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Toxins, Volume 6, Issue 8 (August 2014), Pages 2210-2593

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Editorial

Jump to: Research, Review

Open AccessEditorial Increased Extracellular ATP: An Omen of Bacterial RTX Toxin-Induced Hemolysis?
Toxins 2014, 6(8), 2432-2434; doi:10.3390/toxins6082432
Received: 17 June 2014 / Revised: 6 August 2014 / Accepted: 11 August 2014 / Published: 15 August 2014
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Abstract
Bacterial infection is a major threat to human health. Although pathogenic bacteria vary in their virulence, it has been recognized that many pathogenic bacteria share common mechanisms when attacking host cells and tissues. Some pathogenic bacteria synthesize and secrete polysaccharides to form an
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Bacterial infection is a major threat to human health. Although pathogenic bacteria vary in their virulence, it has been recognized that many pathogenic bacteria share common mechanisms when attacking host cells and tissues. Some pathogenic bacteria synthesize and secrete polysaccharides to form an extracellular capsule. Capsules serve as virulence determinants by multiple mechanisms including facilitation of bacterial adherence, evasion of the immune response, and antibiotic resistance [1]. Moreover, to the exterior of bacterial plasma membranes are certain toxic components (e.g., lipopolysaccharide (LPS) in Gram-negative bacteria, and peptidoglycan fragments and teichoic acids in Gram-positive bacteria) that play key roles in causing bacterial septic shock or multiple organ dysfunction [2]. Significantly, bacteria may secrete proteinaceous or non-proteinaceous molecules, namely exotoxins, capable of directly destroying host cells. The Repeat-in-Toxin (RTX) family is a group of virulence-associated exotoxins that are generated by Gram-negative bacteria and are noted for their ability to form pores on the membrane of host cells including leukocytes [3]. Despite the intense effort that has been input into investigating the interaction between RTX toxins and host cells during bacterial infection, our understanding of how RTX toxins insert into host cell membranes, and in turn, how host cells respond to the challenge of these toxins remains very limited. [...] Full article
(This article belongs to the Section Bacterial Toxins)

Research

Jump to: Editorial, Review

Open AccessArticle Cancer Cell Growth Inhibitory Effect of Bee Venom via Increase of Death Receptor 3 Expression and Inactivation of NF-kappa B in NSCLC Cells
Toxins 2014, 6(8), 2210-2228; doi:10.3390/toxins6082210
Received: 8 April 2014 / Revised: 16 June 2014 / Accepted: 18 July 2014 / Published: 25 July 2014
Cited by 11 | PDF Full-text (1630 KB) | HTML Full-text | XML Full-text
Abstract
Our previous findings have demonstrated that bee venom (BV) has anti-cancer activity in several cancer cells. However, the effects of BV on lung cancer cell growth have not been reported. Cell viability was determined with trypan blue uptake, soft agar formation as well
[...] Read more.
Our previous findings have demonstrated that bee venom (BV) has anti-cancer activity in several cancer cells. However, the effects of BV on lung cancer cell growth have not been reported. Cell viability was determined with trypan blue uptake, soft agar formation as well as DAPI and TUNEL assay. Cell death related protein expression was determined with Western blotting. An EMSA was used for nuclear factor kappaB (NF-κB) activity assay. BV (1–5 μg/mL) inhibited growth of lung cancer cells by induction of apoptosis in a dose dependent manner in lung cancer cell lines A549 and NCI-H460. Consistent with apoptotic cell death, expression of DR3 and DR6 was significantly increased. However, deletion of DRs by small interfering RNA significantly reversed BV induced cell growth inhibitory effects. Expression of pro-apoptotic proteins (caspase-3 and Bax) was concomitantly increased, but the NF-κB activity and expression of Bcl-2 were inhibited. A combination treatment of tumor necrosis factor (TNF)-like weak inducer of apoptosis, TNF-related apoptosis-inducing ligand, docetaxel and cisplatin, with BV synergistically inhibited both A549 and NCI-H460 lung cancer cell growth with further down regulation of NF-κB activity. These results show that BV induces apoptotic cell death in lung cancer cells through the enhancement of DR3 expression and inhibition of NF-κB pathway. Full article
(This article belongs to the Section Animal Venoms)
Open AccessArticle Occurrence of Pre- and Post-Harvest Mycotoxins and Other Secondary Metabolites in Danish Maize Silage
Toxins 2014, 6(8), 2256-2269; doi:10.3390/toxins6082256
Received: 9 June 2014 / Revised: 4 July 2014 / Accepted: 15 July 2014 / Published: 31 July 2014
Cited by 13 | PDF Full-text (586 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Maize silage is a widely used feed product for cattle worldwide, which may be contaminated with mycotoxins, pre- and post-harvest. This concerns both farmers and consumers. To assess the exposure of Danish cattle to mycotoxins from maize silage, 99 samples of whole-crop maize
[...] Read more.
