaccination is reported as the most important prophylactic measure for botulism control, although there are no specific vaccines commercially available for buffaloes in Brazil. This study aimed to evaluate the humoral immune response of buffalo groups vaccinated with three different concentrations of recombinant proteins (100, 200, and 400 µg) against BoNTs serotypes C and D as well as to compare the groups to each other and with a group vaccinated with a bivalent commercial toxoid. The recombinant vaccine with a concentration of 400 μg of proteins induced the highest titers among the tested vaccines and was proven to be the best choice among the formulations evaluated and should be considered as a potential vaccine against botulism in buffalo. Full article
(This article belongs to the Special Issue Botulinum Neurotoxins Antibody and Vaccine)
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Open AccessArticle Asymmetric Cryo-EM Structure of Anthrax Toxin Protective Antigen Pore with Lethal Factor N-Terminal Domain
Toxins 2017, 9(10), 298; doi: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 LFN-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 LFN bound. At pH 7.5, the contributions from the three unstructured LFN 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)
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Open AccessFeature PaperArticle Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain
Toxins 2017, 9(10), 299; doi: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)
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Open AccessArticle Safety Assessment of Lactobacillus helveticus KLDS1.8701 Based on Whole Genome Sequencing and Oral Toxicity Studies
Toxins 2017, 9(10), 301; doi:10.3390/toxins9100301
Received: 2 August 2017 / Revised: 4 September 2017 / Accepted: 20 September 2017 / Published: 24 September 2017
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Abstract
Lactobacillus helveticus KLDS1.8701 isolated from Chinese traditional fermented dairy product has been shown earlier to possess probiotic potentials but it is important to evaluate its safety in view of its possible use as a probiotic. The aim of the present study is to
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Lactobacillus helveticus KLDS1.8701 isolated from Chinese traditional fermented dairy product has been shown earlier to possess probiotic potentials but it is important to evaluate its safety in view of its possible use as a probiotic. The aim of the present study is to critically assess the safety of L. helveticus KLDS1.8701 through multiple perspectives. The complete genome of L. helveticus KLDS1.8701 was sequenced to mine for safety-associated genes. The minimum inhibitory concentrations of 15 antimicrobials and the adverse metabolites were determined. Standard acute oral and subacute toxicity studies were conducted in rats. The results in silico disclosed that the genome of L. helveticus KLDS1.8701 carries no transferable antibiotic resistance genes, no virulence factors and only 3 genes related to adverse metabolites. In vitro results showed that L. helveticus KLDS1.8701 was resistant against 6 antimicrobials and did not raise safety concerns about biogenic amine, D-lactic acid and nitroreductase. The results in vivo revealed that no adverse effects on experimental rats were observed in the oral toxicity tests. Overall, findings from this study suggest that L. helveticus KLDS1.8701 is safe and can be used as a potential probiotic for human consumption. Full article
(This article belongs to the Section Bacterial Toxins)
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Open AccessArticle Rapid Detection and Identification of Mycotoxigenic Fungi and Mycotoxins in Stored Wheat Grain
Toxins 2017, 9(10), 302; doi:10.3390/toxins9100302
Received: 24 August 2017 / Revised: 19 September 2017 / Accepted: 20 September 2017 / Published: 25 September 2017
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Abstract
This study aimed to assess the occurrence of toxigenic fungi and mycotoxin contamination in stored wheat grains by using advanced molecular and analytical techniques. A multiplex polymerase chain reaction (PCR) strategy was established for rapid identification of mycotoxigenic fungi, and an improved analytical
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This study aimed to assess the occurrence of toxigenic fungi and mycotoxin contamination in stored wheat grains by using advanced molecular and analytical techniques. A multiplex polymerase chain reaction (PCR) strategy was established for rapid identification of mycotoxigenic fungi, and an improved analytical method was developed for simultaneous multi-mycotoxin determination in wheat grains by liquid chromatography-tandem mass spectrometry (LC/MS/MS) without the need for any clean-up. The optimized multiplex PCR method was highly specific in detecting fungal species containing species-specific and mycotoxin metabolic pathway genes. The method was applied for evaluation of 34 wheat grain samples collected from storage warehouses for the presence of mycotoxin-producing fungi, and a few samples were found positive for Fusarium and Aspergillus species. Further chemical analysis revealed that 17 samples contained mycotoxins above the level of detection, but only six samples were found to be contaminated over the EU regulatory limits with at least one mycotoxin. Aflatoxin B1, fumonisins, and deoxynivalenol were the most common toxins found in these samples. The results showed a strong correlation between the presence of mycotoxin biosynthesis genes as analyzed by multiplex PCR and mycotoxin detection by LC/MS/MS. The present findings indicate that a combined approach might provide rapid, accurate, and sensitive detection of mycotoxigenic species and mycotoxins in wheat grains. Full article
(This article belongs to the Section Mycotoxins)
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Open AccessArticle Emergence of Nasal Carriage of ST80 and ST152 PVL+ Staphylococcus aureus Isolates from Livestock in Algeria
Toxins 2017, 9(10), 303; doi:10.3390/toxins9100303
Received: 12 August 2017 / Revised: 9 September 2017 / Accepted: 20 September 2017 / Published: 25 September 2017
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Abstract
