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Toxins
Volume 8
Issue 12
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Toxins, Volume 8, Issue 12 (December 2016) – 32 articles

Cover Story (view full-size image): Deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, nivalenol and zearalenone are mycotoxins contaminating wheat dust. Mycotoxins are toxic upon ingestion and considered potentially toxic when inhaled. Whereas dietary exposure to mycotoxins is controlled in food, data on occupational exposure by inhalation by grain workers are scarce. The objectives of this study were to determine the incidence of those mycotoxins in aerosols generated during grain harvesting and unloading and the risk of exposure of workers. Personal sampling revealed that working in a cab was an efficient protective measure. However it was not sufficient to avoid chronic exposure to multiple mycotoxins, in particular during cleaning activities.View this paper.
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6831 KiB  
Article
Transcriptome Analysis to Understand the Toxicity of Latrodectus tredecimguttatus Eggs
by Dehong Xu and Xianchun Wang
Toxins 2016, 8(12), 378; https://doi.org/10.3390/toxins8120378 - 20 Dec 2016
Cited by 15 | Viewed by 6412
Abstract
Latrodectus tredecimguttatus is a kind of highly venomous black widow spider, with toxicity coming from not only venomous glands but also other parts of its body as well as newborn spiderlings and eggs. Up to date, although L. tredecimguttatus eggs have been demonstrated [...] Read more.
Latrodectus tredecimguttatus is a kind of highly venomous black widow spider, with toxicity coming from not only venomous glands but also other parts of its body as well as newborn spiderlings and eggs. Up to date, although L. tredecimguttatus eggs have been demonstrated to be rich in proteinaceous toxins, there is no systematic investigation on such active components at transcriptome level. In this study, we performed a high-throughput transcriptome sequencing of L. tredecimguttatus eggs with Illumina sequencing technology. As a result, 53,284 protein-coding unigenes were identified, of which 14,185 unigenes produced significant hits in the available databases, including 280 unigenes encoding proteins or peptides homologous to known proteinaceous toxins. GO term and KEGG pathway enrichment analyses of the 280 unigenes showed that 375 GO terms and 18 KEGG pathways were significantly enriched. Functional analysis indicated that these unigene-coded toxins have the bioactivities to degrade tissue proteins, inhibit ion channels, block neuromuscular transmission, provoke anaphylaxis, induce apoptosis and hyperalgesia, etc. No known typical proteinaceous toxins in L. tredecimguttatus venomous glands, such as latrotoxins, were identified, suggesting that the eggs have a different toxicity mechanism from that of the venom. Our present transcriptome analysis not only helps to reveal the gene expression profile and toxicity mechanism of the L. tredecimguttatus eggs, but also provides references for the further related researches. Full article
(This article belongs to the Section Animal Venoms)
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Figure 1

Figure 1
<p>GO classification of the unigenes from <span class="html-italic">L. tredecimguttatus</span> eggs. Unigenes were annotated within three categories: biological process (<b>A</b>); molecular function (<b>B</b>); and cellular component (<b>C</b>). The <span class="html-italic">x</span>-axis represents the different categories. The number and percent of the unigenes matching GO annotation terms are presented on the <span class="html-italic">y</span>-axis.</p> Full article ">Figure 2
<p>KOG classification of the egg unigenes: 9528 annotated unigenes were classified into 25 KOG categories. The <span class="html-italic">x</span>-axis represents the different KOG categories. The number and percent of unigenes matching KOG classification are presented on the <span class="html-italic">y</span>-axis.</p> Full article ">Figure 3
<p>KEGG classification of the egg unigenes: 3141 unigenes were classified into 317 pathways. The different colors represent different KEGG categories. The number and percent of the unigenes matching KEGG classification are presented on the upper and lower axes, respectively.</p> Full article ">Figure 4
<p>GO enrichment analysis scatterplot for the 280 unigenes encoding toxins. The size of black circle represents the unigene number. Different colors represent different <span class="html-italic">p</span> values for significance test.</p> Full article ">Figure 5
<p>KEGG enrichment analysis scatterplot for the 280 unigenes encoding toxins. The size of black circle represents the unigene number. Different colors represent different <span class="html-italic">p</span> values for significance test.</p> Full article ">Figure 6
<p>Distribution of the egg unigenes. Left pie graph shows the composition of the egg unigenes. “Not annotated” represents the unigenes not annotated in the available databases. Unigenes annotated to encode the proteins matching known toxins are labeled as “Putative toxins”, and those matching other proteins are labeled as “Non-toxins”. The number of the unigenes in each subcategory is given followed by its percentage shown in the bracket. Right pie graph is further classification of the putative toxins. Putative toxins are further divided into four types. The number of the unigenes in each type is given followed by its percentage shown in the bracket. ICK, inhibitor cystine knot.</p> Full article ">Figure 7
<p>Primary and secondary structure analyses of the predicted ICK toxins. The amino acids forming an alpha helix are colored in pink. Yellow arrows represent β-sheet. Red and green rectangles indicate predicted signal peptides and propeptides, respectively. Blue lines show the connecting pattern of disulfide bonds.</p> Full article ">Figure 8
<p>Sequence alignment and phylogenetic analysis of comp21602_c0_seq2. Alignment was performed by DNAman soft. Strictly conserved cysteins are indicated by deep blue and other less conserved amino acids are marked using different colors. Phylogenetic analysis was implemented in MEGA 3.1 using the neighbor-joining method. ArachnoServer accession numbers precede the species name for each sequence. Numbers at the nodes indicate the bootstrap values based on 10,000 replicates.</p> Full article ">Figure 9
<p>Sequence alignment and phylogenetic analysis of comp25199_c0_seq1. Alignment was performed by DNAman soft. Strictly conserved cysteins are indicated by deep blue and other less conserved amino acids are marked using different colors. Phylogenetic analysis was implemented in MEGA 3.1 using the neighbor-joining method. ArachnoServer accession numbers precede the species name for each sequence. Numbers at the nodes indicate the bootstrap values based on 10,000 replicates.</p> Full article ">Figure 10
<p>Amino acid sequence alignment of comp31734_c0_seq1 and comp19276_c0_seq1: (<b>A</b>) Multisequence alignment of comp31734_c0_seq1 with AP00140 and its precursor (GenBank:GJ19999). The underlined amino acid residues indicate a putative signal peptide sequence. (<b>B</b>) Sequence alignment of comp19276_c0_seq1 with AP2030. Identical residues are shaded in blue in both (<b>A</b>) and (<b>B</b>).</p> Full article ">
3555 KiB  
Article
Probiotic Microorganisms Inhibit Epithelial Cell Internalization of Botulinum Neurotoxin Serotype A
by Tina I. Lam, Christina C. Tam, Larry H. Stanker and Luisa W. Cheng
Toxins 2016, 8(12), 377; https://doi.org/10.3390/toxins8120377 - 16 Dec 2016
Cited by 14 | Viewed by 6517
Abstract
Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins known to man and are threats to public health and safety. Previous work from our laboratory showed that both BoNT serotype A complex and holotoxin can bind and transit through the intestinal [...] Read more.
Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins known to man and are threats to public health and safety. Previous work from our laboratory showed that both BoNT serotype A complex and holotoxin can bind and transit through the intestinal epithelia to disseminate in the blood. The timing of BoNT/A toxin internalization was shown to be comparable in both the Caco-2 in vitro cell culture and in the oral mouse intoxication models. Probiotic microorganisms have been extensively studied for their beneficial effects in not only maintaining the normal gut mucosa but also protection from allergens, pathogens, and toxins. In this study, we evaluate whether probiotic microorganisms will block BoNT/A uptake in the in vitro cell culture system using Caco-2 cells. Several probiotics tested (Saccharomyces boulardii, Lactobacillus acidophilus, Lactobacillus rhamnosus LGG, and Lactobacillus reuteri) blocked BoNT/A uptake in a dose-dependent manner whereas a non-probiotic strain of Escherichia coli did not. We also showed that inhibition of BoNT/A uptake was not due to the degradation of BoNT/A nor by sequestration of toxin via binding to probiotics. These results show for the first time that probiotic treatment can inhibit BoNT/A binding and internalization in vitro and may lead to the development of new therapies. Full article
(This article belongs to the Special Issue Novel Pharmacological Inhibitors for Bacterial Protein Toxins)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Internalization of BoNT/A into Caco-2 cells is significantly reduced in a dose-dependent manner by pre-treatment with the probiotic <span class="html-italic">Saccharomyces boulardii</span>. (<b>A</b>) Caco-2 cells were treated with media (control) or BoNT/A complex for 4 h at 37 °C. Some Caco-2 cells were either pre-treated with <span class="html-italic">Saccharomyces boulardii</span> (SB<span class="html-italic">)</span> for 30 min at 37 °C at either high SB (10<sup>8</sup> CFU) or low SB (10<sup>4</sup> CFU) before the addition of BoNT/A. Cells were fixed and stained with Alexa-488 labeled antibodies to BoNT/A, DAPI (nuclear), and Rhodamine-Phalloidin (actin cytoskeleton). Representative images at 40− magnification are shown; (<b>B</b>) The cellular uptake of BoNT/A was quantified by determining the mean fluorescence of three randomly chosen optical fields from each of four coverslips per experiment using ImageJ software. Values represent means of four independent experiments ± SEM. Statistical significance was determined using two-way ANOVA followed by the Tukey-Kramer test where multiple groups that are compared with <span class="html-italic">p</span>-values &lt; 0.05 are taken to indicate significant differences between groups (*).</p> Full article ">Figure 1 Cont.
<p>Internalization of BoNT/A into Caco-2 cells is significantly reduced in a dose-dependent manner by pre-treatment with the probiotic <span class="html-italic">Saccharomyces boulardii</span>. (<b>A</b>) Caco-2 cells were treated with media (control) or BoNT/A complex for 4 h at 37 °C. Some Caco-2 cells were either pre-treated with <span class="html-italic">Saccharomyces boulardii</span> (SB<span class="html-italic">)</span> for 30 min at 37 °C at either high SB (10<sup>8</sup> CFU) or low SB (10<sup>4</sup> CFU) before the addition of BoNT/A. Cells were fixed and stained with Alexa-488 labeled antibodies to BoNT/A, DAPI (nuclear), and Rhodamine-Phalloidin (actin cytoskeleton). Representative images at 40− magnification are shown; (<b>B</b>) The cellular uptake of BoNT/A was quantified by determining the mean fluorescence of three randomly chosen optical fields from each of four coverslips per experiment using ImageJ software. Values represent means of four independent experiments ± SEM. Statistical significance was determined using two-way ANOVA followed by the Tukey-Kramer test where multiple groups that are compared with <span class="html-italic">p</span>-values &lt; 0.05 are taken to indicate significant differences between groups (*).</p> Full article ">Figure 2
<p>Pre-treatment with <span class="html-italic">Escherichia coli</span> MG1655 does not affect the internalization of BoNT/A into Caco-2. (<b>A</b>) Caco-2 cells were treated with media (control) or BoNT/A complex for 4 h at 37 °C. Some Caco-2 cells were either pre-treated with <span class="html-italic">Escherichia coli</span> (EC) for 30 min at 37 °C at either high EC (10<sup>8</sup> CFU) or low EC (10<sup>4</sup> CFU) before the addition of toxin. Cells were fixed and stained with Alexa-488 labeled antibodies to BoNT/A, DAPI (nuclear), and Rhodamine-Phalloidin (actin cytoskeleton). Representative images at 40− magnification are shown; (<b>B</b>) The cellular uptake of BoNT/A was quantified by determining the mean fluorescence of three randomly chosen optical fields from each of three coverslips per experiment acquired using a Zeiss Axio Observer.Z1 with Apotome.2 and analyzed with Zeiss Zen Pro 2012 software. Values represent means of four independent experiments ± SEM<b>.</b> Statistical significance was determined using two-way ANOVA followed by the Tukey-Kramer test where multiple groups that are compared with <span class="html-italic">p</span>-values &lt; 0.05 are taken to indicate significant differences between groups (*). There is no statistical significance with pretreatment with EC.</p> Full article ">Figure 3
<p>Pre-treatment with the probiotic <span class="html-italic">Lactobacillus acidophilus</span>, <span class="html-italic">Lactobacillus rhamnosus</span>, and <span class="html-italic">Lactobacillus reuteri</span> blocks internalization of BoNT/A into Caco-2 cells. (<b>A</b>) Caco-2 cells were treated with media (control) or with BoNT/A for 4 h at 37 °C. Some Caco-2 cells were either pre-treated with <span class="html-italic">Lactobacillus acidophilus</span> (LA)<span class="html-italic">, Lactobacillus rhamnosus</span> (LGG), <span class="html-italic">or Lactobacillus reuteri</span> (Lr) for 30 min at 37°C at either high (10<sup>8</sup> CFU) or low (10<sup>4</sup> CFU) before addition of BoNT/A complex. Cells were fixed and stained with Alexa-488 labeled antibodies to BoNT/A, DAPI (nuclear), and Rhodamine-Phalloidin (actin cytoskeleton). Representative images at 40− magnification showing BoNT/A fluorescence are shown; (<b>B</b>) The cellular uptake of BoNT/A was quantified by determining the mean fluorescence of three randomly chosen optical fields from each of the four coverslips per strain per experiment using ImageJ software. Values represent means of four independent experiments ± SEM<b>.</b> Statistical significance was determined using two-way ANOVA followed by the Tukey-Kramer test where multiple groups are compared with <span class="html-italic">p</span>-values &lt; 0.05 are taken to indicate significant differences between groups (*).</p> Full article ">Figure 3 Cont.
