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Toxins, Volume 6, Issue 4 (April 2014), Pages 1155-1470

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Open AccessArticle Survey of Mycotoxins in Corn Distillers’ Dried Grains with Solubles from Seventy-Eight Ethanol Plants in Twelve States in the U.S. in 2011
Toxins 2014, 6(4), 1155-1168; doi:10.3390/toxins6041155
Received: 6 February 2014 / Revised: 13 March 2014 / Accepted: 14 March 2014 / Published: 26 March 2014
Cited by 5 | PDF Full-text (249 KB) | HTML Full-text | XML Full-text
Abstract
Fuel ethanol co-products known as distillers’ dried grains with solubles (DDGS) are a significant source of energy, protein, and phosphorous in animal feed. Fuel ethanol production may concentrate mycotoxins present in corn into DDGS. One hundred and forty one corn DDGS lots [...] Read more.
Fuel ethanol co-products known as distillers’ dried grains with solubles (DDGS) are a significant source of energy, protein, and phosphorous in animal feed. Fuel ethanol production may concentrate mycotoxins present in corn into DDGS. One hundred and forty one corn DDGS lots collected in 2011 from 78 ethanol plants located in 12 states were screened for the mycotoxins deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON), nivalenol (NIV), and zearalenone (ZON). DON ranged from <0.50 to 14.62 μg g−1, 15-ADON ranged from <0.10 to 7.55 μg g−1, and ZON ranged from <0.10 to 2.12 μg g−1. None of the DDGS lots contained 3-ADON or NIV. Plants in OH had the highest levels of DON overall (mean of 9.51 μg g−1), and plants in NY, MI, IN, NE, and WI had mean DON levels >1 and <4 μg g−1. Twenty six percent (36/141) of the DDGS lots contained 1.0 to 5.0 μg g−1 DON, 2% (3/141) contained >5.0 and <10.0 μg g−1 DON, and 3% (4/141) contained >10.0 μg g−1 DON. All DDGS lots contaminated with unacceptable levels of DON evaded detection prior to their commercial distribution and were likely sold as feed products. Full article
Open AccessArticle Larvicidal and Cytotoxic Potential of Squamocin on the Midgut of Aedes aegypti (Diptera: Culicidae)
Toxins 2014, 6(4), 1169-1176; doi:10.3390/toxins6041169
Received: 20 January 2014 / Revised: 28 February 2014 / Accepted: 11 March 2014 / Published: 26 March 2014
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Abstract
Acetogenins are secondary metabolites exclusively produced by Annonaceae, which have antitumor, cytotoxic, and pesticide activities. In this study, we evaluated the larvicidal and cytotoxic effect of squamocin from Annona squamosa on Aedes aegypti (Diptera: Culicidae) midgut. The compound was solubilized in 2% [...] Read more.
Acetogenins are secondary metabolites exclusively produced by Annonaceae, which have antitumor, cytotoxic, and pesticide activities. In this study, we evaluated the larvicidal and cytotoxic effect of squamocin from Annona squamosa on Aedes aegypti (Diptera: Culicidae) midgut. The compound was solubilized in 2% Tween 20 at 10, 20, 50, 80 and 100 ppm. The assay was conducted in a completely randomized design with four replications, each with 20 third-instar larvae. Larval mortality was assessed every hour until total mortality, and the data were subjected to Probit analysis. Cellular damage was evaluated every 30 min in groups comprising five larvae subjected to squamocin at 50 and 100 ppm for 240 min. The total larval mortality occurred after 360 min following application of 50, 80, and 100 ppm squamocin, and 600 min after applying other concentrations with LC50 at 6.4 ppm. Both 50 and 100 ppm of squamocin showed cytotoxic activity in the midgut epithelium of A. aegypti after 240 min with 50 ppm resulting in midgut cells with light cytoplasm containing small vacuoles, whereas at 100 ppm were found cells with cytoplasm highly vacuolated, damaged apical surface and cell protrusion toward the gut lumen. In conclusion, squamocin has the potential to control A. aegypti. Full article
Open AccessArticle Analysis of Individual and Combined Effects of Ochratoxin A and Zearalenone on HepG2 and KK-1 Cells with Mathematical Models
Toxins 2014, 6(4), 1177-1192; doi:10.3390/toxins6041177
Received: 25 November 2013 / Revised: 7 March 2014 / Accepted: 11 March 2014 / Published: 26 March 2014
Cited by 7 | PDF Full-text (970 KB) | HTML Full-text | XML Full-text
Abstract
Ochratoxin A (OTA) and Zearalenone (ZEA) are widespread mycotoxins that contaminate foodstuffs simultaneously, but sufficient data regarding their mixed toxicities are lacking. This study aims to analyze the style of combined effects of OTA and ZEA on cells of their target organs. [...] Read more.