The spread of toxinogenic Staphylococcus aureus is a public health problem in Africa. The objectives of the study were to investigate the rate of S. aureus nasal carriage and molecular characteristics of these strains in livestock and humans in three Algerian provinces. Nasal
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The spread of toxinogenic Staphylococcus aureus is a public health problem in Africa. The objectives of the study were to investigate the rate of S. aureus nasal carriage and molecular characteristics of these strains in livestock and humans in three Algerian provinces. Nasal samples were collected from camels, horses, cattle, sheep and monkeys, as well as humans in contact with them. S. aureus isolates were genotyped using DNA microarray. The rate of S. aureus nasal carriage varied between species: camels (53%), humans and monkeys (50%), sheep (44.2%), horses (15.2%) and cattle (15%). Nine methicillin-resistant S. aureus (MRSA) isolates (7.6%) were identified, isolated from camels and sheep. The S. aureus isolates belonged to 15 different clonal complexes. Among them, PVL+ (Panton–Valentine Leukocidin) isolates belonging to ST80-MRSA-IV and ST152-MSSA were identified in camels (n = 3, 13%) and sheep (n = 4, 21.1%). A high prevalence of toxinogenic animal strains was noted containing TSST-1- (22.2%), EDINB- (29.6%) and EtD- (11.1%) encoding genes. This study showed the dispersal of the highly human pathogenic clones ST152-MSSA and ST-80-MRSA in animals. It suggests the ability of some clones to cross the species barrier and jump between humans and several animal species. Full article
(This article belongs to the collection Staphylococcus aureus Toxins)
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Open AccessArticle Characterization of Post-Translational Modifications and Cytotoxic Properties of the Adenylate-Cyclase Hemolysin Produced by Various Bordetella pertussis and Bordetella parapertussis Isolates
Toxins 2017, 9(10), 304; doi: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 PaperArticle Determination of Ochratoxin A in Rye and Rye-Based Products by Fluorescence Polarization Immunoassay
Toxins 2017, 9(10), 305; doi: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 AccessArticle Responses of Microcystis Colonies of Different Sizes to Hydrogen Peroxide Stress
Toxins 2017, 9(10), 306; doi:10.3390/toxins9100306
Received: 29 August 2017 / Revised: 18 September 2017 / Accepted: 23 September 2017 / Published: 27 September 2017
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Abstract
Microcystis blooms have become a ubiquitous phenomenon in freshwater ecosystems, and the size of Microcystis colonies varies widely throughout the year. In the present study, hydrogen peroxide (H2O2) was applied to test the effect of this algaecide on Microcystis
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Microcystis blooms have become a ubiquitous phenomenon in freshwater ecosystems, and the size of Microcystis colonies varies widely throughout the year. In the present study, hydrogen peroxide (H2O2) was applied to test the effect of this algaecide on Microcystis colonies of different sizes and to evaluate the colonies' antioxidant strategy. The results showed that Microcystis populations collapsed under treatment with 5 mg/L H2O2 at colony sizes smaller than 25 μm. A dosage of 20 mg/L H2O2 was necessary to efficiently control Microcystis colonies larger than 25 μm. The enzymatic and non-enzymatic antioxidant systems of different colonies exhibited various strategies to mitigate oxidative stress. In small colonies, superoxide dismutase (SOD) activity was readily stimulated and operated with catalase (CAT) activity to eliminate reactive oxygen species (ROS). In colonies larger than 25 μm, the antioxidant enzyme CAT and antioxidant substance glutathione (GSH) played major roles in mitigating oxidative stress at H2O2 concentrations below 20 mg/L. In addition, application of the algaecide led to the release of intracellular-microcystins (MCs), and oxidatively-driven MCs reached high concentrations when colony size was larger than 100 μm. Algaecide control measures should be implemented before the formation of large colonies to limit the algaecide dosage and MC release. Full article
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Open AccessArticle Alternagin-C (ALT-C), a Disintegrin-Like Cys-Rich Protein Isolated from the Venom of the Snake Rhinocerophis alternatus, Stimulates Angiogenesis and Antioxidant Defenses in the Liver of Freshwater Fish, Hoplias malabaricus
Toxins 2017, 9(10), 307; doi:10.3390/toxins9100307
Received: 29 August 2017 / Revised: 20 September 2017 / Accepted: 26 September 2017 / Published: 28 September 2017
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Abstract
Alternagin-C (ALT-C) is a disintegrin-like protein isolated from Rhinocerophis alternatus snake venom, which induces endothelial cell proliferation and angiogenesis. The aim of this study was to evaluate the systemic effects of a single dose of alternagin-C (0.5 mg·kg−1, via intra-arterial) on
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Alternagin-C (ALT-C) is a disintegrin-like protein isolated from Rhinocerophis alternatus snake venom, which induces endothelial cell proliferation and angiogenesis. The aim of this study was to evaluate the systemic effects of a single dose of alternagin-C (0.5 mg·kg−1, via intra-arterial) on oxidative stress biomarkers, histological alterations, vascular endothelial growth factor (VEGF) production, and the degree of vascularization in the liver of the freshwater fish traíra, Hoplias malabaricus, seven days after the initiation of therapy. ALT-C treatment increased VEGF levels and hepatic angiogenesis. ALT-C also enhanced hepatic antioxidant enzymes activities such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, decreasing the basal oxidative damage to lipids and proteins in the fish liver. These results indicate that ALT-C improved hepatic tissue and may play a crucial role in tissue regeneration mechanisms. Full article
(This article belongs to the Special Issue Venom and Toxin as Targeted Therapy)
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Open AccessArticle Profiling of Extracellular Toxins Associated with Diarrhetic Shellfish Poison in Prorocentrum lima Culture Medium by High-Performance Liquid Chromatography Coupled with Mass Spectrometry
Toxins 2017, 9(10), 308; doi: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
[...] Read more.