<p>Pre-treatment with the probiotic <span class="html-italic">Lactobacillus acidophilus</span>, <span class="html-italic">Lactobacillus rhamnosus</span>, and <span class="html-italic">Lactobacillus reuteri</span> blocks internalization of BoNT/A into Caco-2 cells. (<b>A</b>) Caco-2 cells were treated with media (control) or with BoNT/A for 4 h at 37 °C. Some Caco-2 cells were either pre-treated with <span class="html-italic">Lactobacillus acidophilus</span> (LA)<span class="html-italic">, Lactobacillus rhamnosus</span> (LGG), <span class="html-italic">or Lactobacillus reuteri</span> (Lr) for 30 min at 37°C at either high (10<sup>8</sup> CFU) or low (10<sup>4</sup> CFU) before addition of BoNT/A complex. Cells were fixed and stained with Alexa-488 labeled antibodies to BoNT/A, DAPI (nuclear), and Rhodamine-Phalloidin (actin cytoskeleton). Representative images at 40− magnification showing BoNT/A fluorescence are shown; (<b>B</b>) The cellular uptake of BoNT/A was quantified by determining the mean fluorescence of three randomly chosen optical fields from each of the four coverslips per strain per experiment using ImageJ software. Values represent means of four independent experiments ± SEM<b>.</b> Statistical significance was determined using two-way ANOVA followed by the Tukey-Kramer test where multiple groups are compared with <span class="html-italic">p</span>-values &lt; 0.05 are taken to indicate significant differences between groups (*).</p> Full article ">Figure 4
<p>Decreased cellular uptake of BoNT/A complex is not due to the proteolytic degradation of holotoxin nor binding of toxin to probiotics. BoNT/A was added to either <span class="html-italic">Escherichia coli</span> MG1655, or probiotics and incubated for 4 h at 37 °C. Soluble supernatant (S) and insoluble pellet (P) fractions were precipitated with trichloracetic acid (TCA). Precipitates were solubilized with sample loading buffer and loaded onto 10% Bis-Tris NuPage gels. Gels were transferred onto PVDF membranes and incubated with primary polyclonal antibody to BoNT/A (Metabiologics) and secondary goat anti-rabbit-HRP. Western blot was developed using Pierce SuperSignal ECL substrate. (<b>A</b>) Mean percent signal of BoNT/A in each fraction was quantified from four independent experiments ± SEM using FluorChem SP (Alpha Innotech); (<b>B</b>) Representative Western depicting the presence of full length BoNT/A. Statistical significance was determined by a two-tailed unpaired Student’s <span class="html-italic">t</span>-test, (*) <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">
2134 KiB  
Review
Modified Lipids and Lipoproteins in Chronic Kidney Disease: A New Class of Uremic Toxins
by Nans Florens, Catherine Calzada, Egor Lyasko, Laurent Juillard and Christophe O. Soulage
Toxins 2016, 8(12), 376; https://doi.org/10.3390/toxins8120376 - 16 Dec 2016
Cited by 76 | Viewed by 12232
Abstract
Chronic kidney disease (CKD) is associated with an enhanced oxidative stress and deep modifications in lipid and lipoprotein metabolism. First, many oxidized lipids accumulate in CKD and were shown to exert toxic effects on cells and tissues. These lipids are known to interfere [...] Read more.
Chronic kidney disease (CKD) is associated with an enhanced oxidative stress and deep modifications in lipid and lipoprotein metabolism. First, many oxidized lipids accumulate in CKD and were shown to exert toxic effects on cells and tissues. These lipids are known to interfere with many cell functions and to be pro-apoptotic and pro-inflammatory, especially in the cardiovascular system. Some, like F2-isoprostanes, are directly correlated with CKD progression. Their accumulation, added to their noxious effects, rendered their nomination as uremic toxins credible. Similarly, lipoproteins are deeply altered by CKD modifications, either in their metabolism or composition. These impairments lead to impaired effects of HDL on their normal effectors and may strongly participate in accelerated atherosclerosis and failure of statins in end-stage renal disease patients. This review describes the impact of oxidized lipids and other modifications in the natural history of CKD and its complications. Moreover, this review focuses on the modifications of lipoproteins and their impact on the emergence of cardiovascular diseases in CKD as well as the appropriateness of considering them as actual mediators of uremic toxicity. Full article
(This article belongs to the Special Issue Novel Issues in Uremic Toxicity)
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Figure 1
<p>Main modifications of lipoprotein metabolism induced by chronic kidney disease (CKD). CKD induces a deep modification in lipoprotein metabolism resulting in the accumulation of pro-atherogenic particles such as intermediary density lipoprotein (IDL) and triglyceride-rich lipoproteins (TGRL). Main modifications are listed below: In CKD, ApoA1 and A2 levels are decreased resulting in low level of circulating high density lipoprotein (HDL) (❶). In CKD, modifications of ApoA1 decrease HDL binding to macrophages and participate in the observed impaired cholesterol efflux (❷). Nascent HDL are transformed into discoid HDL-3 and then spherical HDL-2 enriched in cholesterol by the action of lecithin-cholesterol acyltransferase (LCAT). In CKD, LCAT level and activity are impaired (❸), leading to the accumulation of HDL-3 and reduced level of HDL-2 (❹). Thus, low HDL-2 concentration result in less transfer of triglycerides from TGRL to HDL-2 by cholesterol-ester transfer protein (CETP). Moreover, HDL-2 fail to enrich very-low density lipoprotein (VLDL) and chylomicrons with ApoC and E, essential for the binding and activation of lipoprotein lipase (LPL) respectively and such defect, associated with evidence of peripheral LPL lacking in CKD, leads to a reduced release of triglycerides into peripheral tissues and leads to an accumulation of TGRL (❺). IDL and remnants accumulate in CKD because of a down-regulation of LDL receptor protein (LRP) (❻), the lower level of CETP (❹) and the down-regulation of hepatic lipase (HL) expression (❼). A part of VLDL accumulates because of the down-regulation of the VLDL-receptor (VLDL-R) in myocytes and adipocytes (❽). Abbreviations: refer to abbreviation section.</p> Full article ">Figure 2
<p>Major reactive lipid aldehydes derived from poly unsaturated fatty acids (PUFAs) oxidation. Malondialdehyde (MDA) results from the oxidation of various polyunsaturated fatty acids containing more than two double bounds. MDA binds with nucleic acids or lysine amino-groups and creates toxic adducts called advanced peroxidation lipid end products (ALEs). On ApoB, these adducts were associated with atherosclerosis. 4-hydroxy-2-nonenal (4-HNE) and 4-hydroxy-2-hexenal (4-HHE) result respectively from the oxidation of ω6 PUFAs and ω3 PUFAs. They can react with proteins by a Michael addition mechanism and create ALEs. These ALEs disrupt several biological functions and lead to the formation of atherosclerosis and foam cells. Abbreviations: refer to abbreviation section.</p> Full article ">Figure 3
<p>Main effects of oxidized lipids and lipoproteins in chronic kidney disease (CKD). CKD is associated with increased oxidative stress, which promotes covalent modifications of lipids and lipoproteins. Lipid products of this unbalanced metabolism are oxidized phospholipids (oxPLs), fatty acid peroxidation products (FAPPs), oxysterols and F2-isoprostanes. Posttranslational modification derived products (PTMDPs) are the result of an enhanced myeloperoxidase (MPO) activity in CKD, an increased carbamylation and a massive production of advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs). ALEs are derived from lipid aldehydes issued from peroxidation of fatty acids (FAPPs). MPO catalyzes the nitrosilation on phospholipids to create oxPLs (❶). MPO are also involved in carbamylation process by the addition of thiocyanate on proteic residues (❷). Lipoproteins are also modified in CKD. First, triglyceride-rich lipoproteins (TGRL) have an impaired metabolism leading to their accumulation. Low-density lipoproteins (LDL) exhibit large amount of toxic oxidized (oxLDL) and carbamylated (cLDL) forms in CKD. These modifications lead to impaired functions and promote the progression of cardiovascular disease (CVD) especially in hemodialysis (HD) patients. High density lipoproteins (HDL) are also modified in CKD. Their whole metabolism is impaired and this dysregulation leads to many pro-atherosclerotic effects. MPO and carbamylation are greatly responsible for lipoproteins’ modifications and dysfunctions (❸) so are FAPP products that generate ALEs, especially on apolipoproteins A and B (ApoB) (❹). Abbreviations: refer to abbreviation section.</p> Full article ">
1706 KiB  
Article
QuEChERS Purification Combined with Ultrahigh-Performance Liquid Chromatography Tandem Mass Spectrometry for Simultaneous Quantification of 25 Mycotoxins in Cereals
by Juan Sun, Weixi Li, Yan Zhang, Xuexu Hu, Li Wu and Bujun Wang
Toxins 2016, 8(12), 375; https://doi.org/10.3390/toxins8120375 - 15 Dec 2016
Cited by 36 | Viewed by 6747
Abstract
A method based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) purification combined with ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS), was optimized for the simultaneous quantification of 25 mycotoxins in cereals. Samples were extracted with a solution containing 80% [...] Read more.
A method based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) purification combined with ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS), was optimized for the simultaneous quantification of 25 mycotoxins in cereals. Samples were extracted with a solution containing 80% acetonitrile and 0.1% formic acid, and purified with QuEChERS before being separated by a C18 column. The mass spectrometry was conducted by using positive electrospray ionization (ESI+) and multiple reaction monitoring (MRM) models. The method gave good linear relations with regression coefficients ranging from 0.9950 to 0.9999. The detection limits ranged from 0.03 to 15.0 µg·kg−1, and the average recovery at three different concentrations ranged from 60.2% to 115.8%, with relative standard deviations (RSD%) varying from 0.7% to 19.6% for the 25 mycotoxins. The method is simple, rapid, accurate, and an improvement compared with the existing methods published so far. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Response obtained for a fixed concentration of the 25 mycotoxins at different concentrations of the formic acid in water. DON: deoxynivalenol; AF: aflatoxin; ZEN: zearalenone; SMC: sterigmatocystin; OTA: ochratoxin; T-2: T-2 toxin; HT-2: HT-2 toxin; VCG: verruculogen; ENN: enniatin; BEA: beauvericin; FUS-X: fusarenon-X; GLT: gliotoxin; NEO: neosolaniol; DAS: 4,5-diacetoxyscirpenol; FB: fumonisin</p> Full article ">Figure 2
<p>The change of peak shape between ENNs and BEA when using different ratios of formic acid water solution/methanol. MRM: multiple reaction monitoring.</p> Full article ">Figure 3
<p>Multiple reaction monitoring (MRM) chromatograms of mixed solutions of 25 mycotoxins standards by positive electrospray ionization (ESI+).</p> Full article ">Figure 3 Cont.
<p>Multiple reaction monitoring (MRM) chromatograms of mixed solutions of 25 mycotoxins standards by positive electrospray ionization (ESI+).</p> Full article ">Figure 4
<p>Matrix effects of blank cereals (wheat, corn, and rice) on the response of 25 mycotoxins. The two dashed lines show the tolerance level of the matrix effect.</p> Full article ">Figure 5
<p>Multiple mycotoxins determination of cereal samples collected from farmland and local supermarkets in China.</p> Full article ">
232 KiB  
Article
Abobotulinum Toxin A in the Treatment of Chronic Low Back Pain
by Duarte Machado, Aditya Kumar and Bahman Jabbari
Toxins 2016, 8(12), 374; https://doi.org/10.3390/toxins8120374 - 15 Dec 2016
Cited by 13 | Viewed by 5842
Abstract
Chronic low back pain is a debilitating condition with a complex and multifactorial pathophysiology. Botulinum neurotoxins (BoNTs) have strong analgesic effects, as shown in both animal models of pain and in human beings. A randomized, double-blind, placebo-controlled, parallel format study to investigate the [...] Read more.
Chronic low back pain is a debilitating condition with a complex and multifactorial pathophysiology. Botulinum neurotoxins (BoNTs) have strong analgesic effects, as shown in both animal models of pain and in human beings. A randomized, double-blind, placebo-controlled, parallel format study to investigate the efficacy of abobotulinum toxin A (aboA) in chronic low back pain was conducted. The study cohort consisted of 18 patients who received 100 units of aboA into each of the five lumbar extensor spinae muscles unilaterally or bilaterally (total dose 500 to 1000 units), and 19 who received normal saline of the same volume. The level of pain and quality of life were assessed using the visual analogue scale (VAS) and three questionnaires including the Oswestry Low Back Pain Disability Questionnaire (OLBPDQ). Patients’ perception of improvement was recorded via patient global impression of change (PGIC). The primary outcome measure, the proportion of responders with VAS of <4 at 6 weeks, was not met, but the data was significantly in favor of aboA at 4 weeks (p = 0.008). The total Oswestry score representing quality of life improved in the aboA group compared to the placebo group (p = 0.0448). Moreover, significantly more patients reported their low back pain as “much improved” in the abobotulinum toxin A group (0.0293). Full article
(This article belongs to the Collection Botulinum Toxins on Human Pain)
2250 KiB  
Article
Limited Link between Oxidative Stress and Ochratoxin A—Induced Renal Injury in an Acute Toxicity Rat Model
by Liye Zhu, Tao Yu, Xiaozhe Qi, Jing Gao, Kunlun Huang, Xiaoyun He, Haoshu Luo and Wentao Xu
Toxins 2016, 8(12), 373; https://doi.org/10.3390/toxins8120373 - 14 Dec 2016
Cited by 37 | Viewed by 5850
Abstract
Ochratoxin A (OTA) displays nephrotoxicity and hepatotoxicity. However, in the acute toxicity rat model, there is no evidence on the relationship between OTA and nephrotoxicity and hepatotoxicity. Based on this, the integrated analysis of physiological status, damage biomarkers, oxidative stress, and DNA damage [...] Read more.