Ochratoxin A (OTA) and Zearalenone (ZEA) are widespread mycotoxins that contaminate foodstuffs simultaneously, but sufficient data regarding their mixed toxicities are lacking. This study aims to analyze the style of combined effects of OTA and ZEA on cells of their target organs. For this purpose, cytotoxicity was determined in HepG2 and KK-1 cells treated with single and combined forms of OTA and ZEA. Furthermore, we have analyzed the data using two mathematical models based on the concepts of concentration addition (CA) and independent addition (IA). By analyzing data with nonlinear regression, toxins applied singly showed classic sigmoid dose-response curves in HepG2 cells whereas in KK-1 cells hormetic responses were observed. Exposure to equieffective mixtures of OTA and ZEA showed additive effects, irrespective of different nonlinear regression models used. Our results demonstrate that IA is an appropriate concept to account for mixture effects of OTA and ZEA. The results in ROS generation indicate a departure from additivity to antagonism or synergism at different concentrations, probably due to potential interaction during ROS production. This study shows that a risk assessment of mycotoxins should account for mixture effects, and prediction models are valuable tools for mixture assessment. Full article
Open AccessArticle Inhibitory Effects of Respiration Inhibitors on Aflatoxin Production
Toxins 2014, 6(4), 1193-1200; doi:10.3390/toxins6041193
Received: 21 February 2014 / Revised: 13 March 2014 / Accepted: 19 March 2014 / Published: 26 March 2014
Cited by 3 | PDF Full-text (449 KB) | HTML Full-text | XML Full-text
Abstract
Aflatoxin production inhibitors, which do not inhibit the growth of aflatoxigenic fungi, may be used to control aflatoxin without incurring a rapid spread of resistant strains. A respiration inhibitor that inhibits aflatoxin production was identified during a screening process for natural, aflatoxin-production [...] Read more.
Aflatoxin production inhibitors, which do not inhibit the growth of aflatoxigenic fungi, may be used to control aflatoxin without incurring a rapid spread of resistant strains. A respiration inhibitor that inhibits aflatoxin production was identified during a screening process for natural, aflatoxin-production inhibitors. This prompted us to evaluate respiration inhibitors as potential aflatoxin control agents. The inhibitory activities of four natural inhibitors, seven synthetic miticides, and nine synthetic fungicides were evaluated on aflatoxin production in Aspergillus parasiticus. All of the natural inhibitors (rotenone, siccanin, aptenin A5, and antimycin A) inhibited fungal aflatoxin production with IC50 values around 10 µM. Among the synthetic miticides, pyridaben, fluacrypyrim, and tolfenpyrad exhibited strong inhibitory activities with IC50 values less than 0.2 µM, whereas cyflumetofen did not show significant inhibitory activity. Of the synthetic fungicides, boscalid, pyribencarb, azoxystrobin, pyraclostrobin, and kresoxim-methyl demonstrated strong inhibitory activities, with IC50 values less than 0.5 µM. Fungal growth was not significantly affected by any of the inhibitors tested at concentrations used. There was no correlation observed between the targets of respiration inhibitors (complexes I, II, and III) and their IC50 values for aflatoxin-production inhibitory activity. This study suggests that respiration inhibitors, including commonly used pesticides, are useful for aflatoxin control. Full article
Open AccessArticle A qPCR Assay to Detect and Quantify Shiga Toxin-Producing E. coli (STEC) in Cattle and on Farms: A Potential Predictive Tool for STEC Culture-Positive Farms
Toxins 2014, 6(4), 1201-1221; doi:10.3390/toxins6041201
Received: 3 January 2014 / Revised: 3 March 2014 / Accepted: 11 March 2014 / Published: 27 March 2014
Cited by 7 | PDF Full-text (466 KB) | HTML Full-text | XML Full-text
Abstract
Shiga toxin-producing E. coli (STEC), of various serogroups harboring the intimin gene, form a serious threat to human health. They are asymptomatically carried by cattle. In this study, a quantitative real-time PCR (qPCR) method was developed as a molecular method to detect [...] Read more.