Maize silage is a widely used feed product for cattle worldwide, which may be contaminated with mycotoxins, pre- and post-harvest. This concerns both farmers and consumers. To assess the exposure of Danish cattle to mycotoxins from maize silage, 99 samples of whole-crop maize (ensiled and un-ensiled) were analyzed for their contents of 27 mycotoxins and other secondary fungal metabolites by liquid chromatography-tandem mass spectrometry. The method specifically targets the majority of common pre- and post-harvest fungi associated with maize silage in Denmark. Sixty-one samples contained one or more of the 27 analytes in detectable concentrations. The most common mycotoxins were zearalenone, enniatin B nivalenol and andrastin A, found in 34%, 28%, 16% and 15% of the samples, respectively. None of the samples contained mycotoxins above the EU recommended maximum concentrations for Fusarium toxins in cereal-based roughage. Thus, the present study does not indicate that Danish maize silage in general is a cause of acute single mycotoxin intoxications in cattle. However, 31 of the samples contained multiple analytes; two samples as much as seven different fungal metabolites. Feed rations with maize silage may therefore contain complex mixtures of fungal secondary metabolites with unknown biological activity. This emphasizes the need for a thorough examination of the effects of chronic exposure and possible synergistic effects. Full article
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Open AccessArticle Sub-Emetic Toxicity of Bacillus cereus Toxin Cereulide on Cultured Human Enterocyte-Like Caco-2 Cells
Toxins 2014, 6(8), 2270-2290; doi:10.3390/toxins6082270
Received: 2 March 2014 / Revised: 18 July 2014 / Accepted: 22 July 2014 / Published: 4 August 2014
Cited by 6 | PDF Full-text (1084 KB) | HTML Full-text | XML Full-text
Abstract
Cereulide (CER) intoxication occurs at relatively high doses of 8 µg/kg body weight. Recent research demonstrated a wide prevalence of low concentrations of CER in rice and pasta dishes. However, the impact of exposure to low doses of CER has not been studied
[...] Read more.
Cereulide (CER) intoxication occurs at relatively high doses of 8 µg/kg body weight. Recent research demonstrated a wide prevalence of low concentrations of CER in rice and pasta dishes. However, the impact of exposure to low doses of CER has not been studied before. In this research, we investigated the effect of low concentrations of CER on the behavior of intestinal cells using the Caco-2 cell line. The MTT (mitochondrial 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and the SRB (sulforhodamine B) reactions were used to measure the mitochondrial activity and cellular protein content, respectively. Both assays showed that differentiated Caco-2 cells were sensitive to low concentrations of CER (in a MTT reaction of 1 ng/mL after three days of treatment; in an SRB reaction of 0.125 ng/mL after three days of treatment). Cell counts revealed that cells were released from the differentiated monolayer at 0.5 ng/mL of CER. Additionally, 0.5 and 2 ng/mL of CER increased the lactate presence in the cell culture medium. Proteomic data showed that CER at a concentration of 1 ng/mL led to a significant decrease in energy managing and H2O2 detoxification proteins and to an increase in cell death markers. This is amongst the first reports to describe the influence of sub-emetic concentrations of CER on a differentiated intestinal monolayer model showing that low doses may induce an altered enterocyte metabolism and membrane integrity. Full article
(This article belongs to the Section Bacterial Toxins)
Open AccessArticle Genetic Relationships, Carbendazim Sensitivity and Mycotoxin Production of the Fusarium Graminearum Populations from Maize, Wheat and Rice in Eastern China
Toxins 2014, 6(8), 2291-2309; doi:10.3390/toxins6082291
Received: 23 June 2014 / Revised: 18 July 2014 / Accepted: 21 July 2014 / Published: 4 August 2014
Cited by 7 | PDF Full-text (1303 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Members of the Fusarium graminearum species complex (FGSC) are important pathogens on wheat, maize, barley, and rice in China. Harvested grains are often contaminated by mycotoxins, such as the trichothecene nivalenol (NIV) and deoxynivalenol (DON) and the estrogenic mycotoxin zearalenone (ZEN), which is
[...] Read more.