Ochratoxin A (OTA) displays nephrotoxicity and hepatotoxicity. However, in the acute toxicity rat model, there is no evidence on the relationship between OTA and nephrotoxicity and hepatotoxicity. Based on this, the integrated analysis of physiological status, damage biomarkers, oxidative stress, and DNA damage were performed. After OTA treatment, the body weight decreased and AST, ALP, TP, and BUN levels in serum increased. Hydropic degeneration, swelling, vacuolization, and partial drop occurred in proximal tubule epithelial cells. PCNA and Kim-1 were dose-dependently increased in the kidney, but Cox-2 expression and proliferation were not found in the liver. In OTA-treated kidneys, the mRNA expressions of Kim-1, Cox-2, Lcn2, and Clu were dose-dependently increased. The mRNA expressions of Vim and Cox-2 were decreased in OTA-treated livers. Some oxidative stress indicators were altered in the kidneys (ROS and SOD) and livers (SOD and GSH). DNA damage and oxidative DNA damage were not found. In conclusion, there is a limited link between oxidative stress and OTA-induced renal injury in an acute toxicity rat model. Full article
(This article belongs to the Collection Ochratoxins-Collection)
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Figure 1
<p>(<b>A</b>) The serum biochemical parameters of rats in different OTA administration groups; (<b>B</b>) The ratios of kidney (liver) and body weight were detected; (<b>C</b>) The body weights were detected in three groups. Male Wistar rats were treated with OTA (0, 1 or 4 mg/kg b.w.), denoted as CK, L, and H group, respectively for 7 days. The data are presented as the mean ± SD (<span class="html-italic">n</span> = 6). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 2
<p>H &amp; E stained kidney and liver sections after OTA treatment. Rats were treated with OTA (0, 1 or 4 mg/kg b.w.), denoted as CK, L, and H group, respectively for 7 days. Original magnification = 200×.</p> Full article ">Figure 3
<p>Immunohistochemical staining. (<b>A</b>) The immunohistochemical staining of PCNA and Kim-1 in the kidney; (<b>B</b>) The immunohistochemical staining of PCNA and Cox-2 in the liver. Rats were treated with OTA (0, 1 or 4 mg/kg b.w.), denoted as CK, L, and H group, respectively for 7 days. Original magnification = 200×.</p> Full article ">Figure 4
<p>Changes in the mRNA expression of <span class="html-italic">Clu</span>, <span class="html-italic">Lcn2</span>, <span class="html-italic">Vim,</span> and <span class="html-italic">Cox-2</span> in kidneys and livers of male Wistar rats dosed with OTA for 7 days. <span class="html-italic">Kim-1</span> was only detected in kidneys; C: control group (0 mg/kg b.w.); L: low-dose group (1 mg/kg b.w.); H: high-dose group (4 mg/kg b.w.). <b>A</b>: <span class="html-italic">Clu</span>; <b>B</b>: <span class="html-italic">Lcn2</span>; <b>C</b>: <span class="html-italic">Vim</span>; <b>D</b>: <span class="html-italic">Cox-2</span>; <b>E</b>: <span class="html-italic">Kim-1</span>. Data are presented as the means ± SD (<span class="html-italic">n</span> = 6). * <span class="html-italic">p</span> &lt; 0.05</p> Full article ">Figure 5
<p>Expression of ROS, SOD, MDA, and GSH in kidneys and livers of male Wistar rats treated with OTA for 7 days. <b>A</b>: The detection of oxidative stress in the kidney; <b>B</b>: The detection of oxidative stress in the liver. CK: control group (0 mg/kg b.w.); L: low-dose group (1 mg/kg b.w.); H: high-dose group (4 mg/kg b.w.). Data are presented as the means ± SD (<span class="html-italic">n</span> = 6). * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 6
<p>DNA damage and DNA oxidative damage. Male Wistar rats were treated with OTA (0, 1 or 4 mg/kg b.w.), denoted as CK, L, and H group, respectively for 7 days. (<b>A</b>) DNA damage was detected by bone marrow micronucleus test; (<b>B</b>) 8-OHdG levels were determined in kidneys and livers. Data are presented as the means ± SD (<span class="html-italic">n</span> = 6).</p> Full article ">
3303 KiB  
Article
Proteomic Analyses of Agkistrodon contortrix contortrix Venom Using 2D Electrophoresis and MS Techniques
by Aleksandra Bocian, Małgorzata Urbanik, Konrad Hus, Andrzej Łyskowski, Vladimír Petrilla, Zuzana Andrejčáková, Monika Petrillová and Jaroslav Legáth
Toxins 2016, 8(12), 372; https://doi.org/10.3390/toxins8120372 - 13 Dec 2016
Cited by 13 | Viewed by 5628
Abstract
Snake venom is a complex mixture of proteins and peptides which in the Viperidae is mainly hemotoxic. The diversity of these components causes the venom to be an extremely interesting object of study. Discovered components can be used in search for new pharmaceuticals [...] Read more.
Snake venom is a complex mixture of proteins and peptides which in the Viperidae is mainly hemotoxic. The diversity of these components causes the venom to be an extremely interesting object of study. Discovered components can be used in search for new pharmaceuticals used primarily in the treatment of diseases of the cardiovascular system. In order to determine the protein composition of the southern copperhead venom, we have used high resolution two dimensional electrophoresis and MALDI ToF/ToF MS-based identification. We have identified 10 groups of proteins present in the venom, of which phospholipase A2 and metalloprotease and serine proteases constitute the largest groups. For the first time presence of 5′-nucleotidase in venom was found in this group of snakes. Three peptides present in the venom were also identified. Two of them as bradykinin-potentiating agents and one as an inhibitor. Full article
(This article belongs to the Section Animal Venoms)
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Figure 1
<p>Representative 2-D protein maps obtained from southern copperhead venom with identified protein groups shown. 1. Snake venom 5′-nucleotidase; 2. <span class="html-small-caps">l</span>-amino-acid oxidase; 3. Beta-fibrinogenase; 4. Thrombin-like proteins; 5. Protein C activator; 6. Basic phospholipase A2; 7. Cysteine-rich venom protein; 8. Snake venom metalloproteinase; 9. Acidic phospholipase A<sub>2</sub>; 10. C-type lectin. The proteins were separated by isoelectrofocusing at pH range 3–10, then distributed on polyacrylamide gels by SDS-PAGE and stained with colloidal Coomassie Brilliant Blue G-250. Molecular weight (MW) and pH 3–10 scale are shown.</p> Full article ">Figure 2
<p>Representative 2-D protein maps obtained from southern copperhead venom with identified protein groups shown. 1. Snake venom 5′-nucleotidase; 2. <span class="html-small-caps">l</span>-amino-acid oxidase; 3. Beta-fibrinogenase; 4. Thrombin-like proteins; 5. Protein C activator; 6. Basic phospholipase A<sub>2</sub>; 7. Cysteine-rich venom protein; 8. Snake venom metalloproteinase; 9. Acidic phospholipase A<sub>2</sub>; 10. C-type lectin. The proteins were separated by isoelectrofocusing at pH range 3–10, then distributed on polyacrylamide gels by SDS-PAGE and stained with colloidal Coomassie Brilliant Blue G-250. Molecular weight (MW) and pH 3–10 scale are shown.</p> Full article ">Figure 3
<p>Percentage of protein amount in groups of <span class="html-italic">Agkistrodon contortrix contortrix</span> venom calculated on the basis of %Vol of particular spots on gels.</p> Full article ">Figure 4
<p>Mass spectrum of peptidome fraction of southern copperhead venom obtained on MALDI ToF/ToF mass spectrometer.</p> Full article ">
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Article
Harmonized Collaborative Validation of Aflatoxins and Sterigmatocystin in White Rice and Sorghum by Liquid Chromatography Coupled to Tandem Mass Spectrometry
by Hyun Ee Ok, Fei Tian, Eun Young Hong, Ockjin Paek, Sheen-Hee Kim, Dongsul Kim and Hyang Sook Chun
Toxins 2016, 8(12), 371; https://doi.org/10.3390/toxins8120371 - 13 Dec 2016
Cited by 19 | Viewed by 4362
Abstract
An interlaboratory study was performed in eight laboratories to validate a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of aflatoxins and sterigmatocystin (STC) in white rice and sorghum (Sorghum bicolor). Fortified samples (at three different levels) of white [...] Read more.
An interlaboratory study was performed in eight laboratories to validate a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of aflatoxins and sterigmatocystin (STC) in white rice and sorghum (Sorghum bicolor). Fortified samples (at three different levels) of white rice and sorghum were extracted, purified through a solid-phase extraction (SPE) column, and then analyzed by LC/MS/MS. The apparent recoveries (ARs) ranged from 78.8% to 95.0% for aflatoxins and from 85.3% to 96.7% for STC. The relative standard deviations for repeatability (RSDr) and reproducibility (RSDR) of aflatoxins were in the ranges 7.9%–33.8% and 24.4%–81.0%, respectively. For STC, the RSDr ranged from 7.1% to 40.2% and the RSDR ranged from 28.1% to 99.2%. The Horwitz ratio values for the aflatoxins and STC ranged from 0.4 to 1.2 in white rice and from 0.3 to 1.0 in sorghum, respectively. These results validated this method for the simultaneous determination of aflatoxins and STC by LC/MS/MS after SPE column cleanup. The percentages of satisfactory Z-score values (|Z| ≤ 2) were the following: for white rice, 100% for aflatoxins and STC; for sorghum, 100%, except in data from two laboratories for STC (0.3 μg/kg). This validated that the LC/MS/MS method was successfully applied for the determination of aflatoxins and STC in 20 white rice and 20 sorghum samples sourced from Korean markets. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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Article
Frequent Occupational Exposure to Fusarium Mycotoxins of Workers in the Swiss Grain Industry
by Hélène Niculita-Hirzel, Gregoire Hantier, Ferdinand Storti, Gregory Plateel and Thierry Roger
Toxins 2016, 8(12), 370; https://doi.org/10.3390/toxins8120370 - 12 Dec 2016
Cited by 30 | Viewed by 6808
Abstract
Type B trichotecens such as deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and zearalenone (ZEN) are mycotoxins contaminating wheat and wheat dust. Mycotoxins are toxic upon ingestion and considered potentially toxic when inhaled. Whereas dietary exposure to mycotoxins is controlled in food, [...] Read more.
Type B trichotecens such as deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and zearalenone (ZEN) are mycotoxins contaminating wheat and wheat dust. Mycotoxins are toxic upon ingestion and considered potentially toxic when inhaled. Whereas dietary exposure to mycotoxins is controlled in food, data on occupational exposure by inhalation by grain workers are scarce. The objectives of this study were to determine the incidence of DON, 3-ADON, 15-ADON, NIV and ZEN in aerosols generated during grain harvesting and unloading and the risk of exposure of grain workers. Aerosols were collected during the threshing of 78 winter wheat fields and grain unloading of 59 grain lots in six grain terminals in the Vaud region (Switzerland). The samples represented the diversity of the winter wheat cultivar and of the farming system (88 treated with fungicides, 46 untreated). Using a HPLC MS/MS method developed to quantify mycotoxins in aerosols, we report that the mycotoxin content of aerosols was not affected by the wheat cultivars or farming system, but that the incidence of the mycotoxins differed between activities. While wheat harvesting generated on average 28, 20 and 1 ng·m−3 of DON, NIV and ZEN, respectively, grain unloading generated 53, 46 and 4 ng·m−3. Personal sampling revealed that working in a cab was an efficient protective measure. However, it was not sufficient to avoid chronic exposure to multiple mycotoxins. The most exposed activity was the cleaning, exposing workers to DON, NIV and ZEN at concentrations as high as 65, 59 and 3 ng·m−3. These data provide valuable information for future studies of mycotoxin toxicity at relevant concentrations on respiratory health. Full article
(This article belongs to the Special Issue Exposure and Risk Assessment for Mycotoxins)
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Graphical abstract

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<p>Study area in the granary region of Vaud (Switzerland). The white and black dots correspond to the sampled wheat fields and the blue ones to grain terminals. The farming system used in wheat fields is indicated: white circle for organic farming, white square for extensive farming and black square for conventional farming. The background represents the summer rainfall level.</p> Full article ">Figure 2
<p>Co-occurrence of mycotoxins in aerosols. (<b>a</b>–<b>c</b>) The proportions of samples for which the different combinations of mycotoxins were detected in, respectively, overall threshing and grain unloading samples; (<b>d</b>–<b>f</b>) Samples for which all mycotoxins from a particular combination were detected at a quantifiable level.</p> Full article ">
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Article
Venom Gland Transcriptomic and Proteomic Analyses of the Enigmatic Scorpion Superstitionia donensis (Scorpiones: Superstitioniidae), with Insights on the Evolution of Its Venom Components
by Carlos E. Santibáñez-López, Jimena I. Cid-Uribe, Cesar V. F. Batista, Ernesto Ortiz and Lourival D. Possani
Toxins 2016, 8(12), 367; https://doi.org/10.3390/toxins8120367 - 9 Dec 2016
Cited by 41 | Viewed by 7060
Abstract
Venom gland transcriptomic and proteomic analyses have improved our knowledge on the diversity of the heterogeneous components present in scorpion venoms. However, most of these studies have focused on species from the family Buthidae. To gain insights into the molecular diversity of the [...] Read more.