Shiga toxin-producing E. coli (STEC), of various serogroups harboring the intimin gene, form a serious threat to human health. They are asymptomatically carried by cattle. In this study, a quantitative real-time PCR (qPCR) method was developed as a molecular method to detect and quantify Shiga toxin genes stx1 and stx2 and the intimin gene eae. Subsequently, 59 fecal samples from six farms were tested using qPCR and a culture method as a reference. Three farms had contaminated animals as demonstrated by the culture method. Culture-positive farms showed moderate significantly higher stx prevalences than culture-negative farms (p = 0.05). This is the first study which showed preliminary results that qPCR can predict STEC farm contamination, with a specificity of 77% and a sensitivity of 83%, as compared with the culture method. Furthermore, the presence or quantity of stx genes in feces was not correlated to the isolation of STEC from the individual animal. Quantitative data thus did not add value to the results. Finally, the detection of both stx and eae genes within the same fecal sample or farm using qPCR was not correlated with the isolation of an eae-harboring STEC strain from the respective sample or farm using the culture method. Full article
(This article belongs to the Section Bacterial Toxins)
Open AccessArticle Extracellular Hb Enhances Cardiac Toxicity in Endotoxemic Guinea Pigs: Protective Role of Haptoglobin
Toxins 2014, 6(4), 1244-1259; doi:10.3390/toxins6041244
Received: 31 January 2014 / Revised: 18 March 2014 / Accepted: 21 March 2014 / Published: 31 March 2014
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Abstract
Endotoxemia plays a major causative role in the myocardial injury and dysfunction associated with sepsis. Extracellular hemoglobin (Hb) has been shown to enhance the pathophysiology of endotoxemia. In the present study, we examined the myocardial pathophysiology in guinea pigs infused with lipopolysaccharide [...] Read more.
Endotoxemia plays a major causative role in the myocardial injury and dysfunction associated with sepsis. Extracellular hemoglobin (Hb) has been shown to enhance the pathophysiology of endotoxemia. In the present study, we examined the myocardial pathophysiology in guinea pigs infused with lipopolysaccharide (LPS), a Gram-negative bacterial endotoxin, and purified Hb. We also examined whether the administration of the Hb scavenger haptoglobin (Hp) could protect against the effects observed. Here, we show that Hb infusion following LPS administration, but not either insult alone, increased myocardial iron deposition, heme oxygenase-1 expression, phagocyte activation and infiltration, as well as oxidative DNA damage and apoptosis assessed by 8-hydroxy-2'-deoxyguanosine (8-OHdG) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) immunostaining, respectively. Co-administration of Hp significantly attenuated the myocardial events induced by the combination of LPS and Hb. These findings may have relevant therapeutic implications for the management of sepsis during concomitant disease or clinical interventions associated with the increased co-exposures to LPS and Hb, such as trauma, surgery or massive blood transfusions. Full article
(This article belongs to the collection Toxicity and Therapeutic Interventions in the Immune System)
Open AccessArticle Changes in Gene Expression in the Larval Gut of Ostrinia nubilalis in Response to Bacillus thuringiensis Cry1Ab Protoxin Ingestion
Toxins 2014, 6(4), 1274-1294; doi:10.3390/toxins6041274
Received: 11 December 2013 / Revised: 13 March 2014 / Accepted: 26 March 2014 / Published: 3 April 2014
Cited by 6 | PDF Full-text (586 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
We developed a microarray based on 2895 unique transcripts assembled from 15,000 cDNA sequences from the European corn borer (Ostrinia nubilalis) larval gut. This microarray was used to monitor gene expression in early third-instar larvae of Bacillus thuringiensis (Bt)-susceptible O. [...] Read more.
We developed a microarray based on 2895 unique transcripts assembled from 15,000 cDNA sequences from the European corn borer (Ostrinia nubilalis) larval gut. This microarray was used to monitor gene expression in early third-instar larvae of Bacillus thuringiensis (Bt)-susceptible O. nubilalis after 6 h feeding on diet, with or without the Bt Cry1Ab protoxin. We identified 174 transcripts, for which the expression was changed more than two-fold in the gut of the larvae fed Cry1Ab protoxin (p < 0.05), representing 80 down-regulated and 94 up-regulated transcripts. Among 174 differentially expressed transcripts, 13 transcripts putatively encode proteins that are potentially involved in Bt toxicity, and these transcripts include eight serine proteases, three aminopeptidases, one alkaline phosphatase, and one cadherin. The expressions of trypsin-like protease and three aminopeptidase transcripts were variable, but two potential Bt-binding proteins, alkaline phosphatase and cadherin were consistently up-regulated in larvae fed Cry1Ab protoxin. The significantly up and down-regulated transcripts may be involved in Cry1Ab toxicity by activation, degradation, toxin binding, and other related cellular responses. This study is a preliminary survey of Cry1Ab protoxin-induced transcriptional responses in O. nubilalis gut and our results are expected to help with further studies on Bt toxin-insect interactions at the molecular level. Full article
(This article belongs to the Special Issue <i>Bacillus thuringiensis</i> Toxins)
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Open AccessArticle Further Aspects of Ochratoxin A-Cation Interactions: Complex Formation with Zinc Ions and a Novel Analytical Application of Ochratoxin A-Magnesium Interaction in the HPLC-FLD System
Toxins 2014, 6(4), 1295-1307; doi:10.3390/toxins6041295
Received: 14 February 2014 / Revised: 25 March 2014 / Accepted: 1 April 2014 / Published: 10 April 2014
Cited by 3 | PDF Full-text (1089 KB) | HTML Full-text | XML Full-text
Abstract
Ochratoxin A (OTA) is a mycotoxin produced by different Aspergillus and Penicillium species. Since its mechanism of action is not fully understood yet, it is important to gain further insight into different interactions of OTA at the molecular level. OTA is found [...] Read more.