Members of the Fusarium graminearum species complex (FGSC) are important pathogens on wheat, maize, barley, and rice in China. Harvested grains are often contaminated by mycotoxins, such as the trichothecene nivalenol (NIV) and deoxynivalenol (DON) and the estrogenic mycotoxin zearalenone (ZEN), which is a big threat to humans and animals. In this study, 97 isolates were collected from maize, wheat, and rice in Jiangsu and Anhui provinces in 2013 and characterized by species- and chemotype-specific PCR. F. graminearum sensu stricto (s. str.) was predominant on maize, while most of the isolates collected from rice and wheat were identified as F. asiaticum. Fusarium isolates from three hosts varied in trichothecene chemotypes. The 3-acetyldeoxynivalenol (3ADON) chemotype predominated on wheat and rice population, while 15ADON was prevailing in the remaining isolates. Sequence analysis of the translation elongation factor 1α and trichodiene synthase indicated the accuracy of the above conclusion. Additionally, phylogenetic analysis suggested four groups with strong correlation with species, chemotype, and host. These isolates were also evaluated for their sensitivity to carbendazim and mycotoxins production. The maize population was less sensitive than the other two. The DON levels were similar in three populations, while those isolates on maize produced more ZEN. More DON was produced in carbendazim resistant strains than sensitive ones, but it seemed that carbendazim resistance had no effect on ZEN production in wheat culture. Full article
Open AccessArticle Profiling of Amatoxins and Phallotoxins in the Genus Lepiota by Liquid Chromatography Combined with UV Absorbance and Mass Spectrometry
Toxins 2014, 6(8), 2336-2347; doi:10.3390/toxins6082336
Received: 27 March 2014 / Revised: 25 July 2014 / Accepted: 29 July 2014 / Published: 5 August 2014
Cited by 9 | PDF Full-text (972 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Species in the mushroom genus Lepiota can cause fatal mushroom poisonings due to their content of amatoxins such as α-amanitin. Previous studies of the toxin composition of poisonous Lepiota species relied on analytical methods of low sensitivity or resolution. Using liquid chromatography coupled
[...] Read more.
Species in the mushroom genus Lepiota can cause fatal mushroom poisonings due to their content of amatoxins such as α-amanitin. Previous studies of the toxin composition of poisonous Lepiota species relied on analytical methods of low sensitivity or resolution. Using liquid chromatography coupled to UV absorbance and mass spectrometry, we analyzed the spectrum of peptide toxins present in six Italian species of Lepiota, including multiple samples of three of them collected in different locations. Field taxonomic identifications were confirmed by sequencing of the internal transcribed spacer (ITS) regions. For comparison, we also analyzed specimens of Amanita phalloides from Italy and California, a specimen of A. virosa from Italy, and a laboratory-grown sample of Galerina marginata. α-Amanitin, β-amanitin, amanin, and amaninamide were detected in all samples of L. brunneoincarnata, and α-amanitin and γ-amanitin were detected in all samples of L. josserandii. Phallotoxins were not detected in either species. No amatoxins or phallotoxins were detected in L. clypeolaria, L. cristata, L. echinacea, or L. magnispora. The Italian and California isolates of A. phalloides had similar profiles of amatoxins and phallotoxins, although the California isolate contained more β-amanitin relative to α-amanitin. Amaninamide was detected only in A. virosa. Full article
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Open AccessArticle Is Increased Susceptibility to Balkan Endemic Nephropathy in Carriers of Common GSTA1 (*A/*B) Polymorphism Linked with the Catalytic Role of GSTA1 in Ochratoxin A Biotransformation? Serbian Case Control Study and In Silico Analysis
Toxins 2014, 6(8), 2348-2362; doi:10.3390/toxins6082348
Received: 16 June 2014 / Revised: 28 July 2014 / Accepted: 30 July 2014 / Published: 8 August 2014
Cited by 5 | PDF Full-text (874 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Although recent data suggest aristolochic acid as a putative cause of Balkan endemic nephropathy (BEN), evidence also exists in favor of ochratoxin A (OTA) exposure as risk factor for the disease. The potential role of xenobiotic metabolizing enzymes, such as the glutathione transferases
[...] Read more.