Venom gland transcriptomic and proteomic analyses have improved our knowledge on the diversity of the heterogeneous components present in scorpion venoms. However, most of these studies have focused on species from the family Buthidae. To gain insights into the molecular diversity of the venom components of scorpions belonging to the family Superstitioniidae, one of the neglected scorpion families, we performed a transcriptomic and proteomic analyses for the species Superstitionia donensis. The total mRNA extracted from the venom glands of two specimens was subjected to massive sequencing by the Illumina protocol, and a total of 219,073 transcripts were generated. We annotated 135 transcripts putatively coding for peptides with identity to known venom components available from different protein databases. Fresh venom collected by electrostimulation was analyzed by LC-MS/MS allowing the identification of 26 distinct components with sequences matching counterparts from the transcriptomic analysis. In addition, the phylogenetic affinities of the found putative calcins, scorpines, La1-like peptides and potassium channel κ toxins were analyzed. The first three components are often reported as ubiquitous in the venom of different families of scorpions. Our results suggest that, at least calcins and scorpines, could be used as molecular markers in phylogenetic studies of scorpion venoms. Full article
(This article belongs to the Section Animal Venoms)
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Graphical abstract

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<p>(<b>A</b>) Distribution of annotated sequences from the venom gland transcriptome of <span class="html-italic">S. donensis</span> according to Gene Ontology (GO) terms. The category designated by GO as “Biological process” was the most diverse. (<b>B</b>–<b>D</b>) Distribution of the most represented categories within each GO term (GO numbers shown).</p> Full article ">Figure 2
<p>Relative proportion (expressed as percentages) of the Pfam domains of the 135 annotated transcripts, which putatively code for venom components found in the venom gland transcriptome analysis of <span class="html-italic">S. donensis</span>. The category Toxins includes putative Na<sup>+</sup>, K<sup>+</sup> and Ca<sup>2+</sup> toxin channels peptides; the category NDBPs (Non-Disulfide-Bridged Peptides) includes all possible NDBPs peptides even when no Pfam domain was found; the category Protease Inhibitors includes Ascaris-Type and Kunitz-Type inhibitors; the category La1 includes putative La1-type peptides; the category Enzymes includes all possible peptides with venom enzymatic activity; and the category Other Venom Components includes putative venom proteins and possible CAP peptides.</p> Full article ">Figure 3
<p>Sequence alignment of components with identity with sodium channel toxins (cysteine-stabilized α/β motif, CS αβ, indicated as CSab in the toxin names) found in the transcriptome analysis of the venom gland of <span class="html-italic">S. donensis</span> and those that were similar. Unitprot entry numbers precede the toxins’ names: (a) Component sdc14319_g1_i1, translated ORF; (b) CSab-Cer-2 from <span class="html-italic">Ce. squama</span>; (c) CSab-Cer-1 from <span class="html-italic">Ce. squama</span>; (d) CSab-Uro-2 from <span class="html-italic">Urodacus manicatus</span>; and (e) CSab-Iso-3 from <span class="html-italic">Isometroides vescus</span>.</p> Full article ">Figure 4
<p>Amino acid sequences of the translated transcripts showing identity with the αKTx subfamily 6, found in the transcriptome analysis of the venom gland of <span class="html-italic">S. donensis</span>, aligned to similar sequences. Unitprot entry numbers precede the species’ names: C5J896 (potassium channel toxin αKTx 6.16); H2CYS1 (αKTx-like peptide); Q6XLL6 (potassium channel toxin αKTx 6.9); Q6XLL5 (Potassium channel toxin αKTx 6.10); Q6XLL7 (potassium channel toxin αKTx 6.8); Q6XLL8 (Potassium channel toxin αKTx 6.7); and P0DL37 (potassium channel toxin αKTx 6.21). The predicted signal peptide is underlined and the mature peptide is in bold.</p> Full article ">Figure 5
<p>Sequence alignment of components with identity with Scorpines found in the transcriptome analysis of the venom gland of <span class="html-italic">S. donensis</span>. Peptide sequences were generated by translation from the reported transcripts. For comparative purposes, other known sequences are included (Unitprot entry numbers in brackets). Components sdc34997_g1_i1, sdc14222_g4_i1 and sdc14222_g4_i2; Hge scorpine and He scorpine-like 2 from <span class="html-italic">Ho. gertschi</span> (Q0GY40 and P0C8W5 respectively); Scorpine-like peptide Ev37 from <span class="html-italic">E. validus</span> (P0DL47); CSab-Cer-6 from <span class="html-italic">Ce. squama</span> (T1DMR0); β-KTx-like peptide LaIT2 from <span class="html-italic">Liocheles australasiae</span> (C7G3K3); Antimicrobial peptide scorpine-like 2 from <span class="html-italic">U. yaschenkoi</span> (L0GCW2); and Opiscorpine 3 from <span class="html-italic">Op. carinatus</span> (Q5WQZ7). The predicted signal peptide is underlined and the mature peptide is in bold.</p> Full article ">Figure 6
<p>Sequence alignment of components with identity with calcins found in the transcriptome analysis of the venom gland of <span class="html-italic">S. donensis</span> and those that were similar. The transcripts were translated to generate the peptidic precursor sequences. Unitprot entry numbers in brackets. Components sdc9999_g2_i1 and sdc13987_g1_i1; Calcium channel toxin like 20 from <span class="html-italic">Urodacus yaschenkoi</span> (L0GBR1); Hadrucalcin from <span class="html-italic">Hoffmannihadrurus gertschi</span> (B8QG00); ViCaTx1 from <span class="html-italic">Thorellius intrepidus</span> [<a href="#B11-toxins-08-00367" class="html-bibr">11</a>]; β-KTx-like peptide LaIT2 from <span class="html-italic">Liocheles australasiae</span> (C7G3K3); Antimicrobial peptide scorpine-like 2 <span class="html-italic">Urodacus yaschenkoi</span> (L0GCW2); and Opiscorpine 3 from <span class="html-italic">Op. carinatus</span> (Q5WQZ7). The predicted signal peptide is underlined; the mature peptide is in bold and the propeptide is in italics.</p> Full article ">Figure 7
<p>Sequence alignment of components with identity with Non-Disulfide-Bridged Peptides found in the transcriptome analysis of the venom gland of <span class="html-italic">S. donensis</span>. The sequences derived from transcripts were translated to show the precursor peptidic sequences. For comparative purposes other known sequences are included (Unitprot entry numbers in brackets): CYLIP-Uro-1 and CYLIP-Uro-3 from <span class="html-italic">U. manicatus</span> (T1E6X5 and T1DPA6, respectively); and CYLIP-Cer-2 and CYLIP-Cer-3 from <span class="html-italic">Ce. squama</span> (T1E6W7 and T1E7M2, respectively). The predicted signal peptide is underlined and the mature peptide is in bold.</p> Full article ">Figure 8
<p>Phylogenetic tree obtained from the Bayesian analysis of 22 sequences of putative and confirmed calcins from 14 scorpion species belonging to 12 genera and eight families selected from the InterPro database and the available literature. The originally reported names are used (or the UniProt or GenBank accession codes for those lacking a name), followed by the scorpion species (see <a href="#app1-toxins-08-00367" class="html-app">Supplementary Table S3</a>). Posterior probabilities higher than 0.76 are indicated above the branches.</p> Full article ">Figure 9
<p>Phylogenetic tree obtained from the Bayesian analysis of 62 sequences of scorpines and putative scorpines, plus 34 sequences of βKtx or putative βKtx from 34 scorpion species of 22 genera and 10 families, and one sequence as outgroup (αKTx), selected from the InterPro database and the available literature. Terminal names are composed of UniProt or GenBank accession codes and the name of the scorpion species, except for those named as in their original publications (see <a href="#app1-toxins-08-00367" class="html-app">Supplementary Table S4</a>). Posterior probabilities higher than 0.65 are indicated above/below branches. Clades in red represent sequences from species of genus <span class="html-italic">Tityus</span>; in light green sequences from species of genus <span class="html-italic">Mesobuthus</span>; in orange sequences from species of genus <span class="html-italic">Androctonus</span>; in magenta, sequences from species of genus <span class="html-italic">Chaerilus</span>; in yellow sequences from species of genus <span class="html-italic">Lychas</span>; in purple sequences from species of family Vaejovidae; in light blue sequences from species of family Scorpionidae; in dark green sequences from <span class="html-italic">S. donensis</span>, and in dark blue sequences from several non buthid families.</p> Full article ">Figure 10
<p>Phylogenetic tree obtained from the Bayesian analysis of 36 sequences of La1-like peptides or putative La1-like peptides from 23 scorpion species of 18 genera and nine families selected from the InterPro database and the available literature. Terminal names are composed of UniProt or GenBank accession codes and the name of the scorpion species, except for those named as in their original publications (see <a href="#app1-toxins-08-00367" class="html-app">Supplementary Table S5</a>). Posterior probabilities higher than 0.65 are indicated above branches. Colored clades indicate monophyletic groups of La1-like peptides from scorpions of families Buthidae (red), Scorpionidae (green) and Vaejovidae (blue).</p> Full article ">Figure 11
<p>Phylogenetic tree obtained from the Bayesian analysis of 20 sequences of potassium channel κ toxins (κKTxs) from eight scorpion species of four genera and three families; and 12 sequences of potassium channel α toxins and chlorotoxins as outgroup, selected from the InterPro database and available literature (see <a href="#app1-toxins-08-00367" class="html-app">Supplementary Table S6</a>). Posterior probabilities are indicated above branches. Colored clades indicate the monophyletic subfamilies proposed earlier: subfamily 1 (orange); subfamily 2 (blue); subfamily 3 (green); subfamily 4 (red); and subfamily 5 (purple). The name in red shows κ buthitoxin.</p> Full article ">
432 KiB  
Review
Fibroblast Growth Factor-23—A Potential Uremic Toxin
by Piotr Kuczera, Marcin Adamczak and Andrzej Wiecek
Toxins 2016, 8(12), 369; https://doi.org/10.3390/toxins8120369 - 8 Dec 2016
Cited by 30 | Viewed by 7077
Abstract
Fibroblast growth factor-23 (FGF23) is a circulating member of the FGF family produced mainly by the osteocytes and osteoblasts that can act as a hormone. The main action of FGF23 is to lower phosphatemia via the reduction of urinary phosphate reabsorption and the [...] Read more.
Fibroblast growth factor-23 (FGF23) is a circulating member of the FGF family produced mainly by the osteocytes and osteoblasts that can act as a hormone. The main action of FGF23 is to lower phosphatemia via the reduction of urinary phosphate reabsorption and the decrease of 1,25(OH)2-D generation in the kidney. In the course of chronic kidney disease (CKD), plasma FGF23 concentration rises early, most probably to compensate the inability of the deteriorating kidneys to excrete an adequate amount of phosphate. However, this comes at the cost of FGF23-related target organ toxicity. Results of clinical studies suggest that elevated plasma FGF23 concentration is independently associated with the increased risk of CKD progression, occurrence of cardio-vascular complications, and mortality in different stages of CKD. FGF23 also contributes to cardiomyocyte hypertrophy, vascular calcification, and endothelial dysfunction. The impact of FGF23 on heart muscle is not dependent on Klotho, but rather on the PLCγ–calcineurin–NFAT (nuclear factor of activated T-cells) pathway. Among the factors increasing plasma FGF23 concentration, active vitamin D analogues play a significant role. Additionally, inflammation and iron deficiency can contribute to the increase of plasma FGF23. Among the factors decreasing plasma FGF23, dietary phosphate restriction, some intestinal phosphate binders, cinacalcet (and other calcimimetics), and nicotinamide can be enumerated. Anti-FGF23 antibodies have also recently been developed to inhibit the action of FGF23 in target organs. Still, the best way to normalize plasma FGF23 in maintenance hemodialysis patients is restoring kidney function by successful kidney transplantation. Full article
(This article belongs to the Special Issue Novel Issues in Uremic Toxicity)
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<p>Brief summary of fibroblast growth factor-23 toxicity in chronic kidney disease. CKD—chronic kidney disease; FGF23—fibroblast growth factor-23.</p> Full article ">
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Article
Evolution of the Cytolytic Pore-Forming Proteins (Actinoporins) in Sea Anemones
by Jason Macrander and Marymegan Daly
Toxins 2016, 8(12), 368; https://doi.org/10.3390/toxins8120368 - 8 Dec 2016
Cited by 35 | Viewed by 6749
Abstract
Sea anemones (Cnidaria, Anthozoa, and Actiniaria) use toxic peptides to incapacitate and immobilize prey and to deter potential predators. Their toxin arsenal is complex, targeting a variety of functionally important protein complexes and macromolecules involved in cellular homeostasis. Among these, actinoporins are one [...] Read more.