Ochratoxin A (OTA) is a mycotoxin produced by different Aspergillus and Penicillium species. Since its mechanism of action is not fully understood yet, it is important to gain further insight into different interactions of OTA at the molecular level. OTA is found worldwide in many foods and drinks. Moreover, it can also be detected in human and animal tissues and body fluids, as well. Therefore, the development of highly sensitive quantitative methods for the determination of OTA is of utmost importance. OTA most likely forms complexes with divalent cations, both in cells and body fluids. In the present study, the OTA-zinc interaction was investigated and compared to OTA-magnesium complex formation using fluorescence spectroscopy and molecular modeling. Our results show that zinc(II) ion forms a two-fold higher stable complex with OTA than magnesium(II) ion. In addition, based on the enhanced fluorescence emission of OTA in its magnesium-bound form, a novel RP-HPLC-fluorescence detector (FLD) method was also established. Our results highlight that the application of magnesium chloride in alkaline eluents results in an approximately two-fold increase in sensitivity using the HPLC-FLD technique. Full article
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Open AccessArticle Systemic Growth of F. graminearum in Wheat Plants and Related Accumulation of Deoxynivalenol
Toxins 2014, 6(4), 1308-1324; doi:10.3390/toxins6041308
Received: 24 February 2014 / Revised: 28 March 2014 / Accepted: 31 March 2014 / Published: 10 April 2014
Cited by 7 | PDF Full-text (580 KB) | HTML Full-text | XML Full-text
Abstract
Fusarium head blight (FHB) is an important disease of wheat worldwide caused mainly by Fusarium graminearum (syn. Gibberella zeae). This fungus can be highly aggressive and can produce several mycotoxins such as deoxynivalenol (DON), a well known harmful metabolite for humans, [...] Read more.
Fusarium head blight (FHB) is an important disease of wheat worldwide caused mainly by Fusarium graminearum (syn. Gibberella zeae). This fungus can be highly aggressive and can produce several mycotoxins such as deoxynivalenol (DON), a well known harmful metabolite for humans, animals, and plants. The fungus can survive overwinter on wheat residues and on the soil, and can usually attack the wheat plant at their point of flowering, being able to infect the heads and to contaminate the kernels at the maturity. Contaminated kernels can be sometimes used as seeds for the cultivation of the following year. Poor knowledge on the ability of the strains of F. graminearum occurring on wheat seeds to be transmitted to the plant and to contribute to the final DON contamination of kernels is available. Therefore, this study had the goals of evaluating: (a) the capability of F. graminearum causing FHB of wheat to be transmitted from the seeds or soil to the kernels at maturity and the progress of the fungus within the plant at different growth stages; (b) the levels of DON contamination in both plant tissues and kernels. The study has been carried out for two years in a climatic chamber. The F. gramineraum strain selected for the inoculation was followed within the plant by using Vegetative Compatibility technique, and quantified by Real-Time PCR. Chemical analyses of DON were carried out by using immunoaffinity cleanup and HPLC/UV/DAD. The study showed that F. graminearum originated from seeds or soil can grow systemically in the plant tissues, with the exception of kernels and heads. There seems to be a barrier that inhibits the colonization of the heads by the fungus. High levels of DON and F. graminearum were found in crowns, stems, and straw, whereas low levels of DON and no detectable levels of F. graminearum were found in both heads and kernels. Finally, in all parts of the plant (heads, crowns, and stems at milk and vitreous ripening stages, and straw at vitreous ripening), also the accumulation of significant quantities of DON-3-glucoside (DON-3G), a product of DON glycosylation, was detected, with decreasing levels in straw, crown, stems and kernels. The presence of DON and DON-3G in heads and kernels without the occurrence of F. graminearum may be explained by their water solubility that could facilitate their translocation from stem to heads and kernels. The presence of DON-3G at levels 23 times higher than DON in the heads at milk stage without the occurrence of F. graminearum may indicate that an active glycosylation of DON also occurs in the head tissues. Finally, the high levels of DON accumulated in straws are worrisome since they represent additional sources of mycotoxin for livestock. Full article
Open AccessArticle An N-Terminal Fragment of Yeast Ribosomal Protein L3 Inhibits the Cytotoxicity of Pokeweed Antiviral Protein in Saccharomyces cerevisiae
Toxins 2014, 6(4), 1349-1361; doi:10.3390/toxins6041349
Received: 24 February 2014 / Revised: 29 March 2014 / Accepted: 2 April 2014 / Published: 11 April 2014
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Abstract
We have previously shown that ribosomal protein L3 is required for pokeweed antiviral protein (PAP), a type I ribosome inactivating protein, to bind to ribosomes and depurinate the α-sarcin/ricin loop (SRL) in yeast. Co-expression of the N-terminal 99 amino acids of [...] Read more.