Although recent data suggest aristolochic acid as a putative cause of Balkan endemic nephropathy (BEN), evidence also exists in favor of ochratoxin A (OTA) exposure as risk factor for the disease. The potential role of xenobiotic metabolizing enzymes, such as the glutathione transferases (GSTs), in OTA biotransformation is based on OTA glutathione adducts (OTHQ-SG and OTB-SG) in blood and urine of BEN patients. We aimed to analyze the association between common GSTA1, GSTM1, GSTT1, and GSTP1 polymorphisms and BEN susceptibility, and thereafter performed an in silico simulation of particular GST enzymes potentially involved in OTA transformations. GSTA1, GSTM1, GSTT1 and GSTP1 genotypes were determined in 207 BEN patients and 138 non-BEN healthy individuals from endemic regions by polymerase chain reaction (PCR). Molecular modeling in silico was performed for GSTA1 protein. Among the GST polymorphisms tested, only GSTA1 was significantly associated with a higher risk of BEN. Namely, carriers of the GSTA1*B gene variant, associated with lower transcriptional activation, were at a 1.6-fold higher BEN risk than those carrying the homozygous GSTA1*A/*A genotype (OR = 1.6; p = 0.037). In in silico modeling, we found four structures, two OTB-SG and two OTHQ-SG, bound in a GSTA1 monomer. We found that GSTA1 polymorphism was associated with increased risk of BEN, and suggested, according to the in silico simulation, that GSTA1-1 might be involved in catalyzing the formation of OTHQ-SG and OTB-SG conjugates. Full article
Open AccessArticle Synthesis and Analgesic Effects of μ-TRTX-Hhn1b on Models of Inflammatory and Neuropathic Pain
Toxins 2014, 6(8), 2363-2378; doi:10.3390/toxins6082363
Received: 28 March 2014 / Revised: 17 July 2014 / Accepted: 18 July 2014 / Published: 13 August 2014
Cited by 2 | PDF Full-text (1126 KB) | HTML Full-text | XML Full-text
Abstract
μ-TRTX-Hhn1b (HNTX-IV) is a 35-amino acid peptide isolated from the venom of the spider, Ornithoctonus hainana. It inhibits voltage-gated sodium channel Nav1.7, which has been considered as a therapeutic target for pain. The goal of the present study is to elucidate the
[...] Read more.
μ-TRTX-Hhn1b (HNTX-IV) is a 35-amino acid peptide isolated from the venom of the spider, Ornithoctonus hainana. It inhibits voltage-gated sodium channel Nav1.7, which has been considered as a therapeutic target for pain. The goal of the present study is to elucidate the analgesic effects of synthetic μ-TRTX-Hhn1b on animal models of pain. The peptide was first synthesized and then successfully refolded/oxidized. The synthetic peptide had the same inhibitory effect on human Nav1.7 current transiently expressed in HEK 293 cells as the native toxin. Furthermore, the analgesic potentials of the synthetic peptide were examined on models of inflammatory pain and neuropathic pain. μ-TRTX-Hhn1b produced an efficient reversal of acute nociceptive pain in the abdominal constriction model, and significantly reduced the pain scores over the 40-min period in the formalin model. The efficiency of μ-TRTX-Hhn1b on both models was equivalent to that of morphine. In the spinal nerve model, the reversal effect of μ-TRTX-Hhn1b on allodynia was longer and higher than mexiletine. These results demonstrated that μ-TRTX-Hhn1b efficiently alleviated acute inflammatory pain and chronic neuropathic pain in animals and provided an attractive template for further clinical analgesic drug design. Full article
(This article belongs to the Special Issue Ion Channel Neurotoxins)
Open AccessArticle Bioreactor Study Employing Bacteria with Enhanced Activity toward Cyanobacterial Toxins Microcystins
Toxins 2014, 6(8), 2379-2392; doi:10.3390/toxins6082379
Received: 3 July 2014 / Revised: 24 July 2014 / Accepted: 25 July 2014 / Published: 13 August 2014
Cited by 3 | PDF Full-text (845 KB) | HTML Full-text | XML Full-text
Abstract
An important aim of white (grey) biotechnology is bioremediation, where microbes are employed to remove unwanted chemicals. Microcystins (MCs) and other cyanobacterial toxins are not industrial or agricultural pollutants; however, their occurrence as a consequence of human activity and water reservoir eutrophication is
[...] Read more.
An important aim of white (grey) biotechnology is bioremediation, where microbes are employed to remove unwanted chemicals. Microcystins (MCs) and other cyanobacterial toxins are not industrial or agricultural pollutants; however, their occurrence as a consequence of human activity and water reservoir eutrophication is regarded as anthropogenic. Microbial degradation of microcystins is suggested as an alternative to chemical and physical methods of their elimination. This paper describes a possible technique of the practical application of the biodegradation process. The idea relies on the utilization of bacteria with a significantly enhanced MC-degradation ability (in comparison with wild strains). The cells of an Escherichia coli laboratory strain expressing microcystinase (MlrA) responsible for the detoxification of MCs were immobilized in alginate beads. The degradation potency of the tested bioreactors was monitored by HPLC detection of linear microcystin LR (MC-LR) as the MlrA degradation product. An open system based on a column filled with alginate-entrapped cells was shown to operate more efficiently than a closed system (alginate beads shaken in a glass container). The maximal degradation rate calculated per one liter of carrier was 219.9 µg h−1 of degraded MC-LR. A comparison of the efficiency of the described system with other biological and chemo-physical proposals suggests that this new idea presents several advantages and is worth investigating in future studies. Full article
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Open AccessArticle Protective Antigen-Specific Memory B Cells Persist Years after Anthrax Vaccination and Correlate with Humoral Immunity
Toxins 2014, 6(8), 2424-2431; doi:10.3390/toxins6082424
Received: 4 June 2014 / Revised: 11 July 2014 / Accepted: 17 July 2014 / Published: 13 August 2014
Cited by 1 | PDF Full-text (290 KB) | HTML Full-text | XML Full-text
Abstract
Anthrax Vaccine Adsorbed (AVA) generates short-lived protective antigen (PA) specific IgG that correlates with in vitro toxin neutralization and protection from Bacillus anthracis challenge. Animal studies suggest that when PA-specific IgG has waned, survival after spore challenge correlates with an activation of PA-specific
[...] Read more.