Sea anemones (Cnidaria, Anthozoa, and Actiniaria) use toxic peptides to incapacitate and immobilize prey and to deter potential predators. Their toxin arsenal is complex, targeting a variety of functionally important protein complexes and macromolecules involved in cellular homeostasis. Among these, actinoporins are one of the better characterized toxins; these venom proteins form a pore in cellular membranes containing sphingomyelin. We used a combined bioinformatic and phylogenetic approach to investigate how actinoporins have evolved across three superfamilies of sea anemones (Actinioidea, Metridioidea, and Actinostoloidea). Our analysis identified 90 candidate actinoporins across 20 species. We also found clusters of six actinoporin-like genes in five species of sea anemone (Nematostella vectensis, Stomphia coccinea, Epiactis japonica, Heteractis crispa, and Diadumene leucolena); these actinoporin-like sequences resembled actinoporins but have a higher sequence similarity with toxins from fungi, cone snails, and Hydra. Comparative analysis of the candidate actinoporins highlighted variable and conserved regions within actinoporins that may pertain to functional variation. Although multiple residues are involved in initiating sphingomyelin recognition and membrane binding, there is a high rate of replacement for a specific tryptophan with leucine (W112L) and other hydrophobic residues. Residues thought to be involved with oligomerization were variable, while those forming the phosphocholine (POC) binding site and the N-terminal region involved with cell membrane penetration were highly conserved. Full article
(This article belongs to the Collection Evolution of Venom Systems)
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Figure 1
<p>(<b>A</b>) Maximum Likelihood tree of actinoporins and actinoporin-like proteins produced in FastTree2. Numbers on branches represent bootstrap values of 1000 replicates. Bootstrapping values greater than 50 are shown at the nodes. Branch labels of clustered gene groups represent lineage common names followed by percent identity (identical amino acid residues) within gene cluster and percent identity when compared to actinoporins in bold. Labeled individual branches show GenBank accession followed by species, and protein name (if applicable). Labels denoted with PREDICTED or GENOME indicate that they were derived bioinformatically in GenBank and are not validated proteins. Branch labels with Genbank accession and species for sea anemones are indicated in bold. The colored box indicates which actinoporin sequences were used in subsequent analyses; (<b>B</b>) Phylogenetic tree with branch colors depicting superfamily associations (Blue: Edwardsioidea, Yellow: Actinostoloidea, Red: Actinioidea, Green: Metridioidea) [<a href="#B42-toxins-08-00368" class="html-bibr">42</a>].</p> Full article ">Figure 2
<p>(<b>A</b>) Functionally important residues identified on EqII [<a href="#B43-toxins-08-00368" class="html-bibr">43</a>]. Functional sites are as follows: (B) site of bend when N-terminus comes into contact with the cell membrane, (POC) residues involved with the POC binding site, (O) residues involved with oligomerization, (S) key sphingomyelin binding site. Colors are used to aid in determining the orientation between the two views shown; (<b>B</b>) Characterization of amino acid variation for the different gene clusters (Clade 1, Clade 2M, Clade 2A) identified in our analysis. SeqLogo graphs for residues that have been identified previously as functionally important above the alignment with the size of each amino acid residue representing the frequency in which these residues occurred in the alignment. Numbers along the bottom correspond to positions of specific amino acid residues in EqII; (<b>C</b>) Maximum Likelihood actinoporin gene tree produced in FastTree2. Colored branches depict superfamily associations (see <a href="#toxins-08-00368-f001" class="html-fig">Figure 1</a>, Yellow: Actinostoloidea, Red: Actinioidea, Green: Metridioidea). Bootstrapping values greater than 50 are shown at the nodes. Branch labels include GenBank ID (when applicable) and the species from which the toxin gene was derived. Bold labels indicate that the mature protein sequence was recovered. Sequences derived from genomic data are indicated with “G” following species in sequence IDs. The superfamily association for <span class="html-italic">Actineria villosa</span> may be incorrect and is noted with an asterisk.</p> Full article ">Figure 3
<p>Edmundson wheel projections were determined in HeliQuest. Associated Sequence IDs are shown below the species name.</p> Full article ">Figure 3 Cont.
<p>Edmundson wheel projections were determined in HeliQuest. Associated Sequence IDs are shown below the species name.</p> Full article ">
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Communication
Yeast Reporter Assay to Identify Cellular Components of Ricin Toxin A Chain Trafficking
by Björn Becker, Tina Schnöder and Manfred J. Schmitt
Toxins 2016, 8(12), 366; https://doi.org/10.3390/toxins8120366 - 6 Dec 2016
Cited by 6 | Viewed by 5060
Abstract
RTA, the catalytic A-subunit of the ribosome inactivating A/B toxin ricin, inhibits eukaryotic protein biosynthesis by depurination of 28S rRNA. Although cell surface binding of ricin holotoxin is mainly mediated through its B-subunit (RTB), sole application of RTA is also toxic, albeit to [...] Read more.
RTA, the catalytic A-subunit of the ribosome inactivating A/B toxin ricin, inhibits eukaryotic protein biosynthesis by depurination of 28S rRNA. Although cell surface binding of ricin holotoxin is mainly mediated through its B-subunit (RTB), sole application of RTA is also toxic, albeit to a significantly lower extent, suggesting alternative pathways for toxin uptake and transport. Since ricin toxin trafficking in mammalian cells is still not fully understood, we developed a GFP-based reporter assay in yeast that allows rapid identification of cellular components required for RTA uptake and subsequent transport through a target cell. We hereby show that Ypt6p, Sft2p and GARP-complex components play an important role in RTA transport, while neither the retromer complex nor COPIB vesicles are part of the transport machinery. Analyses of yeast knock-out mutants with chromosomal deletion in genes whose products regulate ADP-ribosylation factor GTPases (Arf-GTPases) and/or retrograde Golgi-to-ER (endoplasmic reticulum) transport identified Sso1p, Snc1p, Rer1p, Sec22p, Erv46p, Gea1p and Glo3p as novel components in RTA transport, suggesting the developed reporter assay as a powerful tool to dissect the multistep processes of host cell intoxication in yeast. Full article
(This article belongs to the Special Issue Ribosome Inactivating Toxins)
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<p>(<b>a</b>) Schematic overview of the reporter plasmid used to screen yeast for proteins involved in ricin toxin A chain (RTA) transport. Yeast enhanced GFP extended by an <span class="html-italic">N</span>-terminal signal peptide (SP) derived from K28 preprotoxin [<a href="#B34-toxins-08-00366" class="html-bibr">34</a>] is placed under transcriptional control of the <span class="html-italic">GAL1</span> promoter and <span class="html-italic">CYC1</span> terminator, allowing inducible expression in the presence of galactose. Indicated numbers correspond to amino acid position in GFP; (<b>b</b>) Experimental assay setup. After transformation of a particular yeast strain with pRS315-K28<sub>SP</sub>-GFP and cell wall removal by zymolyase treatment, 2 × 10<sup>7</sup> spheroplasts were seeded in 96 microtiter plates and GFP expression was induced by the addition of 3% galactose. Simultaneously, RTA or the negative control sample was added and fluorescence development was measured over a time window of 20 h; (<b>c</b>) Relative fluorescence emission of 2 × 10<sup>7</sup> wild-type yeast spheroplasts expressing GFP from the reporter plasmid pRS315-K28<sub>SS</sub>-GFP under induced (3% galactose, GFP<sub>ind.</sub>) and non-induced (2% raffinose, GFP<sub>repr.</sub>) culture conditions. Yeast spheroplasts were also incubated in the presence of RTA (5 µM), G418 (300 µg/mL) or the negative control (NC) over 20 h. Standard deviation is indicated; all measurements were repeated 6 to 12 times as independent experiments without technical replicates; (<b>d</b>) Time course of GFP fluorescence development of wild-type yeast spheroplasts expressing GFP from the reporter plasmid pRS315-K28<sub>SS</sub>-GFP in the presence (RTA) or absence (NC) of 5 µM RTA.</p> Full article ">Figure 2
<p>(<b>a</b>) Assay validation. Relative fluorescence of <span class="html-italic">∆der</span>1, <span class="html-italic">∆hrd</span>1, <span class="html-italic">∆yos</span>9 and <span class="html-italic">∆nup</span>120 knock-out mutant spheroplasts is indicated. Measurements were performed in the presence of RTA (5 µM) under induced culture conditions (3% galactose) over 20 h. Shown fluorescence values at 20 h were normalized to the fluorescence of spheroplasts treated with the negative control sample; (<b>b</b>) Impact of the indicated yeast deletion mutants on RTA trafficking. Fluorescence values of 2 × 10<sup>7</sup> yeast spheroplasts of each mutant were displayed after 20 h induction (3% galactose) in the presence of RTA (5 µM). Values shown in (<b>a</b>,<b>b</b>) were normalized to the fluorescence of negative control spheroplasts, mean values and standard deviations are indicated. All measurements were at least repeated 3 to 12 times as independent experiments without technical replicates. The dotted line in (<b>a</b>) and (<b>b</b>) indicates the chosen threshold of 75% for a positive hit.</p> Full article ">Figure 3
<p>Overview of cellular components involved in RTA uptake and intracellular trafficking in yeast. Proteins involved in RTA transport are shown in blue, non-involved proteins in red; proteins marked with an asterisk (*) are essential for cell viability in yeast; proteins shown in black were not tested. EE, early endosome; PM, plasma membrane; TGN, trans-Golgi network; ER, endoplasmic reticulum; ERGIC, ER/Golgi intermediate compartment; COPI, coat protein I vesicle.</p> Full article ">
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Article
ATP Release from Human Airway Epithelial Cells Exposed to Staphylococcus aureus Alpha-Toxin
by Romina Baaske, Mandy Richter, Nils Möller, Sabine Ziesemer, Ina Eiffler, Christian Müller and Jan-Peter Hildebrandt
Toxins 2016, 8(12), 365; https://doi.org/10.3390/toxins8120365 - 6 Dec 2016
Cited by 13 | Viewed by 6179
Abstract
Airway epithelial cells reduce cytosolic ATP content in response to treatment with S. aureus alpha-toxin (hemolysin A, Hla). This study was undertaken to investigate whether this is due to attenuated ATP generation or to release of ATP from the cytosol and extracellular ATP [...] Read more.