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 AccessArticle The Influence of Resiniferatoxin (RTX) and Tetrodotoxin (TTX) on the Distribution, Relative Frequency, and Chemical Coding of Noradrenergic and Cholinergic Nerve Fibers Supplying the Porcine Urinary Bladder Wall
Toxins 2017, 9(10), 310; doi:10.3390/toxins9100310
Received: 29 August 2017 / Revised: 20 September 2017 / Accepted: 1 October 2017 / Published: 3 October 2017
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Abstract
The present study investigated the influence of intravesically instilled resiniferatoxin (RTX) or tetrodotoxin (TTX) on the distribution, number, and chemical coding of noradrenergic and cholinergic nerve fibers (NF) supplying the urinary bladder in female pigs. Samples from the bladder wall were processed for
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The present study investigated the influence of intravesically instilled resiniferatoxin (RTX) or tetrodotoxin (TTX) on the distribution, number, and chemical coding of noradrenergic and cholinergic nerve fibers (NF) supplying the urinary bladder in female pigs. Samples from the bladder wall were processed for double-labelling immunofluorescence with antibodies against cholinergic and noradrenergic markers and some other neurotransmitter substances. Both RTX and TTX caused a significant decrease in the number of cholinergic NF in the urinary bladder wall (in the muscle coat, submucosa, and beneath the urothelium). RTX instillation resulted in a decrease in the number of noradrenergic NF in the submucosa and urothelium, while TTX treatment caused a significant increase in the number of these axons in all the layers. The most remarkable changes in the chemical coding of the NF comprised a distinct decrease in the number of the cholinergic NF immunoreactive to CGRP (calcitonin gene-related peptide), nNOS (neuronal nitric oxide synthase), SOM (somatostatin) or VIP (vasoactive intestinal polypeptide), and an increase in the number of noradrenergic NF immunopositive to GAL (galanin) or nNOS, both after RTX or TTX instillation. The present study is the first to suggest that both RTX and TTX can modify the number of noradrenergic and cholinergic NF supplying the porcine urinary bladder. Full article
(This article belongs to the Special Issue Toxins in Drug Discovery and Pharmacology)
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Open AccessArticle Target-Specificity in Scorpions; Comparing Lethality of Scorpion Venoms across Arthropods and Vertebrates
Toxins 2017, 9(10), 312; doi: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 AccessArticle Variation and Distribution of L-A Helper Totiviruses in Saccharomyces sensu stricto Yeasts Producing Different Killer Toxins
Toxins 2017, 9(10), 313; doi:10.3390/toxins9100313
Received: 14 September 2017 / Revised: 2 October 2017 / Accepted: 6 October 2017 / Published: 11 October 2017
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Abstract
Yeasts within the Saccharomyces sensu stricto cluster can produce different killer toxins. Each toxin is encoded by a medium size (1.5–2.4 Kb) M dsRNA virus, maintained by a larger helper virus generally called L-A (4.6 Kb). Different types of L-A are found associated
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Yeasts within the Saccharomyces sensu stricto cluster can produce different killer toxins. Each toxin is encoded by a medium size (1.5–2.4 Kb) M dsRNA virus, maintained by a larger helper virus generally called L-A (4.6 Kb). Different types of L-A are found associated to specific Ms: L-A in K1 strains and L-A-2 in K2 strains. Here, we extend the analysis of L-A helper viruses to yeasts other than S. cerevisiae, namely S. paradoxus, S. uvarum and S. kudriavzevii. Our sequencing data from nine new L-A variants confirm the specific association of each toxin-producing M and its helper virus, suggesting co-evolution. Their nucleotide sequences vary from 10% to 30% and the variation seems to depend on the geographical location of the hosts, suggesting cross-species transmission between species in the same habitat. Finally, we transferred by genetic methods different killer viruses from S. paradoxus into S. cerevisiae or viruses from S. cerevisiae into S. uvarum or S. kudriavzevii. In the foster hosts, we observed no impairment for their stable transmission and maintenance, indicating that the requirements for virus amplification in these species are essentially the same. We also characterized new killer toxins from S. paradoxus and constructed “superkiller” strains expressing them. Full article
(This article belongs to the Special Issue Yeast Killer Toxins)
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Open AccessFeature PaperArticle The Aspergillus flavus Homeobox Gene, hbx1, Is Required for Development and Aflatoxin Production
Toxins 2017, 9(10), 315; doi: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
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Open AccessArticle Response of Intestinal Bacterial Flora to the Long-term Feeding of Aflatoxin B1 (AFB1) in Mice
Toxins 2017, 9(10), 317; doi:10.3390/toxins9100317
Received: 9 August 2017 / Revised: 28 September 2017 / Accepted: 30 September 2017 / Published: 12 October 2017
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Abstract
In order to investigate the influence of aflatoxin B1 (AFB1) on intestinal bacterial flora, 24 Kunming mice (KM mice) were randomly placed into four groups, which were labeled as control, low-dose, medium-dose, and high-dose groups. They were fed intragastrically with 0.4 mL of
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In order to investigate the influence of aflatoxin B1 (AFB1) on intestinal bacterial flora, 24 Kunming mice (KM mice) were randomly placed into four groups, which were labeled as control, low-dose, medium-dose, and high-dose groups. They were fed intragastrically with 0.4 mL of 0 mg/L, 2.5 mg/L, 4 mg/L, or 10 mg/L of AFB1 solutions, twice a day for 2 months. The hypervariable region V3 + V4 on 16S rDNA of intestinal bacterial flora was sequenced by the use of a high-flux sequencing system on a Miseq Illumina platform; then, the obtained sequences were analyzed. The results showed that, when compared with the control group, both genera and phyla of intestinal bacteria in the three treatment groups decreased. About one third of the total genera and one half of the total phyla remained in the high-dose group. The dominant flora were Lactobacillus and Bacteroides in all groups. There were significant differences in the relative abundance of intestinal bacterial flora among groups. Most bacteria decreased as a whole from the control to the high-dose groups, but several beneficial and pathogenic bacterial species increased significantly with increasing dose of AFB1. Thus, the conclusion was that intragastric feeding with 2.5~10 mg/mL AFB1 for 2 months could decrease the majority of intestinal bacterial flora and induce the proliferation of some intestinal bacteria flora. Full article
(This article belongs to the Special Issue Effects of Mycotoxins on the Intestine)
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Open AccessArticle Palladium Nanoparticles-Based Fluorescence Resonance Energy Transfer Aptasensor for Highly Sensitive Detection of Aflatoxin M1 in Milk