Anthrax Vaccine Adsorbed (AVA) generates short-lived protective antigen (PA) specific IgG that correlates with in vitro toxin neutralization and protection from Bacillus anthracis challenge. Animal studies suggest that when PA-specific IgG has waned, survival after spore challenge correlates with an activation of PA-specific memory B cells. Here, we characterize the quantity and the longevity of AVA-induced memory B cell responses in humans. Peripheral blood mononuclear cells (PBMCs) from individuals vaccinated ≥3 times with AVA (n = 50) were collected early (3–6 months, n = 27) or late after their last vaccination (2–5 years, n = 23), pan-stimulated, and assayed by ELISPOT for total and PA-specific memory B cells differentiated into antibody secreting cells (ASCs). PA-specific ASC percentages ranged from 0.02% to 6.25% (median: 1.57%) and did not differ between early and late post-vaccination individuals. PA-specific ASC percentages correlated with plasma PA-specific IgG (r = 0.42, p = 0.03) and toxin neutralization (r = 0.52, p = 0.003) early post vaccination. PA-specific ASC percentages correlated with supernatant anti-PA both early (r = 0.60, p = 0.001) and late post vaccination (r = 0.71, p < 0.0001). These data suggest PA-specific memory B cell responses are long-lived and can be estimated after recent vaccination by the magnitude and neutralization capacity of the humoral response. Full article
(This article belongs to the Section Bacterial Toxins)
Open AccessArticle Selection and Characterization of a Novel DNA Aptamer for Label-Free Fluorescence Biosensing of Ochratoxin A
Toxins 2014, 6(8), 2435-2452; doi:10.3390/toxins6082435
Received: 16 June 2014 / Revised: 25 July 2014 / Accepted: 30 July 2014 / Published: 15 August 2014
Cited by 19 | PDF Full-text (1170 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Nucleic acid aptamers are emerging as useful molecular recognition tools for food safety monitoring. However, practical and technical challenges limit the number and diversity of available aptamer probes that can be incorporated into novel sensing schemes. This work describes the selection of novel
[...] Read more.
Nucleic acid aptamers are emerging as useful molecular recognition tools for food safety monitoring. However, practical and technical challenges limit the number and diversity of available aptamer probes that can be incorporated into novel sensing schemes. This work describes the selection of novel DNA aptamers that bind to the important food contaminant ochratoxin A (OTA). Following 15 rounds of in vitro selection, sequences were analyzed for OTA binding. Two of the isolated aptamers demonstrated high affinity binding and selectivity to this mycotoxin compared to similar food adulterants. These sequences, as well as a truncated aptamer (minimal sequence required for binding), were incorporated into a SYBR® Green I fluorescence-based OTA biosensing scheme. This label-free detection platform is capable of rapid, selective, and sensitive OTA quantification with a limit of detection of 9 nM and linear quantification up to 100 nM. Full article
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Open AccessArticle The Correlation of the Presence and Expression Levels of cry Genes with the Insecticidal Activities against Plutella xylostella for Bacillus thuringiensis Strains
Toxins 2014, 6(8), 2453-2470; doi:10.3390/toxins6082453
Received: 23 April 2014 / Revised: 11 August 2014 / Accepted: 11 August 2014 / Published: 19 August 2014
Cited by 1 | PDF Full-text (971 KB) | HTML Full-text | XML Full-text
Abstract
The use of Bacillus thuringiensis (Bt) strains with high insecticidal activity is essential for the preparation of bioinsecticide. In this study, for 60 Bt strains isolated in Taiwan, their genotypes and the correlation of some cry genes as well as the
[...] Read more.