Airway epithelial cells reduce cytosolic ATP content in response to treatment with S. aureus alpha-toxin (hemolysin A, Hla). This study was undertaken to investigate whether this is due to attenuated ATP generation or to release of ATP from the cytosol and extracellular ATP degradation by ecto-enzymes. Exposure of cells to rHla did result in mitochondrial calcium uptake and a moderate decline in mitochondrial membrane potential, indicating that ATP regeneration may have been attenuated. In addition, ATP may have left the cells through transmembrane pores formed by the toxin or through endogenous release channels (e.g., pannexins) activated by cellular stress imposed on the cells by toxin exposure. Exposure of cells to an alpha-toxin mutant (H35L), which attaches to the host cell membrane but does not form transmembrane pores, did not induce ATP release from the cells. The Hla-mediated ATP-release was completely blocked by IB201, a cyclodextrin-inhibitor of the alpha-toxin pore, but was not at all affected by inhibitors of pannexin channels. These results indicate that, while exposure of cells to rHla may somewhat reduce ATP production and cellular ATP content, a portion of the remaining ATP is released to the extracellular space and degraded by ecto-enzymes. The release of ATP from the cells may occur directly through the transmembrane pores formed by alpha-toxin. Full article
(This article belongs to the Collection Staphylococcus aureus Toxins)
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<p>Residual intracellular ATP and extracellular ATP in airway epithelial cells exposed to <span class="html-italic">S. aureus</span> α-toxin (rHla), rHla-H35L or a blocker of the rHla-pore, IB201. The amounts of intracellular (<b>A</b>,<b>C</b>) or extracellular (<b>B</b>,<b>D</b>) ATP were determined using a luminometric assay in suspended 16HBE14o- cells (<b>light grey</b> bars) or S9 cells (<b>dark grey</b> bars) exposed for 1 h (<b>A</b>,<b>B</b>) or 2 h (<b>C</b>,<b>D</b>) to 1000 or 2000 ng/mL rHla or to the pore formation-deficient mutant rHla-H35L, or to the rHla pore blocker IB201 (1 µmol/L), respectively. Assays supplemented with phosphate-buffered saline (PBS) as a vehicle for rHla or dimethyl sulfoxide (DMSO) as a vehicle for IB201, respectively, served as controls. Data are presented as means ± S.D. (numbers of independent experiments as indicated by the numbers next to the bars). Testing the data series for acceptance of the H<sub>0</sub> hypothesis (no differences of means of all data series) using ANOVA revealed that this hypothesis had to be declined (<span class="html-italic">p</span> &gt; 0.05) for all series except for the data on extracellular ATP in 16HBE14o- cells treated for 2 h ((<b>D</b>), <span class="html-italic">p</span> &lt; 0.05). Comparisons of individual means (experimental vs. PBS controls): * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 2
<p>Exposure of airway epithelial cells to <span class="html-italic">S. aureus</span> α-toxin results in the reduction of inner mitochondrial membrane potential and calcium uptake into the matrix. 16HBE14o- or S9 cells were loaded for 1 h with 5 nmol/L of the fluorescent indicator dye tetramethylrhodamine methyl ester (TMRM<sup>+</sup>), which shows different subcellular localization and fluorescence intensities with changing values of the inner mitochondrial membrane potential. (<b>A</b>,<b>B</b>) examples of fluorescent images obtained in TMRM<sup>+</sup>-loaded 16HBE14o- cells kept under control conditions for 1 h (<b>A</b>) or after exposure to 2000 ng/mL rHla for 1 h (<b>B</b>); (<b>C</b>,<b>D</b>) TMRM<sup>+</sup> fluorescence intensities in 16HBE14o- cells (<b>C</b>) or S9 cells (<b>D</b>) measured after 1 h of cell exposure to the vehicle (PBS, control), 2000 ng/mL rHla or rHla-H35L, respectively. Data are presented as means ± standard deviation (S.D.) (<span class="html-italic">n</span> = 4). Testing the data series for acceptance of the H<sub>0</sub> hypothesis (no differences of means of all data series) using ANOVA revealed that this hypothesis had to be declined (<span class="html-italic">p</span> &lt; 0.05) for 16HBE14o- cells (<a href="#toxins-08-00365-f002" class="html-fig">Figure 2</a>C) and accepted for S9 cells (<a href="#toxins-08-00365-f002" class="html-fig">Figure 2</a>D, <span class="html-italic">p</span> &gt; 0.05). Comparisons of individual means (experimental vs. PBS controls) of data obtained using 16HBE14o- cells, however, did not reveal any significant differences; (<b>E</b>) Rhod 2-fluorescence indicating concentrations of free calcium ions in the mitochrondrial matrix of suspended and dye-loaded 16HBE14o- cells in the absence (control) or presence of 2000 ng/mL rHla (addition of vehicle or rHla at 5 min). Data are presented as means ± S.D. (<span class="html-italic">n</span> = 3).</p> Full article ">Figure 3
<p>Degradation of ATP added to the extracellular medium of cultured 16HBE14o- or S9 airway epithelial cells. The amount of residual ATP was measured luminometrically in medium samples taken from confluent cultures of airway epithelial cells ((<b>A</b>) 16HBE14o-; (<b>B</b>) S9) after initial spiking of the medium with 0.3 µmol/L ATP. Results of assays performed in the presence of cells are indicated by dots, and those of assays performed in the absence of cells (controls) are indicated by triangles. Data are presented as means ± S.D. (<span class="html-italic">n</span> = 3). Significant differences of means compared with the controls: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>Residual intracellular ATP and extracellular ATP in airway epithelial cells exposed to <span class="html-italic">S. aureus</span> rHla in the absence or in the presence of Panx1 channel blockers. The amounts of intracellular (<b>A</b>,<b>C</b>) or extracellular (<b>B</b>,<b>D</b>) ATP were determined using a luminometric assay in suspended 16HBE14o- cells (<b>light grey</b> bars) or S9 cells (<b>dark grey</b> bars) exposed for 1 h (<b>A</b>,<b>B</b>) or 2 h (<b>C</b>,<b>D</b>) to 2000 ng/mL rHla or to the Panx1 pore blockers brilliant blue FCF (5 µmol/L) or carbenoxolone (10 µmol/L), respectively. Assays supplemented with PBS as the vehicle for these agents served as controls. Data are presented as means ± S.D. (numbers of independent experiments as indicated by the numbers next to the bars). Testing the data series for acceptance of the H<sub>0</sub> hypothesis (no differences of means of all data series) using ANOVA revealed that this hypothesis had to be declined (<span class="html-italic">p</span> &gt; 0.05) for all series except for the data on extracellular ATP in cells (16HBE14o- as well as S9) treated for 2 h ((<b>D</b>) <span class="html-italic">p</span> &lt; 0.05). Comparisons of individual means (experimental vs. PBS controls): * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">
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Article
Co-Occurrence of Regulated, Masked and Emerging Mycotoxins and Secondary Metabolites in Finished Feed and Maize—An Extensive Survey
by Paula Kovalsky, Gregor Kos, Karin Nährer, Christina Schwab, Timothy Jenkins, Gerd Schatzmayr, Michael Sulyok and Rudolf Krska
Toxins 2016, 8(12), 363; https://doi.org/10.3390/toxins8120363 - 6 Dec 2016
Cited by 176 | Viewed by 11297
Abstract
Global trade of agricultural commodities (e.g., animal feed) requires monitoring for fungal toxins. Also, little is known about masked and emerging toxins and metabolites. 1926 samples from 52 countries were analysed for toxins and metabolites. Of 162 compounds detected, up to 68 metabolites [...] Read more.
Global trade of agricultural commodities (e.g., animal feed) requires monitoring for fungal toxins. Also, little is known about masked and emerging toxins and metabolites. 1926 samples from 52 countries were analysed for toxins and metabolites. Of 162 compounds detected, up to 68 metabolites were found in a single sample. A subset of 1113 finished feed, maize and maize silage samples containing 57 compounds from 2012 to 2015 from 44 countries was investigated using liquid chromatography and mass spectrometry. Deoxynivalenol (DON), zearalenone (ZEN) and fumonisins showed large increases of annual medians in Europe. Within a region, distinct trends were observed, suggesting importance of local meteorology and cultivars. In 2015, median DON concentrations increased to 1400 μ g·kg − 1 in Austria, but were stable in Germany at 350 μ g·kg − 1 . In 2014, enniatins occurred at median concentrations of 250 μ g·kg − 1 in Europe, at levels similar to DON and ZEN. The latter were frequently correlated with DON-3-glucoside and ZEN-14-sulfate. Co-occurrence of regulated toxins was frequent with e.g., enniatins, and moniliformin. Correlation was observed between DON and DON-3-glucoside and with beauvericin. Results indicate that considerably more than 25% of agricultural commodities could be contaminated with mycotoxins as suggested by FAO, although this is at least partly due to the lower limits of detection in the current survey. Observed contamination percentages ranged from 7.1 to 79% for B trichothecenes and 88% for ZEN. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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<p>Simplified global maize trade for 2015. Map shows the largest importer (red) and exporter (blue) on each continent with the five largest countries of origin and destination, respectively [<a href="#B7-toxins-08-00363" class="html-bibr">7</a>].</p> Full article ">Figure 2
<p>Survey results for regulated toxins and toxins with guidance levels in 335 finished feed samples in Central Europe above defined thresholds listed in <a href="#toxins-08-00363-t001" class="html-table">Table 1</a>. <span class="html-italic">n</span> provides number of samples. Boxplots follow definition by McGill et al. [<a href="#B77-toxins-08-00363" class="html-bibr">77</a>].</p> Full article ">Figure 3
<p>Survey results for regulated toxins in finished feed samples in Central Europe. (<b>a</b>) Percentage of samples with concentrations above thresholds; see <a href="#toxins-08-00363-t001" class="html-table">Table 1</a> for details; (<b>b</b>) and (<b>c</b>) Yearly median concentrations from 2012 to 2015 (missing point indicates that no data were available). Error bars reflect the Wilcoxon confidence interval (CI). Lower error were replaced with the median, if the Wilcoxon CI would have resulted in negative concentrations. Significance codes show differences between yearly medians from a Kruskal–Wallis test result. Different letters indicate a significant difference between the groups. Data points were offset on the <span class="html-italic">x</span>-axis for clarity. Sample numbers for calculation of the median of each year are availale in <a href="#toxins-08-00363-t003" class="html-table">Table 3</a>.</p> Full article ">Figure 4
<p>Survey results for (<b>a</b>) masked and (<b>b</b>) emerging toxins in finished feed samples from Central Europe (335 samples) above threshold concentrations; see <a href="#toxins-08-00363-t001" class="html-table">Table 1</a> for details.</p> Full article ">Figure 5
<p>(<b>a</b>) Survey results for masked (DON-3-glucoside and ZEN-14-sulfate) and emerging toxins in finished feed samples from Central Europe above threshold levels; see <a href="#toxins-08-00363-t001" class="html-table">Table 1</a> for details. Subfigures (<b>b</b>) masked, (<b>c</b>) and (<b>d</b>) show yearly median data for emerging toxins for the years 2012–2015 in Central Europe. Error bars reflect the Wilcoxon confidence interval (CI). Lower error were replaced with the median, if the Wilcoxon CI would have resulted in negative concentrations. Significance codes show differences between yearly medians from a Kruskal–Wallis test result. Different letters indicate a significant difference between the groups. Data points were offset on the <span class="html-italic">x</span>-axis for clarity. Sample numbers for calculation of the median of each year are available in <a href="#toxins-08-00363-t003" class="html-table">Table 3</a>.</p> Full article ">Figure 6
<p>Correlation of (<b>a</b>) DON and DON-3-glucoside in Central Europe; (<b>b</b>) DON with BEA (Eastern Europe); (<b>c</b>) DON with DON-3-glucoside (Eastern Europe) and (<b>d</b>) sum of T-2 and HT-2 toxins with BEA in finished feed samples (Eastern Europe).</p> Full article ">Figure 7
<p>Yearly median concentrations of regulated toxins and compounds with guidance levels in finished feed from (<b>a</b>) and (<b>b</b>) Austria, (<b>c</b>) and (<b>d</b>) Germany from 2012 to 2015. Error bars reflect the Wilcoxon confidence interval (CI). Lower error were replaced with the median, if the Wilcoxon CI would have resulted in negative concentrations. Significance codes show differences between yearly medians from a Kruskal–Wallis test result. Different letters indicate a significant difference between the groups. Data points were offset on the <span class="html-italic">x</span>-axis for clarity. Sample numbers for calculation of the median of each year are availale in <a href="#toxins-08-00363-t003" class="html-table">Table 3</a>. Yearly median concentrations of regulated toxins and compounds with guidance levels in finished feed from (<b>e</b>) and (<b>f</b>) Italy, and (<b>g</b>) and (<b>h</b>) The Netherlands from 2012 to 2015. Error bars reflect the Wilcoxon confidence interval (CI). Lower error were replaced with the median, if the Wilcoxon CI would have resulted in negative concentrations. Significance codes show differences between yearly medians from a Kruskal–Wallis test result. Different letters indicate a significant difference between the groups. Data points were offset on the <span class="html-italic">x</span>-axis for clarity. Sample numbers for calculation of the median of each year are available in <a href="#toxins-08-00363-t003" class="html-table">Table 3</a>.</p> Full article ">Figure 7 Cont.
<p>Yearly median concentrations of regulated toxins and compounds with guidance levels in finished feed from (<b>a</b>) and (<b>b</b>) Austria, (<b>c</b>) and (<b>d</b>) Germany from 2012 to 2015. Error bars reflect the Wilcoxon confidence interval (CI). Lower error were replaced with the median, if the Wilcoxon CI would have resulted in negative concentrations. Significance codes show differences between yearly medians from a Kruskal–Wallis test result. Different letters indicate a significant difference between the groups. Data points were offset on the <span class="html-italic">x</span>-axis for clarity. Sample numbers for calculation of the median of each year are availale in <a href="#toxins-08-00363-t003" class="html-table">Table 3</a>. Yearly median concentrations of regulated toxins and compounds with guidance levels in finished feed from (<b>e</b>) and (<b>f</b>) Italy, and (<b>g</b>) and (<b>h</b>) The Netherlands from 2012 to 2015. Error bars reflect the Wilcoxon confidence interval (CI). Lower error were replaced with the median, if the Wilcoxon CI would have resulted in negative concentrations. Significance codes show differences between yearly medians from a Kruskal–Wallis test result. Different letters indicate a significant difference between the groups. Data points were offset on the <span class="html-italic">x</span>-axis for clarity. Sample numbers for calculation of the median of each year are available in <a href="#toxins-08-00363-t003" class="html-table">Table 3</a>.</p> Full article ">Figure 8
<p>Survey results for type A and type B trichotheces from (<b>a</b>) South Africa and (<b>b</b>) Central Europe in finished feed samples above threshold concentrations; see <a href="#toxins-08-00363-t001" class="html-table">Table 1</a> for details.</p> Full article ">Figure 9
<p>Number of samples n in the investigated data set from a (<b>a</b>) Global and (<b>b</b>) European perspective.</p> Full article ">
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Article
Metabolism of HT-2 Toxin and T-2 Toxin in Oats
by Jacqueline Meng-Reiterer, Christoph Bueschl, Justyna Rechthaler, Franz Berthiller, Marc Lemmens and Rainer Schuhmacher
Toxins 2016, 8(12), 364; https://doi.org/10.3390/toxins8120364 - 5 Dec 2016
Cited by 31 | Viewed by 7222
Abstract
The Fusarium mycotoxins HT-2 toxin (HT2) and T-2 toxin (T2) are frequent contaminants in oats. These toxins, but also their plant metabolites, may contribute to toxicological effects. This work describes the use of 13C-assisted liquid chromatography–high-resolution mass spectrometry for the first comprehensive [...] Read more.