Toxins 2017, 9(10), 318; doi: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 AccessArticle Shiga Toxins Induce Apoptosis and ER Stress in Human Retinal Pigment Epithelial Cells
Toxins 2017, 9(10), 319; doi:10.3390/toxins9100319
Received: 29 May 2017 / Revised: 6 October 2017 / Accepted: 6 October 2017 / Published: 13 October 2017
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Abstract
Shiga toxins (Stxs) produced by Shiga toxin-producing bacteria Shigella dysenteriae serotype 1 and select serotypes of Escherichia coli are the most potent known virulence factors in the pathogenesis of hemorrhagic colitis progressing to potentially fatal systemic complications such as acute renal failure, blindness
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Shiga toxins (Stxs) produced by Shiga toxin-producing bacteria Shigella dysenteriae serotype 1 and select serotypes of Escherichia coli are the most potent known virulence factors in the pathogenesis of hemorrhagic colitis progressing to potentially fatal systemic complications such as acute renal failure, blindness and neurological abnormalities. Although numerous studies have defined apoptotic responses to Shiga toxin type 1 (Stx1) or Shiga toxin type 2 (Stx2) in a variety of cell types, the potential significance of Stx-induced apoptosis of photoreceptor and pigmented cells of the eye following intoxication is unknown. We explored the use of immortalized human retinal pigment epithelial (RPE) cells as an in vitro model of Stx-induced retinal damage. To the best of our knowledge, this study is the first report that intoxication of RPE cells with Stxs activates both apoptotic cell death signaling and the endoplasmic reticulum (ER) stress response. Using live-cell imaging analysis, fluorescently labeled Stx1 or Stx2 were internalized and routed to the RPE cell endoplasmic reticulum. RPE cells were significantly sensitive to wild type Stxs by 72 h, while the cells survived challenge with enzymatically deficient mutant toxins (Stx1A or Stx2A). Upon exposure to purified Stxs, RPE cells showed activation of a caspase-dependent apoptotic program involving a reduction of mitochondrial transmembrane potential (Δψm), increased activation of ER stress sensors IRE1, PERK and ATF6, and overexpression CHOP and DR5. Finally, we demonstrated that treatment of RPE cells with Stxs resulted in the activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), suggesting that the ribotoxic stress response may be triggered. Collectively, these data support the involvement of Stx-induced apoptosis in ocular complications of intoxication. The evaluation of apoptotic responses to Stxs by cells isolated from multiple organs may reveal unique functional patterns of the cytotoxic actions of these toxins in the systemic complications that follow ingestion of toxin-producing bacteria. Full article
(This article belongs to the Special Issue Ribosome Inactivating Toxins)
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Open AccessArticle Abrin Toxicity and Bioavailability after Temperature and pH Treatment
Toxins 2017, 9(10), 320; doi:10.3390/toxins9100320
Received: 1 September 2017 / Revised: 7 October 2017 / Accepted: 10 October 2017 / Published: 13 October 2017
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Abstract
Abrin, one of most potent toxins known to man, is derived from the rosary pea (jequirity pea), Abrus precatorius and is a potential bioterror weapon. The temperature and pH stability of abrin was evaluated with an in vitro cell free translation (CFT) assay,
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Abrin, one of most potent toxins known to man, is derived from the rosary pea (jequirity pea), Abrus precatorius and is a potential bioterror weapon. The temperature and pH stability of abrin was evaluated with an in vitro cell free translation (CFT) assay, a Vero cell culture cytotoxicity assay, and an in vivo mouse bioassay. pH treatment of abrin had no detrimental effect on its stability and toxicity as seen either in vitro or in vivo. Abrin exposure to increasing temperatures did not completely abrogate protein translation. In both the cell culture cytotoxicity model and the mouse bioassay, abrin’s toxic effects were completely abrogated if the toxin was exposed to temperatures of 74 °C or higher. In the cell culture model, 63 °C-treated abrin had a 30% reduction in cytotoxicity which was validated in the in vivo mouse bioassay with all mice dying but with a slight time-to-death delay as compared to the non-treated abrin control. Since temperature inactivation did not affect abrin’s ability to inhibit protein synthesis (A-chain), we hypothesize that high temperature treatment affected abrin’s ability to bind to cellular receptors (affecting B-chain). Our results confirm the absolute need to validate in vitro cytotoxicity assays with in vivo mouse bioassays. Full article
(This article belongs to the Special Issue Ribosome Inactivating Toxins)
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Open AccessArticle Anti-Salmonella Activity Modulation of Mastoparan V1—A Wasp Venom Toxin—Using Protease Inhibitors, and Its Efficient Production via an Escherichia coli Secretion System
Toxins 2017, 9(10), 321; doi:10.3390/toxins9100321
Received: 15 September 2017 / Revised: 10 October 2017 / Accepted: 11 October 2017 / Published: 13 October 2017
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Abstract
A previous study highlighted that mastoparan V1 (MP-V1), a mastoparan from the venom of the social wasp Vespula vulgaris, is a potent antimicrobial peptide against Salmonella infection, which causes enteric diseases. However, there exist some limits for its practical application due to
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A previous study highlighted that mastoparan V1 (MP-V1), a mastoparan from the venom of the social wasp Vespula vulgaris, is a potent antimicrobial peptide against Salmonella infection, which causes enteric diseases. However, there exist some limits for its practical application due to the loss of its activity in an increased bacterial density and the difficulty of its efficient production. In this study, we first modulated successfully the antimicrobial activity of synthetic MP-V1 against an increased Salmonella population using protease inhibitors, and developed an Escherichia coli secretion system efficiently producing active MP-V1. The protease inhibitors used, except pepstatin A, significantly increased the antimicrobial activity of the synthetic MP-V1 at minimum inhibitory concentrations (determined against 106 cfu/mL of population) against an increased population (108 cfu/mL) of three different Salmonella serotypes, Gallinarum, Typhimurium and Enteritidis. Meanwhile, the E. coli strain harboring OmpA SS::MP-V1 was identified to successfully secrete active MP-V1 into cell-free supernatant, whose antimicrobial activity disappeared in the increased population (108 cfu/mL) of Salmonella Typhimurium recovered by adding a protease inhibitor cocktail. Therefore, it has been concluded that our challenge using the E. coli secretion system with the protease inhibitors is an attractive strategy for practical application of peptide toxins, such as MP-V1. Full article
(This article belongs to the Special Issue Toxins in Drug Discovery and Pharmacology)