The use of Bacillus thuringiensis (Bt) strains with high insecticidal activity is essential for the preparation of bioinsecticide. In this study, for 60 Bt strains isolated in Taiwan, their genotypes and the correlation of some cry genes as well as the expression levels of cry1 genes, with their insecticidal activities against Plutella xylostella, were investigated. Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) results revealed that the genotypes of these Bt strains are highly diversified. Also, a considerable number of the Bt strains isolated in Taiwan were found to have high insecticidal activities. Since strains that showed individual combined patterns of PFGE and RAPD exhibited distinct insecticidal activities against P. xylostella, thus, these genotypes may be useful for the identification of the new Bt strains and those which have been used in bioinsecticides. In addition, although the presence of cry2Aa1 may have a greater effect on the insecticidal activity of Bt strains in bioassay than other cry genes, only high expression level of cry1 genes plays a key role to determine the insecticidal activity of Bt strains. In conclusion, both RAPD and PFGE are effective in the differentiation of Bt strains. The presence of cry2Aa1 and, especially, the expression level of cry1 genes are useful for the prediction of the insecticidal activities of Bt strains against P. xylostella. Full article
(This article belongs to the Special Issue Bacillus thuringiensis Toxins)
Open AccessArticle Immunological Cross-Reactivity and Neutralisation of European Viper Venoms with the Monospecific Vipera berus Antivenom ViperaTAb
Toxins 2014, 6(8), 2471-2482; doi:10.3390/toxins6082471
Received: 12 June 2014 / Revised: 13 August 2014 / Accepted: 13 August 2014 / Published: 19 August 2014
Cited by 1 | PDF Full-text (818 KB) | HTML Full-text | XML Full-text
Abstract
Medically important cases of snakebite in Europe are predominately caused by European vipers of the genus Vipera. The mainstay of snakebite therapy is polyclonal antibody therapy, referred to as antivenom. Here we investigate the capability of the monospecific V. berus antivenom,
[...] Read more.
Medically important cases of snakebite in Europe are predominately caused by European vipers of the genus Vipera. The mainstay of snakebite therapy is polyclonal antibody therapy, referred to as antivenom. Here we investigate the capability of the monospecific V. berus antivenom, ViperaTAb®, to cross-react with, and neutralise lethality induced by, a variety of European vipers. Using ELISA and immunoblotting, we find that ViperaTAb® antibodies recognise and bind to the majority of toxic components found in the venoms of the Vipera species tested at comparably high levels to those observed with V. berus. Using in vivo pre-clinical efficacy studies, we demonstrate that ViperaTAb® effectively neutralises lethality induced by V. berus, V. aspis, V. ammodytes and V. latastei venoms and at much higher levels than those outlined by regulatory pharmacopoeial guidelines. Notably, venom neutralisation was found to be superior to (V. berus, V. aspis and V. latastei), or as equally effective as (V. ammodytes), the monospecific V. ammodytes “Zagreb antivenom”, which has long been successfully used for treating European snake envenomings. This study suggests that ViperaTAb® may be a valuable therapeutic product for treating snakebite by a variety of European vipers found throughout the continent. Full article
(This article belongs to the Special Issue Antivenom and Venom Therapeutics)
Open AccessArticle The Activation Effect of Hainantoxin-I, a Peptide Toxin from the Chinese Spider, Ornithoctonus hainana, on Intermediate-Conductance Ca2+-Activated K+ Channels
Toxins 2014, 6(8), 2568-2579; doi:10.3390/toxins6082568
Received: 26 May 2014 / Revised: 28 July 2014 / Accepted: 14 August 2014 / Published: 21 August 2014
PDF Full-text (749 KB) | HTML Full-text | XML Full-text
Abstract
Intermediate-conductance Ca2+-activated K+ (IK) channels are calcium/calmodulin-regulated voltage-independent K+ channels. Activation of IK currents is important in vessel and respiratory tissues, rendering the channels potential drug targets. A variety of small organic molecules have been synthesized and found to
[...] Read more.
Intermediate-conductance Ca2+-activated K+ (IK) channels are calcium/calmodulin-regulated voltage-independent K+ channels. Activation of IK currents is important in vessel and respiratory tissues, rendering the channels potential drug targets. A variety of small organic molecules have been synthesized and found to be potent activators of IK channels. However, the poor selectivity of these molecules limits their therapeutic value. Venom-derived peptides usually block their targets with high specificity. Therefore, we searched for novel peptide activators of IK channels by testing a series of toxins from spiders. Using electrophysiological experiments, we identified hainantoxin-I (HNTX-I) as an IK-channel activator. HNTX-I has little effect on voltage-gated Na+ and Ca2+ channels from rat dorsal root ganglion neurons and on the heterologous expression of voltage-gated rapidly activating delayed rectifier K+ channels (human ether-à-go-go-related gene; human ERG) in HEK293T cells. Only 35.2% ± 0.4% of the currents were activated in SK channels, and there was no effect on BK channels. We demonstrated that HNTX-I was not a phrenic nerve conduction blocker or acutely toxic. This is believed to be the first report of a peptide activator effect on IK channels. Our study suggests that the activity and selectivity of HNTX-I on IK channels make HNTX-I a promising template for designing new drugs for cardiovascular diseases. Full article
(This article belongs to the Special Issue Antivenom and Venom Therapeutics)
Open AccessArticle Characterization of Fumonisin A-Series by High-Resolution Liquid Chromatography-Orbitrap Mass Spectrometry
Toxins 2014, 6(8), 2580-2593; doi:10.3390/toxins6082580
Received: 4 July 2014 / Revised: 8 August 2014 / Accepted: 12 August 2014 / Published: 21 August 2014
Cited by 8 | PDF Full-text (1045 KB) | HTML Full-text | XML Full-text
Abstract
Fumonisin A-series (FAs) in a reference material of corn sample that was naturally contaminated with fumonisins was characterized using high-resolution liquid chromatography-Orbitrap mass spectrometry (LC-Orbitap MS). Peaks for fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3), in addition to three peaks
[...] Read more.