We have previously shown that ribosomal protein L3 is required for pokeweed antiviral protein (PAP), a type I ribosome inactivating protein, to bind to ribosomes and depurinate the α-sarcin/ricin loop (SRL) in yeast. Co-expression of the N-terminal 99 amino acids of yeast L3 (L3Δ99) with PAP in transgenic tobacco plants completely abolished the toxicity of PAP. In this study, we investigated the interaction between PAP and L3Δ99 in Saccharomyces cerevisiae. Yeast cells co-transformed with PAP and L3Δ99 showed markedly reduced growth inhibition and reduced rRNA depurination by PAP, compared to cells transformed with PAP alone. Co-transformation of yeast with PAP and L3Δ21 corresponding to the highly conserved N-terminal 21 amino acids of L3Δ99, reduced the cytotoxicity of PAP. PAP mRNA and protein levels were elevated and L3Δ99 or L3Δ21 mRNA and protein levels were reduced in yeast co-transformed with PAP and L3Δ99 or with PAP and L3Δ21, respectively. PAP interacted with L3Δ21 in yeast cells in vivo and by Biacore analysis in vitro, suggesting that the interaction between L3Δ21 and PAP may inhibit PAP-mediated depurination of the SRL, leading to a reduction in the cytotoxicity of PAP. Full article
(This article belongs to the Section Bacterial Toxins)
Open AccessArticle Trophic State and Toxic Cyanobacteria Density in Optimization Modeling of Multi-Reservoir Water Resource Systems
Toxins 2014, 6(4), 1366-1384; doi:10.3390/toxins6041366
Received: 8 January 2014 / Revised: 23 March 2014 / Accepted: 24 March 2014 / Published: 22 April 2014
Cited by 3 | PDF Full-text (998 KB) | HTML Full-text | XML Full-text
Abstract
The definition of a synthetic index for classifying the quality of water bodies is a key aspect in integrated planning and management of water resource systems. In previous works [1,2], a water system optimization modeling approach that requires a single quality index [...] Read more.
The definition of a synthetic index for classifying the quality of water bodies is a key aspect in integrated planning and management of water resource systems. In previous works [1,2], a water system optimization modeling approach that requires a single quality index for stored water in reservoirs has been applied to a complex multi-reservoir system. Considering the same modeling field, this paper presents an improved quality index estimated both on the basis of the overall trophic state of the water body and on the basis of the density values of the most potentially toxic Cyanobacteria. The implementation of the index into the optimization model makes it possible to reproduce the conditions limiting water use due to excessive nutrient enrichment in the water body and to the health hazard linked to toxic blooms. The analysis of an extended limnological database (1996–2012) in four reservoirs of the Flumendosa-Campidano system (Sardinia, Italy) provides useful insights into the strengths and limitations of the proposed synthetic index. Full article
(This article belongs to the collection Marine and Freshwater Toxins)
Open AccessArticle Recombinant Clostridium difficile Toxin Fragments as Carrier Protein for PSII Surface Polysaccharide Preserve Their Neutralizing Activity
Toxins 2014, 6(4), 1385-1396; doi:10.3390/toxins6041385
Received: 6 February 2014 / Revised: 6 March 2014 / Accepted: 9 April 2014 / Published: 22 April 2014
Cited by 4 | PDF Full-text (471 KB) | HTML Full-text | XML Full-text
Abstract
Clostridium difficile is a Gram-positive bacterium and is the most commonly diagnosed cause of hospital-associated and antimicrobial-associated diarrhea. Despite the emergence of epidemic C. difficile strains having led to an increase in the incidence of the disease, a vaccine against this pathogen [...] Read more.