The Fusarium mycotoxins HT-2 toxin (HT2) and T-2 toxin (T2) are frequent contaminants in oats. These toxins, but also their plant metabolites, may contribute to toxicological effects. This work describes the use of 13C-assisted liquid chromatography–high-resolution mass spectrometry for the first comprehensive study on the biotransformation of HT2 and T2 in oats. Using this approach, 16 HT2 and 17 T2 metabolites were annotated including novel glycosylated and hydroxylated forms of the toxins, hydrolysis products, and conjugates with acetic acid, putative malic acid, malonic acid, and ferulic acid. Further targeted quantitative analysis was performed to study toxin metabolism over time, as well as toxin and conjugate mobility within non-treated plant tissues. As a result, HT2-3-O-β-d-glucoside was identified as the major detoxification product of both parent toxins, which was rapidly formed (to an extent of 74% in HT2-treated and 48% in T2-treated oats within one day after treatment) and further metabolised. Mobility of the parent toxins appeared to be negligible, while HT2-3-O-β-d-glucoside was partly transported (up to approximately 4%) through panicle side branches and stem. Our findings demonstrate that the presented combination of untargeted and targeted analysis is well suited for the comprehensive elucidation of mycotoxin metabolism in plants. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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<p>Overlay of extracted ion chromatograms (EICs) of HT2/T2 plant metabolites. (<b>a</b>) Overlaid EICs of HT2 metabolites based on HT2-treated oat sample (time point full-ripening) and <a href="#toxins-08-00364-t001" class="html-table">Table 1</a>; (<b>b</b>) overlaid EICs of T2 metabolites based on T2-treated oat samples (time point full-ripening and accumulated time points marked with an asterisk) and <a href="#toxins-08-00364-t002" class="html-table">Table 2</a>. Oat panicles were treated with a 1/1 mixture of non-labelled and uniformly <sup>13</sup>C-labelled toxin, extracted and analysed by LC-Orbitrap-MS in positive and negative ion mode and MetExtract II software. Non-labelled metabolite form is depicted with positive intensity (up) and corresponding <sup>13</sup>C-labelled metabolite form with negative intensity (down). HT2, HT-2 toxin; T2, T-2 toxin.</p> Full article ">Figure 2
<p>Time courses of native toxins and major plant-derived metabolites. (<b>a</b>) Time course of HT2 (HT-2 toxin) metabolism; (<b>b</b>) time course of T2 (T-2 toxin) metabolism. Oat panicles were treated with 200 μg non-labelled HT2 and T2 and harvested directly (zero), and one, three, or seven days after treatment, or at full-ripening stage (for each time point toxins were applied on separate panicles in five biological replicates). Quantification was performed on a LC-Q-TOF-system; absolute amounts were calculated in μmol/treated part and put in relation to the amounts of recovered HT2 and T2 at time point zero days (percent mean value ± standard deviation is illustrated). HT2-Glc, HT2-3-<span class="html-italic">O</span>-<span class="html-italic">β</span>-<span class="html-small-caps">d</span>-glucoside.</p> Full article ">Figure 3
<p>Relative time courses of plant-derived metabolites. (<b>a</b>) Relative time courses of HT2 (HT-2 toxin) metabolites; (<b>b</b>) relative time courses of T2 (T-2 toxin) metabolites. Oat panicles were treated with 200 μg non-labelled HT2 and T2 and harvested directly (zero), and one, three, or seven days after treatment, or at the full-ripening stage (for each time point toxins were applied on separate panicles in five biological replicates). Relative quantification was performed on a LC-Q-TOF-system; peak areas of ammonium adducts were normalised by respective weight of treated plant part (area mean value ± standard deviation is illustrated). Glc, glucoside; HexGlc, hexosylglucoside.</p> Full article ">Figure 4
<p>Proposed metabolic fate of HT2 (HT-2 toxin) and T2 (T-2 toxin) in oats. Analysis was performed by liquid chromatography–high-resolution mass spectrometry (LC-HRMS). Structure annotation was based on accurate masses, number of parent toxin-derived C-atoms, LC-HRMS/MS spectra, and on assessment of retention times. Structure identification was based on comparisons with available standards. Glc, glucoside; MalGlc, malonylglucoside; HexGlc, hexosylglucoside; isoval acid, isovaleric acid.</p> Full article ">
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Article
A P-Glycoprotein Is Linked to Resistance to the Bacillus thuringiensis Cry3Aa Toxin in a Leaf Beetle
by Yannick Pauchet, Anne Bretschneider, Sylvie Augustin and David G. Heckel
Toxins 2016, 8(12), 362; https://doi.org/10.3390/toxins8120362 - 5 Dec 2016
Cited by 51 | Viewed by 7500
Abstract
Chrysomela tremula is a polyvoltine oligophagous leaf beetle responsible for massive attacks on poplar trees. This beetle is an important model for understanding mechanisms of resistance to Bacillus thuringiensis (Bt) insecticidal toxins, because a resistant C. tremula strain has been found that can [...] Read more.
Chrysomela tremula is a polyvoltine oligophagous leaf beetle responsible for massive attacks on poplar trees. This beetle is an important model for understanding mechanisms of resistance to Bacillus thuringiensis (Bt) insecticidal toxins, because a resistant C. tremula strain has been found that can survive and reproduce on transgenic poplar trees expressing high levels of the Cry3Aa Bt toxin. Resistance to Cry3Aa in this strain is recessive and is controlled by a single autosomal locus. We used a larval midgut transcriptome for C. tremula to search for candidate resistance genes. We discovered a mutation in an ABC protein, member of the B subfamily homologous to P-glycoprotein, which is genetically linked to Cry3Aa resistance in C. tremula. Cultured insect cells heterologously expressing this ABC protein swell and lyse when incubated with Cry3Aa toxin. In light of previous findings in Lepidoptera implicating A subfamily ABC proteins as receptors for Cry2A toxins and C subfamily proteins as receptors for Cry1A and Cry1C toxins, this result suggests that ABC proteins may be targets of insecticidal three-domain Bt toxins in Coleoptera as well. Full article
(This article belongs to the Special Issue The Insecticidal Bacterial Toxins in Modern Agriculture)
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<p>Diagram of the CtABCB1 protein structure and location of the mutation present in resistant <span class="html-italic">C. tremula</span> individuals. Predicted glycosylation sites on two of the extracellular loops are represented by “Y.” Two highly conserved ATP nucleotide binding folds (NBF1, NBF2) that include the transporter signature motifs 1 and 2 (TpM1, TpM2) are present in the intracellular environment. The structure of CtABCB1consists of two transmembrane domains (TMD 1, TMD 2), each of them made of six transmembrane helices (TM I-VI in TMD 1; TM VII-XII in TMD 2). The approximate position of the four-base-pair deletion discovered in resistant individuals is indicated by a red arrow.</p> Full article ">Figure 2
<p>Genotyping of the mutation in CtABCB1 in backcrosses between susceptible and resistant individuals. Crosses (mating pairs) between individuals of the susceptible and the resistant strains were set up in 2015 (panels <b>a</b>,<b>b</b>) and in 2011 (panel <b>c</b>). The progeny of these crosses (F1) were backcrossed to individuals of the resistant strains also in mating pairs. (<b>a</b>) Phenotype and genotype for backcross family 48; (<b>b</b>) Phenotype and genotype for backcross family 58; (<b>c</b>) Phenotype and genotype for the backcrosses set up in 2011 which correspond to the offspring from seven backcross families having all the same pair of grandparents but different pairs of parents. The offspring of these backcrosses were selected for four days on leaves of Bt poplars. During this time, individuals found dead were considered susceptible to Cry3Aa (phenotype S) and the ones that survived and actively fed were considered resistant to Cry3Aa (phenotype R). Genotyping of each individual was performed by amplifying by PCR the region where the deletion was discovered followed by Sanger sequencing. Individuals with genotype “<span class="html-italic">rr</span>” are homozygous for the presence of the four-base-pair deletion on CtABCB1, whereas individuals with genotype “<span class="html-italic">rs</span>” are heterozygous for the presence of this mutation. “No data” indicates that the genotyping did not work, neither at the PCR level nor at the sequencing level.</p> Full article ">Figure 3
<p>Heterologous expression of CtABCB1 in insect <span class="html-italic">Sf</span>9 cells. (<b>a</b>) Western blot with a V5 epitope-specific antiserum of both cytosoluble fraction (C) and crude membrane fraction (M) prepared from untransfected and transfected <span class="html-italic">Sf</span>9 cells; (<b>b</b>) Effect of the Cry3Aa toxin on cell viability (±SD). Trypsin-activated Cry3Aa was used in concentrations ranging from 10<sup>−12</sup> M to 3.10<sup>−7</sup> M and cells were treated for 24 h. Blue squares: untransfected <span class="html-italic">Sf</span>9 cells. Red squares: CtABCB1-expressing <span class="html-italic">Sf</span>9 cells. The data are based on a MTT assay (<span class="html-italic">N</span> = 6). Values over 100% are due to increase in cell number due to cell division over time in the untransfected <span class="html-italic">Sf</span>9 cells; (<b>c</b>) Morphological changes of <span class="html-italic">Sf</span>9 cells treated with 30 nM trypsin-activated Cry3Aa. Cells were observed for eight hours and pictures were taken every two hours. Scale bars: 10 µm.</p> Full article ">
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Article
Higher Levels of Aflatoxin M1 Contamination and Poorer Composition of Milk Supplied by Informal Milk Marketing Chains in Pakistan
by Naveed Aslam, Muhammad Yasin Tipu, Muhammad Ishaq, Ann Cowling, David McGill, Hassan Mahmood Warriach and Peter Wynn
Toxins 2016, 8(12), 347; https://doi.org/10.3390/toxins8120347 - 5 Dec 2016
Cited by 24 | Viewed by 5496
Abstract
The present study was conducted to observe the seasonal variation in aflatoxin M1 and nutritional quality of milk along informal marketing chains. Milk samples (485) were collected from three different chains over a period of one year. The average concentrations of aflatoxin M1 [...] Read more.
The present study was conducted to observe the seasonal variation in aflatoxin M1 and nutritional quality of milk along informal marketing chains. Milk samples (485) were collected from three different chains over a period of one year. The average concentrations of aflatoxin M1 during the autumn and monsoon seasons (2.60 and 2.59 ppb) were found to be significantly higher (standard error of the difference, SED = 0.21: p = 0.003) than in the summer (1.93 ppb). The percentage of added water in milk was significantly lower (SED = 1.54: p < 0.001) in summer (18.59%) than in the monsoon season (26.39%). There was a significantly different (SED = 2.38: p < 0.001) mean percentage of water added by farmers (6.23%), small collectors (14.97%), large collectors (27.96%) and retailers (34.52%). This was reflected in changes in milk quality along the marketing chain. There was no difference (p = 0.178) in concentration of aflatoxin M1 in milk collected from the farmers (2.12 ppb), small collectors (2.23 ppb), large collectors (2.36 ppb) and retailers (2.58 ppb). The high levels of contamination found in this study, which exceed the standards set by European Union (0.05 ppb) and USFDA (0.5 ppb), demand radical intervention by regulatory authorities and mass awareness of the consequences for consumer health and safety. Full article
(This article belongs to the Collection Understanding Mycotoxin Occurrence in Food and Feed Chains)
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<p>Variation in milk composition and total addition of water at different levels in informal milk marketing chains averaged across seasons for the year (data presented as means with least significant differences for each parameter). Note: ο, +, ∆, x and ◊ describe the values of solids not fat (SNF) lactose fat protein and added water respectively.</p> Full article ">Figure 2
<p>Variation in milk constituents and contaminants with season in informal milk supply chains. Note: ο, +, ∆, x and ◊ describe the values of SNF, lactose, fat, protein and added water respectively.</p> Full article ">Figure 3
<p>A typical structure for an informal milk marketing chain in Pakistan.</p> Full article ">
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Review
Recent Advances and Future Challenges in Modified Mycotoxin Analysis: Why HRMS Has Become a Key Instrument in Food Contaminant Research
by Laura Righetti, Giuseppe Paglia, Gianni Galaverna and Chiara Dall’Asta
Toxins 2016, 8(12), 361; https://doi.org/10.3390/toxins8120361 - 2 Dec 2016
Cited by 55 | Viewed by 8240
Abstract
Mycotoxins are secondary metabolites produced by pathogenic fungi in crops worldwide. These compounds can undergo modification in plants, leading to the formation of a large number of possible modified forms, whose toxicological relevance and occurrence in food and feed is still largely unexplored. [...] Read more.
Mycotoxins are secondary metabolites produced by pathogenic fungi in crops worldwide. These compounds can undergo modification in plants, leading to the formation of a large number of possible modified forms, whose toxicological relevance and occurrence in food and feed is still largely unexplored. The analysis of modified mycotoxins by liquid chromatography–mass spectrometry remains a challenge because of their chemical diversity, the large number of isomeric forms, and the lack of analytical standards. Here, the potential benefits of high-resolution and ion mobility mass spectrometry as a tool for separation and structure confirmation of modified mycotoxins have been investigated/reviewed. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Workflow for targeted analysis with mycotoxin reference standards, targeted screening without analytical standards, and untargeted screening for unexpected unknowns (adapted from Krauss et al. [<a href="#B25-toxins-08-00361" class="html-bibr">25</a>]). CCS = Collision Cross Section.</p> Full article ">Figure 2
<p>Extracted ion chromatogram for DON di glucosides determined in malt (<b>A1</b>) and beer (<b>A2</b>) by using HILIC phase chromatography coupled to HRMS (Orbitrap). (Reproduced with permission from [<a href="#B29-toxins-08-00361" class="html-bibr">29</a>], copyright (2016) American Chemical Society).</p> Full article ">Figure 3
<p>ZEL-MalGlc putative identification steps: UHPLC-Q-Exactive (<b>A</b>) full scan extracted ion chromatogram (resolving power 70,000 FWHM, extraction window 5 ppm); (<b>B</b>) molecular formula assignment of parent ion; theoretical and experimental isotopic pattern comparison (<b>C</b>,<b>D</b>) high resolution fragmentation pathways obtained by using DDA acquisition.</p> Full article ">Figure 4
<p>MS and MS/MS cleaner mass spectra obtained by using LC-IM-QTOF (<b>b</b>) compared to those obtained by LC-QTOF (<b>a</b>) (from Paglia et al. [<a href="#B86-toxins-08-00361" class="html-bibr">86</a>]).</p> Full article ">
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Correction
Correction: Chen, S., et al. Identification of an Essential Region for Translocation of Clostridium difficile Toxin B. Toxins 2016, 8, 241
by Shuyi Chen, Haiying Wang, Huawei Gu, Chunli Sun, Shan Li, Hanping Feng and Jufang Wang
Toxins 2016, 8(12), 352; https://doi.org/10.3390/toxins8120352 - 2 Dec 2016
Cited by 1 | Viewed by 4329
Abstract
n/a Full article
(This article belongs to the Collection Clostridium difficile/Clostridium sordellii and Clostridium perfringens Toxins)
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Review
Snake Genome Sequencing: Results and Future Prospects
by Harald M. I. Kerkkamp, R. Manjunatha Kini, Alexey S. Pospelov, Freek J. Vonk, Christiaan V. Henkel and Michael K. Richardson
Toxins 2016, 8(12), 360; https://doi.org/10.3390/toxins8120360 - 1 Dec 2016
Cited by 26 | Viewed by 8740
Abstract
Snake genome sequencing is in its infancy—very much behind the progress made in sequencing the genomes of humans, model organisms and pathogens relevant to biomedical research, and agricultural species. We provide here an overview of some of the snake genome projects in progress, [...] Read more.