Fumonisin A-series (FAs) in a reference material of corn sample that was naturally contaminated with fumonisins was characterized using high-resolution liquid chromatography-Orbitrap mass spectrometry (LC-Orbitap MS). Peaks for fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3), in addition to three peaks corresponding to unknown compounds I, II, and III, were detected in the chromatogram for the corn sample. Fragment ion analysis for FB1, FB2, and FB3 showed that while the ions formed at m/z values of 200–800 were similar to those formed by the cleavage of the tricarballylic acids and the hydroxyl groups, the fragmentation patterns at m/z values of 50–200 varied depending on the hydroxyl group locations in the compounds. Fragment ion analysis of compounds I–III revealed structural similarities to FBs, only differing by an additional C2H2O in the unknown compounds. Using these results and by comparing the product ion mass spectra of compound I with fumonisin A1 (FA1) synthesized from FB1 standards, compounds I–III were hypothesized to be N-acetyl analogs of FBs: fumonisins A1 (FA1), A2 (FA2), and A3 (FA3). The method for determining concentrations was validated with FA1, FB1, FB2, and FB3 standards and applied to analyze the reference material. The FB1, FB2, and FB3 analytical levels were within acceptance limits and the amount of FA1 in the material was ~15% of FB1 amount at 4.2 mg/kg. Full article
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Review

Jump to: Editorial, Research

Open AccessReview Thuringiensin: A Thermostable Secondary Metabolite from Bacillus thuringiensis with Insecticidal Activity against a Wide Range of Insects
Toxins 2014, 6(8), 2229-2238; doi:10.3390/toxins6082229
Received: 14 April 2014 / Revised: 16 July 2014 / Accepted: 18 July 2014 / Published: 25 July 2014
Cited by 7 | PDF Full-text (871 KB) | HTML Full-text | XML Full-text
Abstract
Thuringiensin (Thu), also known as β-exotoxin, is a thermostable secondary metabolite secreted by Bacillus thuringiensis. It has insecticidal activity against a wide range of insects, including species belonging to the orders Diptera, Coleoptera, Lepidoptera, Hymenoptera, Orthoptera, and Isoptera, and several nematode species.
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Thuringiensin (Thu), also known as β-exotoxin, is a thermostable secondary metabolite secreted by Bacillus thuringiensis. It has insecticidal activity against a wide range of insects, including species belonging to the orders Diptera, Coleoptera, Lepidoptera, Hymenoptera, Orthoptera, and Isoptera, and several nematode species. The chemical formula of Thu is C22H32O19N5P, and it is composed of adenosine, glucose, phosphoric acid, and gluconic diacid. In contrast to the more frequently studied insecticidal crystal protein, Thu is not a protein but a small molecule oligosaccharide. In this review, a detailed and updated description of the characteristics, structure, insecticidal mechanism, separation and purification technology, and genetic determinants of Thu is provided. Full article
(This article belongs to the Special Issue Bacillus thuringiensis Toxins)
Open AccessReview Quorum Sensing in Bacillus thuringiensis Is Required for Completion of a Full Infectious Cycle in the Insect
Toxins 2014, 6(8), 2239-2255; doi:10.3390/toxins6082239
Received: 29 May 2014 / Revised: 10 July 2014 / Accepted: 15 July 2014 / Published: 31 July 2014
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Abstract
Bacterial cell-cell communication or quorum sensing (QS) is a biological process commonly described as allowing bacteria belonging to a same pherotype to coordinate gene expression to cell density. In Gram-positive bacteria, cell-cell communication mainly relies on cytoplasmic sensors regulated by secreted and re-imported
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Bacterial cell-cell communication or quorum sensing (QS) is a biological process commonly described as allowing bacteria belonging to a same pherotype to coordinate gene expression to cell density. In Gram-positive bacteria, cell-cell communication mainly relies on cytoplasmic sensors regulated by secreted and re-imported signaling peptides. The Bacillus quorum sensors Rap, NprR, and PlcR were previously identified as the first members of a new protein family called RNPP. Except for the Rap proteins, these RNPP regulators are transcription factors that directly regulate gene expression. QS regulates important biological functions in bacteria of the Bacillus cereus group. PlcR was first characterized as the main regulator of virulence in B. thuringiensis and B. cereus. More recently, the PlcR-like regulator PlcRa was characterized for its role in cysteine metabolism and in resistance to oxidative stress. The NprR regulator controls the necrotrophic properties allowing the bacteria to survive in the infected host. The Rap proteins negatively affect sporulation via their interaction with a phosphorelay protein involved in the activation of Spo0A, the master regulator of this differentiation pathway. In this review we aim at providing a complete picture of the QS systems that are sequentially activated during the lifecycle of B. cereus and B. thuringiensis in an insect model of infection. Full article