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Open AccessArticle Temperature Influences the Production and Transport of Saxitoxin and the Expression of sxt Genes in the Cyanobacterium Aphanizomenon gracile
Toxins 2017, 9(10), 322; doi:10.3390/toxins9100322
Received: 28 September 2017 / Revised: 7 October 2017 / Accepted: 9 October 2017 / Published: 13 October 2017
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Abstract
The cyanobacterium Aphanizomenon gracile is the most widely distributed producer of the potent neurotoxin saxitoxin in freshwaters. In this work, total and extracellular saxitoxin and the transcriptional response of three genes linked to saxitoxin biosynthesis (sxtA) and transport (sxtM,
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The cyanobacterium Aphanizomenon gracile is the most widely distributed producer of the potent neurotoxin saxitoxin in freshwaters. In this work, total and extracellular saxitoxin and the transcriptional response of three genes linked to saxitoxin biosynthesis (sxtA) and transport (sxtM, sxtPer) were assessed in Aphanizomenon gracile UAM529 cultures under temperatures covering its annual cycle (12 °C, 23 °C, and 30 °C). Temperature influenced saxitoxin production being maximum at high temperatures (30 °C) above the growth optimum (23 °C), concurring with a 4.3-fold increased sxtA expression at 30 °C. Extracellular saxitoxin transport was temperature-dependent, with maxima at extremes of temperature (12 °C with 16.9% extracellular saxitoxin; and especially 30 °C with 53.8%) outside the growth optimum (23 °C), coinciding with a clear upregulation of sxtM at both 12 °C and 30 °C (3.8–4.1 fold respectively), and yet with just a slight upregulation of sxtPer at 30 °C (2.1-fold). Nitrate depletion also induced a high extracellular saxitoxin release (51.2%), although without variations of sxtM and sxtPer transcription, and showing evidence of membrane damage. This is the first study analysing the transcriptional response of sxtPer under environmental gradients, as well as the effect of temperature on putative saxitoxin transporters (sxtM and sxtPer) in cyanobacteria in general. Full article
(This article belongs to the Special Issue Selected Papers from the 5th Iberoamerican Cyanotoxins Meeting)
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Open AccessArticle Combined Venom Gland Transcriptomic and Venom Peptidomic Analysis of the Predatory Ant Odontomachus monticola
Toxins 2017, 9(10), 323; doi: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 AccessArticle Risk Levels of Toxic Cyanobacteria in Portuguese Recreational Freshwaters
Toxins 2017, 9(10), 327; doi:10.3390/toxins9100327
Received: 31 July 2017 / Revised: 13 October 2017 / Accepted: 16 October 2017 / Published: 18 October 2017
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Abstract
Portuguese freshwater reservoirs are important socio-economic resources, namely for recreational use. National legislation concerning bathing waters does not include mandatory levels or guidelines for cyanobacteria and cyanotoxins. This is an issue of concern since cyanotoxin-based evidence is insufficient to change the law, and
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Portuguese freshwater reservoirs are important socio-economic resources, namely for recreational use. National legislation concerning bathing waters does not include mandatory levels or guidelines for cyanobacteria and cyanotoxins. This is an issue of concern since cyanotoxin-based evidence is insufficient to change the law, and the collection of scientific evidence has been hampered by the lack of regulatory levels for cyanotoxins in bathing waters. In this work, we evaluate the profile of cyanobacteria and microcystins (MC) in eight freshwater reservoirs from the center of Portugal, used for bathing/recreation, in order to determine the risk levels concerning toxic cyanobacteria occurrence. Three of the reservoirs did not pose a risk of MC contamination. However, two reservoirs presented a high risk in 7% of the samples according to the World Health Organization (WHO) guidelines for MC in bathing waters (above 20 µg/L). In the remaining three reservoirs, the risk concerning microcystins occurrence was low. However, they exhibited recurrent blooms and persistent contamination with MC up to 4 µg/L. Thus, the risk of exposure to MC and potential acute and/or chronic health outcomes should not be disregarded in these reservoirs. These results contribute to characterize the cyanobacterial blooms profile and to map the risk of toxic cyanobacteria and microcystins occurrence in Portuguese inland waters. Full article
(This article belongs to the Special Issue Selected Papers from the 5th Iberoamerican Cyanotoxins Meeting)
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Open AccessArticle A Monoclonal–Monoclonal Antibody Based Capture ELISA for Abrin
Toxins 2017, 9(10), 328; doi:10.3390/toxins9100328
Received: 1 September 2017 / Revised: 9 October 2017 / Accepted: 13 October 2017 / Published: 18 October 2017
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Abstract
Abrin, one of the most highly potent toxins in the world, is derived from the plant, Abrus precatorius. Because of its high toxicity, it poses potential bioterror risks. Therefore, a need exists for new reagents and technologies that would be able to
[...] Read more.
Abrin, one of the most highly potent toxins in the world, is derived from the plant, Abrus precatorius. Because of its high toxicity, it poses potential bioterror risks. Therefore, a need exists for new reagents and technologies that would be able to rapidly detect abrin contamination as well as lead to new therapeutics. We report here a group of abrin-specific monoclonal antibodies (mAbs) that recognize abrin A-chain, intact A–B chain toxin, and agglutinin by Western blot. Additionally, these mAbs were evaluated for their ability to serve as capture antibodies for a sandwich (capture) ELISA. All possible capture–detector pairs were evaluated and the best antibody pair identified and optimized for a capture ELISA. The capture ELISA based on this capture–detector mAb pair had a limit of detection (L.O.D) of ≈1 ng/mL measured using three independent experiments. The assay did not reveal any false positives with extracts containing other potential ribosome-inactivating proteins (RIPs). Thus, this new capture ELISA uses mAbs for both capture and detection; has no cross-reactivity against other plant RIPs; and has a sensitivity comparable to other reported capture ELISAs using polyclonal antibodies as either capture or detector. Full article
(This article belongs to the Special Issue Ribosome Inactivating Toxins)
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Open AccessArticle Production, Characterisation and Testing of an Ovine Antitoxin against Ricin; Efficacy, Potency and Mechanisms of Action
Toxins 2017, 9(10), 329; doi:10.3390/toxins9100329
Received: 4 September 2017 / Revised: 10 October 2017 / Accepted: 13 October 2017 / Published: 18 October 2017
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Abstract
Ricin is a type II ribosome-inactivating toxin that catalytically inactivates ribosomes ultimately leading to cell death. The toxicity of ricin along with the prevalence of castor beans (its natural source) has led to its increased notoriety and incidences of nefarious use. Despite these
[...] Read more.