Clostridium difficile is a Gram-positive bacterium and is the most commonly diagnosed cause of hospital-associated and antimicrobial-associated diarrhea. Despite the emergence of epidemic C. difficile strains having led to an increase in the incidence of the disease, a vaccine against this pathogen is not currently available. C. difficile strains produce two main toxins (TcdA and TcdB) and express three highly complex cell-surface polysaccharides (PSI, PSII and PSIII). PSII is the more abundantly expressed by most C. difficile ribotypes offering the opportunity of the development of a carbohydrate-based vaccine. In this paper, we evaluate the efficacy, in naive mice model, of PSII glycoconjugates where recombinant toxins A and B fragments (TcdA_B2 and TcdB_GT respectively) have been used as carriers. Both glycoconjugates elicited IgG titers anti-PSII although only the TcdB_GT conjugate induced a response comparable to that obtained with CRM197. Moreover, TcdA_B2 and TcdB_GT conjugated to PSII retained the ability to elicit IgG with neutralizing activity against the respective toxins. These results are a crucial proof of concept for the development of glycoconjugate vaccines against C. difficile infection (CDI) that combine different C. difficile antigens to potentially prevent bacterial colonization of the gut and neutralize toxin activity. Full article
(This article belongs to the Special Issue Toxin-Antibody Interactions)
Open AccessArticle Immunogenicity of a West Nile Virus DIII-Cholera Toxin A2/B Chimera after Intranasal Delivery
Toxins 2014, 6(4), 1397-1418; doi:10.3390/toxins6041397
Received: 16 November 2013 / Revised: 9 April 2014 / Accepted: 9 April 2014 / Published: 22 April 2014
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Abstract
West Nile virus (WNV) causes potentially fatal neuroinvasive disease and persists at endemic levels in many parts of the world. Despite advances in our understanding of WNV pathogenesis, there remains a significant need for a human vaccine. The domain III (DIII) region [...] Read more.
West Nile virus (WNV) causes potentially fatal neuroinvasive disease and persists at endemic levels in many parts of the world. Despite advances in our understanding of WNV pathogenesis, there remains a significant need for a human vaccine. The domain III (DIII) region of the WNV envelope protein contains epitopes that are the target of neutralizing antibodies. We have constructed a chimeric fusion of the non-toxic cholera toxin (CT) CTA2/B domains to DIII for investigation as a novel mucosally-delivered WNV vaccine. Purification and assembly of the chimera, as well as receptor-binding and antigen delivery, were verified by western blot, GM1 ELISA and confocal microscopy. Groups of BALB/c mice were immunized intranasally with DIII-CTA2/B, DIII, DIII mixed with CTA2/B, or CTA2/B control, and boosted at 10 days. Analysis of serum IgG after 14 and 45 days revealed that mucosal immunization with DIII-CTA2/B induced significant DIII-specific humoral immunity and drove isotype switching to IgG2a. The DIII-CTA2/B chimera also induced antigen-specific IgM and IgA responses. Bactericidal assays indicate that the DIII-CTA2/B immunized mice produced DIII-specific antibodies that can trigger complement-mediated killing. A dose escalation resulted in increased DIII-specific serum IgG titers on day 45. DIII antigen alone, in the absence of adjuvant, also induced significant systemic responses after intranasal delivery. Our results indicate that the DIII-CTA2/B chimera is immunogenic after intranasal delivery and merits further investigation as a novel WNV vaccine candidate. Full article
(This article belongs to the Section Bacterial Toxins)
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Open AccessArticle Recombinant Expression and Functional Characterization of Martentoxin: A Selective Inhibitor for BK Channel (α + β4)
Toxins 2014, 6(4), 1419-1433; doi:10.3390/toxins6041419
Received: 18 February 2014 / Revised: 27 March 2014 / Accepted: 1 April 2014 / Published: 22 April 2014
Cited by 5 | PDF Full-text (1505 KB) | HTML Full-text | XML Full-text
Abstract
Martentoxin (MarTX), a 37-residue peptide purified from the venom of East-Asian scorpion (Buthus martensi Karsch), was capable of blocking large-conductance Ca2+-activated K+ (BK) channels. Here, we report an effective expression and purification approach for this toxin. The cDNA [...] Read more.