(This article belongs to the Special Issue Bacillus thuringiensis Toxins)
Open AccessReview sRNA Antitoxins: More than One Way to Repress a Toxin
Toxins 2014, 6(8), 2310-2335; doi:10.3390/toxins6082310
Received: 30 June 2014 / Revised: 15 July 2014 / Accepted: 17 July 2014 / Published: 4 August 2014
Cited by 12 | PDF Full-text (357 KB) | HTML Full-text | XML Full-text
Abstract
Bacterial toxin-antitoxin loci consist of two genes: one encodes a potentially toxic protein, and the second, an antitoxin to repress its function or expression. The antitoxin can either be an RNA or a protein. For type I and type III loci, the antitoxins
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Bacterial toxin-antitoxin loci consist of two genes: one encodes a potentially toxic protein, and the second, an antitoxin to repress its function or expression. The antitoxin can either be an RNA or a protein. For type I and type III loci, the antitoxins are RNAs; however, they have very different modes of action. Type I antitoxins repress toxin protein expression through interacting with the toxin mRNA, thereby targeting the mRNA for degradation or preventing its translation or both; type III antitoxins directly bind to the toxin protein, sequestering it. Along with these two very different modes of action for the antitoxin, there are differences in the functions of the toxin proteins and the mobility of these loci between species. Within this review, we discuss the major differences as to how the RNAs repress toxin activity, the potential consequences for utilizing different regulatory strategies, as well as the confirmed and potential biological roles for these loci across bacterial species. Full article
(This article belongs to the Special Issue Toxin-Antitoxin System)
Open AccessReview Molecular Approaches to Improve the Insecticidal Activity of Bacillus thuringiensis Cry Toxins
Toxins 2014, 6(8), 2393-2423; doi:10.3390/toxins6082393
Received: 14 May 2014 / Revised: 23 June 2014 / Accepted: 27 June 2014 / Published: 13 August 2014
Cited by 3 | PDF Full-text (7741 KB) | HTML Full-text | XML Full-text
Abstract
Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect
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Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect pests in agriculture, particularly because of its specificity, which reflects directly on their lack of cytotoxicity to human health, non-target organisms and the environment. Since the introduction of transgenic plants expressing Bt genes in the mid-1980s, numerous methodologies have been used to search for and improve toxins derived from native Bt strains. These improvements directly influence the increase in productivity and the decreased use of chemical insecticides on Bt-crops. Recently, DNA shuffling and in silico evaluations are emerging as promising tools for the development and exploration of mutant Bt toxins with enhanced activity against target insect pests. In this report, we describe natural and in vitro evolution of Cry toxins, as well as their relevance in the mechanism of action for insect control. Moreover, the use of DNA shuffling to improve two Bt toxins will be discussed together with in silico analyses of the generated mutations to evaluate their potential effect on protein structure and cytotoxicity. Full article
(This article belongs to the Special Issue Bacillus thuringiensis Toxins)
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Open AccessReview Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery
Toxins 2014, 6(8), 2483-2540; doi:10.3390/toxins6082483
Received: 2 June 2014 / Revised: 8 August 2014 / Accepted: 8 August 2014 / Published: 21 August 2014
Cited by 6 | PDF Full-text (1816 KB) | HTML Full-text | XML Full-text
Abstract
To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body
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To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications. Full article
(This article belongs to the Special Issue Intracellular Traffic and Transport of Bacterial Protein Toxins)
Open AccessReview Engineering Venom’s Toxin-Neutralizing Antibody Fragments and Its Therapeutic Potential
Toxins 2014, 6(8), 2541-2567; doi:10.3390/toxins6082541
Received: 4 June 2014 / Revised: 16 July 2014 / Accepted: 4 August 2014 / Published: 21 August 2014
Cited by 7 | PDF Full-text (4302 KB) | HTML Full-text | XML Full-text
Abstract
Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be
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Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety. Full article
(This article belongs to the Special Issue Antivenom and Venom Therapeutics)

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