Ricin is a type II ribosome-inactivating toxin that catalytically inactivates ribosomes ultimately leading to cell death. The toxicity of ricin along with the prevalence of castor beans (its natural source) has led to its increased notoriety and incidences of nefarious use. Despite these concerns, there are no licensed therapies available for treating ricin intoxication. Here, we describe the development of a F(ab’)2 polyclonal ovine antitoxin against ricin and demonstrate the efficacy of a single, post-exposure, administration in an in vivo murine model of intoxication against aerosolised ricin. We found that a single dose of antitoxin afforded a wide window of opportunity for effective treatment with 100% protection observed in mice challenged with aerosolised ricin when given 24 h after exposure to the toxin and 75% protection when given at 30 h. Treated mice had reduced weight loss and clinical signs of intoxication compared to the untreated control group. Finally, using imaging flow cytometry, it was found that both cellular uptake and intracellular trafficking of ricin toxin to the Golgi apparatus was reduced in the presence of the antitoxin suggesting both actions can contribute to the therapeutic mechanism of a polyclonal antitoxin. Collectively, the research highlights the significant potential of the ovine F(ab’)2 antitoxin as a treatment for ricin intoxication. Full article
(This article belongs to the Special Issue Ribosome Inactivating Toxins)
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Open AccessArticle Mycotoxin Analysis of Human Urine by LC-MS/MS: A Comparative Extraction Study
Toxins 2017, 9(10), 330; doi:10.3390/toxins9100330 (registering DOI)
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 AccessArticle P2X-Receptor Antagonists Inhibit the Interaction of S. aureus Hemolysin A with Membranes
Toxins 2017, 9(10), 332; doi:10.3390/toxins9100332 (registering DOI)
Received: 7 September 2017 / Revised: 8 October 2017 / Accepted: 15 October 2017 / Published: 19 October 2017
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Abstract
The pore forming hemolysin A, Hla, is a major virulence factor of Staphylococcus aureus. Apparently, 1–2 pore(s) per cell suffice(s) to cause cell death. Accumulated experimental evidence points towards a major role of ATP-gated purinergic receptors (P2XR) for hemolysis caused by Hla,
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The pore forming hemolysin A, Hla, is a major virulence factor of Staphylococcus aureus. Apparently, 1–2 pore(s) per cell suffice(s) to cause cell death. Accumulated experimental evidence points towards a major role of ATP-gated purinergic receptors (P2XR) for hemolysis caused by Hla, complement and other pore forming proteins, presumably by increasing membrane permeability. Indeed, in experiments employing rabbit erythrocytes, inhibitory concentrations of frequently employed P2XR-antagonists were in a similar range as previously reported for erythrocytes of other species and other toxins. However, Hla-dependent hemolysis was not enhanced by extracellular ATP, and oxidized adenosinetriphosphate (oxATP) had only a minor inhibitory effect. Unexpectedly, P2XR-inhibitors also prevented Hla-induced lysis of pure lipid membranes, demonstrating that the inhibition did not even depend on the presence of P2XR. Fluorescence microscopy and gel-electrophoresis clearly revealed that P2XR-inhibitors interfere with binding and subsequent oligomerisation of Hla with membranes. Similar results were obtained employing HaCaT-cells. Furthermore, calorimetric data and hemolysis experiments with Hla pre-treated with pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) showed that this compound directly binds to Hla. Our results call for a critical re-assessment of the appealing concept, which suggests that P2XR are general amplifiers of damage by pore-forming proteins. Full article
(This article belongs to the Special Issue Toxins and Ion Channels)
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Open AccessArticle Chronic Exposure to the Fusarium Mycotoxin Deoxynivalenol: Impact on Performance, Immune Organ, and Intestinal Integrity of Slow-Growing Chickens
Toxins 2017, 9(10), 334; doi:10.3390/toxins9100334 (registering DOI)
Received: 31 August 2017 / Revised: 13 October 2017 / Accepted: 15 October 2017 / Published: 20 October 2017
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Abstract
This study investigates the long-term effects of deoxynivalenol (DON) consumption on avian growth performance, on the proliferation, apoptosis, and DNA damage of spleen cells, and on intestinal integrity. Two hundred and eight 5-day-old black-feathered Taiwan country chickens were fed diets containing 0, 2,
[...] Read more.
This study investigates the long-term effects of deoxynivalenol (DON) consumption on avian growth performance, on the proliferation, apoptosis, and DNA damage of spleen cells, and on intestinal integrity. Two hundred and eight 5-day-old black-feathered Taiwan country chickens were fed diets containing 0, 2, 5, and 10 mg/kg of DON for 16 weeks. Body weight gain of male birds in the 2 mg/kg group was significantly lower than that in the 5 mg/kg group. At the end of trial, feeding DON-contaminated diets of 5 mg/kg resulted in heavier spleens. Moreover, the increase in DON induced cellular proliferation, apoptosis, and DNA damage signals in the spleen, the exception being female birds fed 10 mg/kg of DON showing reduced proliferation. Expression of claudin-5 was increased in jejunum of female birds fed 2 and 5 mg/kg of DON, whereas decreased expression levels were found in male birds. In conclusion, our results verified that DON may cause a disturbance to the immune system and alter the intestinal barrier in Taiwan country chickens, and may also lead to discrepancies in growth performances in a dose- and sex-dependent manner. Full article
(This article belongs to the collection Fusarium Toxins – Relevance for Human and Animal Health)
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Open AccessReview Invasion of Dendritic Cells, Macrophages and Neutrophils by the Bordetella Adenylate Cyclase Toxin: A Subversive Move to Fool Host Immunity
Toxins 2017, 9(10), 293; doi:10.3390/toxins9100293
Received: 25 August 2017 / Revised: 14 September 2017 / Accepted: 15 September 2017 / Published: 21 September 2017
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Abstract
Adenylate cyclase toxin (CyaA) is released in the course of B. pertussis infection in the host’s respiratory tract in order to suppress its early innate and subsequent adaptive immune defense. CD11b-expressing dendritic cells (DC), macrophages and neutrophils are professional phagocytes and key players
[...] Read more.