Martentoxin (MarTX), a 37-residue peptide purified from the venom of East-Asian scorpion (Buthus martensi Karsch), was capable of blocking large-conductance Ca2+-activated K+ (BK) channels. Here, we report an effective expression and purification approach for this toxin. The cDNA encoding martentoxin was expressed by the prokaryotic expression system pGEX-4T-3 which was added an enterokinase cleavage site by PCR. The fusion protein (GST-rMarTX) was digested by enterokinase to release hetero-expressed toxin and further purified via reverse-phase HPLC. The molecular weight of the hetero-expressed rMarTX was 4059.06 Da, which is identical to that of the natural peptide isolated from scorpion venom. Functional characterization through whole-cell patch clamp showed that rMarTX selectively and potently inhibited the currents of neuronal BK channels (α + β4) (IC50 = 186 nM), partly inhibited mKv1.3, but hardly having any significant effect on hKv4.2 and hKv3.1a even at 10 μM. Successful expression of martentoxin lays basis for further studies of structure-function relationship underlying martentoxin or other potassium-channel specific blockers. Full article
(This article belongs to the Special Issue Ion Channel Neurotoxins)
Open AccessArticle Comments on Environmental and Sanitary Aspects of the Scorpionism by Tityus trivittatus in Buenos Aires City, Argentina
Toxins 2014, 6(4), 1434-1452; doi:10.3390/toxins6041434
Received: 20 January 2014 / Revised: 21 March 2014 / Accepted: 3 April 2014 / Published: 22 April 2014
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Abstract
Deaths by venomous animals are medical emergencies that can lead to death and thus constitute sanitary problems in some regions of the world. In the South of America, the accidents by these animals are a common sanitary problem especially in warm, tropical [...] Read more.
Deaths by venomous animals are medical emergencies that can lead to death and thus constitute sanitary problems in some regions of the world. In the South of America, the accidents by these animals are a common sanitary problem especially in warm, tropical or subtropical regions, related with rural work in several countries. Argentina is located in the extreme South of South America and a minor part of the continental surface is in tropical or subtropical regions, where most of the accidents by venomous animals happen. However, in the big cities in the center and South of the country, with no relation to rural work, scorpionism, mostly due to the synanthropic and facultative parthenogenetic scorpion Tityus trivittatus, has become a sanitary problem in the last few decades. This scorpion is present in the biggest cities of Argentina and in the last decades has killed over 20 children in provinces of the center and north of the country, mostly in big cities. In addition, it seems that this species is growing and spreading in new regions of the cities. In this revision, some characteristics of this scorpion regarding its habitat, spreading in Buenos Aires city, combat measures and available treatments are discussed. Full article
(This article belongs to the Special Issue Scorpion Toxins)
Open AccessArticle The Use of Recently Developed Histochemical Markers for Localizing Neurotoxicant Induced Regional Brain Pathologies
Toxins 2014, 6(4), 1453-1470; doi:10.3390/toxins6041453
Received: 7 March 2014 / Revised: 16 April 2014 / Accepted: 17 April 2014 / Published: 24 April 2014
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Abstract
Neuronal and vascular brain components are interrelated morphologically, physiologically and developmentally. Due to this close interrelationship, it is often difficult to understand the cause and effect relationship between neuronal vs. vascular dysfunction and pathology. This review will discuss four of the more [...] Read more.
Neuronal and vascular brain components are interrelated morphologically, physiologically and developmentally. Due to this close interrelationship, it is often difficult to understand the cause and effect relationship between neuronal vs. vascular dysfunction and pathology. This review will discuss four of the more promising recent developments for detecting vascular pathology, and will compare them with the labeling pattern seen with markers of glial and neuronal pathology; following exposure to well characterized neurotoxicants. To detect the vascular dysfunction in the brain, we recently developed a Fluoro-Turquoise gelatin conjugate (FT-gel), a fluorescent probe that helps to delineate between healthy vs. sclerotic vessels. Similarly, we have investigated the potential for Fluoro-Gold to label in vivo all the endothelial cells in the brain as they co-localize with RECA, an endothelial cell marker. We have also developed Amylo-Glo, a fluorescent tracer that can detect neurotoxic A-beta aggregates in the brain. In this article, we will discuss the potential use of these novel histochemical markers to study the neurotoxicant induced brain. We will also discuss neurovascular strategies that may offer novel therapeutic opportunities for neurodegenerative disorders. Full article
(This article belongs to the collection Marine and Freshwater Toxins)

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Open AccessReview Bacillus thuringiensis subsp. israelensis and Its Dipteran-Specific Toxins
Toxins 2014, 6(4), 1222-1243; doi:10.3390/toxins6041222
Received: 28 January 2014 / Revised: 10 March 2014 / Accepted: 14 March 2014 / Published: 28 March 2014
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Abstract
Bacillus thuringiensis subsp. israelensis (Bti) is the first Bacillus thuringiensis to be found and used as an effective biological control agent against larvae of many mosquito and black fly species around the world. Its larvicidal activity resides in four major [...] Read more.