Adenylate cyclase toxin (CyaA) is released in the course of B. pertussis infection in the host’s respiratory tract in order to suppress its early innate and subsequent adaptive immune defense. CD11b-expressing dendritic cells (DC), macrophages and neutrophils are professional phagocytes and key players of the innate immune system that provide a first line of defense against invading pathogens. Recent findings revealed the capacity of B. pertussis CyaA to intoxicate DC with high concentrations of 3′,5′-cyclic adenosine monophosphate (cAMP), which ultimately skews the host immune response towards the expansion of Th17 cells and regulatory T cells. CyaA-induced cAMP signaling swiftly incapacitates opsonophagocytosis, oxidative burst and NO-mediated killing of bacteria by neutrophils and macrophages. The subversion of host immune responses by CyaA after delivery into DC, macrophages and neutrophils is the subject of this review. Full article
(This article belongs to the Special Issue Adenylate Cyclase (CyaA) Toxin)
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Open AccessFeature PaperReview Understanding the Mechanism of Translocation of Adenylate Cyclase Toxin across Biological Membranes
Toxins 2017, 9(10), 295; doi:10.3390/toxins9100295
Received: 22 August 2017 / Revised: 13 September 2017 / Accepted: 15 September 2017 / Published: 21 September 2017
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Abstract
Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding
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Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ≈400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called “two-step model” and the recently-proposed “toroidal pore model”, will be considered. Full article
(This article belongs to the Special Issue Adenylate Cyclase (CyaA) Toxin)
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Open AccessReview Structure–Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes
Toxins 2017, 9(10), 300; doi:10.3390/toxins9100300
Received: 29 August 2017 / Revised: 19 September 2017 / Accepted: 21 September 2017 / Published: 24 September 2017
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Abstract
Bordetellae, pathogenic to mammals, produce an immunomodulatory adenylate cyclase toxin–hemolysin (CyaA, ACT or AC-Hly) that enables them to overcome the innate immune defense of the host. CyaA subverts host phagocytic cells by an orchestrated action of its functional domains, where an extremely
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Bordetellae, pathogenic to mammals, produce an immunomodulatory adenylate cyclase toxin–hemolysin (CyaA, ACT or AC-Hly) that enables them to overcome the innate immune defense of the host. CyaA subverts host phagocytic cells by an orchestrated action of its functional domains, where an extremely catalytically active adenylyl cyclase enzyme is delivered into phagocyte cytosol by a pore-forming repeat-in-toxin (RTX) cytolysin moiety. By targeting sentinel cells expressing the complement receptor 3, known as the CD11b/CD18 (αMβ2) integrin, CyaA compromises the bactericidal functions of host phagocytes and supports infection of host airways by Bordetellae. Here, we review the state of knowledge on structural and functional aspects of CyaA toxin action, placing particular emphasis on signaling mechanisms by which the toxin-produced 3′,5′-cyclic adenosine monophosphate (cAMP) subverts the physiology of phagocytic cells. Full article
(This article belongs to the Special Issue Adenylate Cyclase (CyaA) Toxin)
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Open AccessReview The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies
Toxins 2017, 9(10), 309; doi:10.3390/toxins9100309
Received: 11 August 2017 / Revised: 15 September 2017 / Accepted: 16 September 2017 / Published: 2 October 2017
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Abstract
The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT) A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and
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The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT) A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and LC) of the three BoNT serotypes were targeted to achieve a synergistic effect (oligoclonal antibodies). For antibody isolation, macaques were immunized with the recombinant and non-toxic BoNT/A, B or E, HC or LC, followed by the generation of immune phage-display libraries. Antibodies were selected from these libraries against the holotoxin and further analyzed in in vitro and ex vivo assays. For each library, the best ex vivo neutralizing antibody fragments were germline-humanized and expressed as immunoglobulin G (IgGs). The IgGs were tested in vivo, in a standardized model of protection, and challenged with toxins obtained from collections of Clostridium strains. Protective antibody combinations against BoNT/A and BoNT/B were evidenced and for BoNT/E, the anti-LC antibody alone was found highly protective. The combination of these five antibodies as an oligoclonal antibody cocktail can be clinically and regulatorily developed while their high “humanness” predicts a high tolerance in humans. Full article
(This article belongs to the Special Issue Botulinum Neurotoxins Antibody and Vaccine)
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Open AccessReview Treatments for Pulmonary Ricin Intoxication: Current Aspects and Future Prospects
Toxins 2017, 9(10), 311; doi:10.3390/toxins9100311
Received: 6 September 2017 / Revised: 26 September 2017 / Accepted: 29 September 2017 / Published: 3 October 2017
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Abstract
Ricin, a plant-derived toxin originating from the seeds of Ricinus communis (castor beans), is one of the most lethal toxins known, particularly if inhaled. Ricin is considered a potential biological threat agent due to its high availability and ease of production. The clinical
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Ricin, a plant-derived toxin originating from the seeds of Ricinus communis (castor beans), is one of the most lethal toxins known, particularly if inhaled. Ricin is considered a potential biological threat agent due to its high availability and ease of production. The clinical manifestation of pulmonary ricin intoxication in animal models is closely related to acute respiratory distress syndrome (ARDS), which involves pulmonary proinflammatory cytokine upregulation, massive neutrophil infiltration and severe edema. Currently, the only post-exposure measure that is effective against pulmonary ricinosis at clinically relevant time-points following intoxication in pre-clinical studies is passive immunization with anti-ricin neutralizing antibodies. The efficacy of this antitoxin treatment depends on antibody affinity and the time of treatment initiation within a limited therapeutic time window. Small-molecule compounds that interfere directly with the toxin or inhibit its intracellular trafficking may also be beneficial against ricinosis. Another approach relies on the co-administration of antitoxin antibodies with immunomodulatory drugs, thereby neutralizing the toxin while attenuating lung injury. Immunomodulators and other pharmacological-based treatment options should be tailored according to the particular pathogenesis pathways of pulmonary ricinosis. This review focuses on the current treatment options for pulmonary ricin intoxication using anti-ricin antibodies, disease-modifying countermeasures, anti-ricin small molecules and their various combinations. Full article
(This article belongs to the Special Issue Ribosome Inactivating Toxins)
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Open AccessReview Plant Ribosome-Inactivating Proteins: Progesses, Challenges and Biotechnological Applications (and a Few Digressions)
Toxins 2017, 9(10), 314; doi:10.3390/toxins9100314
Received: 31 August 2017 / Revised: 29 September 2017 / Accepted: 3 October 2017 / Published: 12 October 2017
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Abstract
Plant ribosome-inactivating protein (RIP) toxins are EC3.2.2.22 N-glycosidases, found among most plant species encoded as small gene families, distributed in several tissues being endowed with defensive functions against fungal or viral infections. The two main plant RIP classes include type I (monomeric) and
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Plant ribosome-inactivating protein (RIP) toxins are EC3.2.2.22 N-glycosidases, found among most plant species encoded as small gene families, distributed in several tissues being endowed with defensive functions against fungal or viral infections. The two main plant RIP classes include type I (monomeric) and type II (dimeric) as the prototype ricin holotoxin from Ricinus communis that is composed of a catalytic active A chain linked via a disulphide bridge to a B-lectin domain that mediates efficient endocytosis in eukaryotic cells. Plant RIPs can recognize a universally conserved stem-loop, known as the α-sarcin/ ricin loop or SRL structure in 23S/25S/28S rRNA. By depurinating a single adenine (A4324 in 28S rat rRNA), they can irreversibly arrest protein translation and trigger cell death in the intoxicated mammalian cell. Besides their useful application as potential weapons against infected/tumor cells, ricin was also used in bio-terroristic attacks and, as such, constitutes a major concern. In this review, we aim to summarize past studies and more recent progresses made studying plant RIPs and discuss successful approaches that might help overcoming some of the bottlenecks encountered during the development of their biomedical applications. Full article
(This article belongs to the Special Issue Ribosome Inactivating Toxins)
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Open AccessReview Helicobacter pylori Vacuolating Toxin and Gastric Cancer
Toxins 2017, 9(10), 316; doi:10.3390/toxins9100316
Received: 13 September 2017 / Revised: 3 October 2017 / Accepted: 5 October 2017 / Published: 12 October 2017
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Abstract
Helicobacter pylori VacA is a channel-forming toxin unrelated to other known bacterial toxins. Most H. pylori strains contain a vacA gene, but there is marked variation among strains in VacA toxin activity. This variation is attributable to strain-specific variations in VacA amino acid
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Helicobacter pylori VacA is a channel-forming toxin unrelated to other known bacterial toxins. Most H. pylori strains contain a vacA gene, but there is marked variation among strains in VacA toxin activity. This variation is attributable to strain-specific variations in VacA amino acid sequences, as well as variations in the levels of VacA transcription and secretion. In this review, we discuss epidemiologic studies showing an association between specific vacA allelic types and gastric cancer, as well as studies that have used animal models to investigate VacA activities relevant to gastric cancer. We also discuss the mechanisms by which VacA-induced cellular alterations may contribute to the pathogenesis of gastric cancer. Full article
(This article belongs to the Special Issue H. pylori Virulence Factors in the Induction of Gastric Cancer)
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Open AccessReview Recent Advances in Mycotoxin Determination for Food Monitoring via Microchip
Toxins 2017, 9(10), 324; doi:10.3390/toxins9100324
Received: 12 September 2017 / Revised: 30 September 2017 / Accepted: 9 October 2017 / Published: 14 October 2017
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Abstract
Mycotoxins are one of the main factors impacting food safety. Mycotoxin contamination has threatened the health of humans and animals. Conventional methods for the detection of mycotoxins are gas chromatography (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS), or enzyme-linked immunosorbent
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Mycotoxins are one of the main factors impacting food safety. Mycotoxin contamination has threatened the health of humans and animals. Conventional methods for the detection of mycotoxins are gas chromatography (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS), or enzyme-linked immunosorbent assay (ELISA). However, all these methods are time-consuming, require large-scale instruments and skilled technicians, and consume large amounts of hazardous regents and solvents. Interestingly, a microchip requires less sample consumption and short analysis time, and can realize the integration, miniaturization, and high-throughput detection of the samples. Hence, the application of a microchip for the detection of mycotoxins can make up for the deficiency of the conventional detection methods. This review focuses on the application of a microchip to detect mycotoxins in foods. The toxicities of mycotoxins and the materials of the microchip are firstly summarized in turn. Then the application of a microchip that integrates various kinds of detection methods (optical, electrochemical, photo-electrochemical, and label-free detection) to detect mycotoxins is reviewed in detail. Finally, challenges and future research directions in the development of a microchip to detect mycotoxins are previewed. Full article
(This article belongs to the collection Biorecognition Assays for Mycotoxins)
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Open AccessReview Animal Toxins Providing Insights into TRPV1 Activation Mechanism
Toxins 2017, 9(10), 326; doi:10.3390/toxins9100326
Received: 28 September 2017 / Revised: 13 October 2017 / Accepted: 13 October 2017 / Published: 16 October 2017
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Abstract
Beyond providing evolutionary advantages, venoms offer unique research tools, as they were developed to target functionally important proteins and pathways. As a key pain receptor in the nociceptive pathway, transient receptor potential vanilloid 1 (TRPV1) of the TRP superfamily has been shown to
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Beyond providing evolutionary advantages, venoms offer unique research tools, as they were developed to target functionally important proteins and pathways. As a key pain receptor in the nociceptive pathway, transient receptor potential vanilloid 1 (TRPV1) of the TRP superfamily has been shown to be a target for several toxins, as a way of producing pain to deter predators. Importantly, TRPV1 is involved in thermoregulation, inflammation, and acute nociception. As such, toxins provide tools to understand TRPV1 activation and modulation, a critical step in advancing pain research and the development of novel analgesics. Indeed, the phytotoxin capsaicin, which is the spicy chemical in chili peppers, was invaluable in the original cloning and characterization of TRPV1. The unique properties of each subsequently characterized toxin have continued to advance our understanding of functional, structural, and biophysical characteristics of TRPV1. By building on previous reviews, this work aims to provide a comprehensive summary of the advancements made in TRPV1 research in recent years by employing animal toxins, in particular DkTx, RhTx, BmP01, Echis coloratus toxins, APHCs and HCRG21. We examine each toxin’s functional aspects, behavioral effects, and structural features, all of which have contributed to our current knowledge of TRPV1. We additionally discuss the key features of TRPV1’s outer pore domain, which proves to be the target of the currently discussed toxins. Full article
(This article belongs to the Special Issue Toxins in Drug Discovery and Pharmacology)
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