Bacillus thuringiensis subsp. israelensis (Bti) is the first Bacillus thuringiensis to be found and used as an effective biological control agent against larvae of many mosquito and black fly species around the world. Its larvicidal activity resides in four major (of 134, 128, 72 and 27 kDa) and at least two minor (of 78 and 29 kDa) polypeptides encoded respectively by cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba, all mapped on the 128 kb plasmid known as pBtoxis. These six δ-endotoxins form a complex parasporal crystalline body with remarkably high, specific and different toxicities to Aedes, Culex and Anopheles larvae. Cry toxins are composed of three domains (perforating domain I and receptor binding II and III) and create cation-selective channels, whereas Cyts are composed of one domain that acts as well as a detergent-like membrane perforator. Despite the low toxicities of Cyt1Aa and Cyt2Ba alone against exposed larvae, they are highly synergistic with the Cry toxins and hence their combinations prevent emergence of resistance in the targets. The lack of significant levels of resistance in field mosquito populations treated for decades with Bti-bioinsecticide suggests that this bacterium will be an effective biocontrol agent for years to come. Full article
(This article belongs to the Special Issue <i>Bacillus thuringiensis</i> Toxins)
Open AccessReview Tracing Monotreme Venom Evolution in the Genomics Era
Toxins 2014, 6(4), 1260-1273; doi:10.3390/toxins6041260
Received: 28 January 2014 / Revised: 17 March 2014 / Accepted: 27 March 2014 / Published: 2 April 2014
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Abstract
The monotremes (platypuses and echidnas) represent one of only four extant venomous mammalian lineages. Until recently, monotreme venom was poorly understood. However, the availability of the platypus genome and increasingly sophisticated genomic tools has allowed us to characterize platypus toxins, and provides [...] Read more.
The monotremes (platypuses and echidnas) represent one of only four extant venomous mammalian lineages. Until recently, monotreme venom was poorly understood. However, the availability of the platypus genome and increasingly sophisticated genomic tools has allowed us to characterize platypus toxins, and provides a means of reconstructing the evolutionary history of monotreme venom. Here we review the physiology of platypus and echidna crural (venom) systems as well as pharmacological and genomic studies of monotreme toxins. Further, we synthesize current ideas about the evolution of the venom system, which in the platypus is likely to have been retained from a venomous ancestor, whilst being lost in the echidnas. We also outline several research directions and outstanding questions that would be productive to address in future research. An improved characterization of mammalian venoms will not only yield new toxins with potential therapeutic uses, but will also aid in our understanding of the way that this unusual trait evolves. Full article
(This article belongs to the collection Evolution of Venom Systems)
Figures

Open AccessReview Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins
Toxins 2014, 6(4), 1325-1348; doi:10.3390/toxins6041325
Received: 10 February 2014 / Revised: 10 March 2014 / Accepted: 28 March 2014 / Published: 11 April 2014
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Abstract
Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding [...] Read more.
Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced. Full article
(This article belongs to the Special Issue Toxin-Antibody Interactions)

Other

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Open AccessComment Comment on Hurley, J.C. Towards Clinical Application of Anti-endotoxin Antibodies; A Re-Appraisal of the Disconnect. Toxins 2013, 5, 2589-2620
Toxins 2014, 6(4), 1362-1363; doi:10.3390/toxins6041362
Received: 20 March 2014 / Accepted: 9 April 2014 / Published: 11 April 2014
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Abstract I have read with interest James C. Hurley’s very good review [1]. Full article
Open AccessReply Response to John G. Brock-Utne. Comment on Hurley, J.C. Towards Clinical Application of Anti-endotoxin Antibodies; A Re-Appraisal of the Disconnect
Toxins 2014, 6(4), 1364-1365; doi:10.3390/toxins6041364
Received: 8 April 2014 / Accepted: 9 April 2014 / Published: 11 April 2014
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Abstract I appreciate the thoughtful comments from Dr. Brock-Utne [1] on my recent review of the disconnect between animal studies and clinical experience with anti-endotoxemia therapies [2]. Full article

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