Snake genome sequencing is in its infancy—very much behind the progress made in sequencing the genomes of humans, model organisms and pathogens relevant to biomedical research, and agricultural species. We provide here an overview of some of the snake genome projects in progress, and discuss the biological findings, with special emphasis on toxinology, from the small number of draft snake genomes already published. We discuss the future of snake genomics, pointing out that new sequencing technologies will help overcome the problem of repetitive sequences in assembling snake genomes. Genome sequences are also likely to be valuable in examining the clustering of toxin genes on the chromosomes, in designing recombinant antivenoms and in studying the epigenetic regulation of toxin gene expression. Full article
(This article belongs to the Special Issue Snake Venom Metalloproteinases)
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<p>Syntenic comparisons of venom genes in the king cobra with other vertebrates revealing toxin recruitment by hijacking/modification and gene duplication. (<b>A</b>) Modification of PLBD1 gene found in the green anole lizard (<span class="html-italic">Anolis carolinensis</span>) and the chicken (<span class="html-italic">Gallus gallus</span>) results in the venom gland expressed phospholipase-B (PLB). Note that PLB is found split across two king cobra genome scaffolds; (<b>B</b>) Modification of HYALP1 gene found in the mouse (<span class="html-italic">Mus musculus</span>) results in the venom gland expressed hyaluronidase (HYAL); (<b>C</b>) Duplication of the non-venom gland expressed ADAM gene in the king cobra results in a venom gland expressed snake venom metalloproteinase (SVMP) gene. The ADAM gene in the green anole is flanked on both sides by non-SVMP genes, demonstrating the absence of gene duplication in this species. Note that subsequent downstream duplication of the SVMP gene in the king cobra results in multiple venom gland expressed SVMP isoforms. Based on Figure S5 from [<a href="#B3-toxins-08-00360" class="html-bibr">3</a>].</p> Full article ">Figure 2
<p>Preliminary analysis of three finger toxin isoforms in the king cobra genome. (<b>a</b>) Phylogeny showing isoform numbers; (<b>b</b>) Expression level (transcript abundance) in the venom gland; (<b>c</b>) Apparent copy number in genome. One hypothesis consistent with the figure is that the more recently expanded paralogues tend to be more highly expressed. The figure is an unpublished analysis by one of us (Christiaan Henkel) based on data in Ref. [<a href="#B3-toxins-08-00360" class="html-bibr">3</a>]. See <a href="#toxins-08-00360-t004" class="html-table">Table 4</a> for corresponding genome sequencing and accession codes of the three finger toxin isoforms.</p> Full article ">
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Review
The Emergence and Epidemiology of Haff Disease in China
by Thomas Y. K. Chan
Toxins 2016, 8(12), 359; https://doi.org/10.3390/toxins8120359 - 1 Dec 2016
Cited by 22 | Viewed by 8470
Abstract
Haff disease is a rare syndrome of unexplained myalgia and rhabdomyolysis occurring within 24 h of consumption of certain types of cooked freshwater fish or crustacean. It is caused by a yet unidentified heat-stable toxin. In the present review of published case studies [...] Read more.
Haff disease is a rare syndrome of unexplained myalgia and rhabdomyolysis occurring within 24 h of consumption of certain types of cooked freshwater fish or crustacean. It is caused by a yet unidentified heat-stable toxin. In the present review of published case studies and official press releases, the main objective is to report the emergence and epidemiology of Haff disease in China. Haff disease first occurred in Beijing in 2000 and in Lianzhou and Liannan, Guangdong Province in 2009. Subsequent outbreaks mostly occurred in the Jiangsu Province—Nanjing, Yangzhou, Huai’an, and Yancheng. Isolated outbreaks occurred in other cities since 2010—Shijiazhuang, Yueyang, Shanghai, Wuhu, Baoding, Shenzhen, and Hong Kong (imported cases from Shenzhen). Outbreaks occurred predominately in the summer. Crayfish accounted for almost all the outbreaks. Two large outbreaks occurred in Lianzhou and Liannan in 2009 (n = 54) after eating pomfrets and in Nanjing in 2010 (n = 42) after eating crayfish. Other reports or outbreaks involved only 1–9 subjects (median 2 subjects). Variability in individual susceptibility and attack rates were noted, with many subjects remaining asymptomatic despite sharing the same seafood meal as the index cases. Adults were predominately involved. Symptoms occurred within 3–20 h of seafood ingestion, including myalgia, weakness, and, less frequently, nausea, vomiting, abdominal pain, and diarrhea. Myalgia and muscle weakness should normally subside within 2–3 days. Serum creatine phosphokinase became normal within 5–6 days. Abnormal renal function was uncommon. Serious complications (renal failure, multi-organ failure, and prolonged myopathy) and death were rare. In any subjects with unexplained myalgia and rhabdomyolysis, seafood consumption should be included in the history. All suspected cases of Haff disease, including milder presentations, should be reported to public health authorities. Full article
(This article belongs to the Section Marine and Freshwater Toxins)
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<p>The cities and provinces of China where Haff disease has been reported since 2000.</p> Full article ">
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Review
Indoxyl Sulfate—Review of Toxicity and Therapeutic Strategies
by Sheldon C. Leong and Tammy L. Sirich
Toxins 2016, 8(12), 358; https://doi.org/10.3390/toxins8120358 - 30 Nov 2016
Cited by 189 | Viewed by 9582
Abstract
Indoxyl sulfate is an extensively studied uremic solute. It is a small molecule that is more than 90% bound to plasma proteins. Indoxyl sulfate is derived from the breakdown of tryptophan by colon microbes. The kidneys achieve high clearances of indoxyl sulfate by [...] Read more.
Indoxyl sulfate is an extensively studied uremic solute. It is a small molecule that is more than 90% bound to plasma proteins. Indoxyl sulfate is derived from the breakdown of tryptophan by colon microbes. The kidneys achieve high clearances of indoxyl sulfate by tubular secretion, a function not replicated by hemodialysis. Clearance by hemodialysis is limited by protein binding since only the free, unbound solute can diffuse across the membrane. Since the dialytic clearance is much lower than the kidney clearance, indoxyl sulfate accumulates to relatively high plasma levels in hemodialysis patients. Indoxyl sulfate has been most frequently implicated as a contributor to renal disease progression and vascular disease. Studies have suggested that indoxyl sulfate also has adverse effects on bones and the central nervous system. The majority of studies have assessed toxicity in cultured cells and animal models. The toxicity in humans has not yet been proven, as most data have been from association studies. Such toxicity data, albeit inconclusive, have prompted efforts to lower the plasma levels of indoxyl sulfate through dialytic and non-dialytic means. The largest randomized trial showed no benefit in renal disease progression with AST-120. No trials have yet tested cardiovascular or mortality benefit. Without such trials, the toxicity of indoxyl sulfate cannot be firmly established. Full article
(This article belongs to the Special Issue Novel Issues in Uremic Toxicity)
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Article
A Topographical Atlas of Shiga Toxin 2e Receptor Distribution in the Tissues of Weaned Piglets
by Daniel Steil, Robert Bonse, Iris Meisen, Gottfried Pohlentz, German Vallejo, Helge Karch and Johannes Müthing
Toxins 2016, 8(12), 357; https://doi.org/10.3390/toxins8120357 - 30 Nov 2016
Cited by 15 | Viewed by 5013
Abstract
Shiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) is the primary virulence factor in the development of pig edema disease shortly after weaning. Stx2e binds to the globo-series glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer), the latter acting as the [...] Read more.
Shiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) is the primary virulence factor in the development of pig edema disease shortly after weaning. Stx2e binds to the globo-series glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer), the latter acting as the preferential Stx2e receptor. We determined Stx receptor profiles of 25 different tissues of a male and a female weaned piglet using immunochemical solid phase binding assays combined with mass spectrometry. All probed tissues harbored GSL receptors, ranging from high (category I) over moderate (category II) to low content (category III). Examples of Gb4Cer expression in category I tissues are small intestinal ileum, kidney pelvis and whole blood, followed by colon, small intestinal duodenum and jejunum belonging to category II, and kidney cortex, cerebrum and cerebellum as members of category III organs holding true for both genders. Dominant Gb3Cer and Gb4Cer lipoforms were those with ceramides carrying constant sphingosine (d18:1) and a variable C16:0, C22:0 or C24:1/C24:0 fatty acid. From the mapping data, we created a topographical atlas for Stx2e receptors in piglet tissues and organs, which might be helpful to further investigations on the molecular and cellular mechanisms that underlie infections of Stx2e-producing STEC in pigs and their zoonotic potential for humans. Full article
(This article belongs to the Collection Shiga Toxins)
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<p>Detection of Stx2e receptors with anti-Gb3Cer antibody in tissues and organs of the male piglet. Aliquots of the GSL extracts of 25 tissues and organs (see <a href="#toxins-08-00357-t001" class="html-table">Table 1</a>) were applied to TLC separation, followed by TLC overlay assays with polyclonal anti-Gb3Cer antibody. A reference GSL mixture (R) containing equimolar quantities of Gb3Cer, Gb4Cer and Forssman GSL served as positive control. Left-hand and right-hand references correspond to 0.1 µg and 0.02 µg of the GSL mixture, respectively.</p> Full article ">Figure 2
<p>Overview mass spectra of Gb3Cer lipoforms derived from: duodenum (<b>A</b>); and colon (<b>B</b>) of the male piglet. The MS<sup>1</sup> spectra display the <span class="html-italic">m</span>/<span class="html-italic">z</span> range between 1020 and 1180 comprising Gb3Cer lipoforms with variable fatty acid from C16:0 up to C24:1/C24:0 as assigned in the spectrum. All Gb3Cer variants carry constant sphingosine (d18:1) in their respective ceramide moiety. The asterisks point to mass shifts of 15.99 u indicating presence of an additional OH-group in the ceramide moiety, most likely linked to the fatty acyl chain, of Gb3Cer variants in the male: duodenum (<b>A</b>); and colon (<b>B</b>).</p> Full article ">Figure 3
<p>Overview mass spectra of Gb3Cer lipoforms derived from: cortex (<b>A</b>); medulla (<b>B</b>); and pelvis (<b>C</b>) of the male piglet. The MS<sup>1</sup> spectra depict the <span class="html-italic">m</span>/<span class="html-italic">z</span> range from 1020 to 1180 encompassing Gb3Cer variants with variable fatty acid from C16:0 up to C24:1/C24:0 as marked in the spectrum. All Gb3Cer variants harbor constant sphingosine (d18:1) in their respective ceramide moiety. The asterisks indicate hydroxylation of the ceramide portion, most likely of the fatty acid, of Gb3Cer variants in the male: cortex (<b>A</b>); medulla (<b>B</b>); and pelvis (<b>C</b>).</p> Full article ">Figure 4
<p>Detection of Stx2e receptors with: anti-Gb4Cer (<b>A</b>); and anti-Forssman GSL antibody (<b>B</b>) in tissues and organs of the male piglet. Aliquots of the GSL extracts of 25 tissues and organs (see <a href="#toxins-08-00357-t001" class="html-table">Table 1</a>) were applied to TLC separation, followed by TLC overlay assays with: polyclonal anti-Gb4Cer antibody (<b>A</b>); or monoclonal anti-Forssman GSL antibody (<b>B</b>). A reference GSL mixture (R) containing equimolar quantities of Gb3Cer, Gb4Cer and Forssman GSL was used as positive control. References on the left and the right correspond: to 0.1 µg and 0.02 µg of the GSL mixture, respectively, for the anti-Gb4Cer assay (<b>A</b>); and to 0.015 µg and 0.003 µg of the GSL mixture, respectively, for anti-Forssman GSL detection (<b>B</b>).</p> Full article ">Figure 5
<p>Overview mass spectra of Gb4Cer lipoforms derived from: duodenum (<b>A</b>); and colon (<b>B</b>) of the male piglet. The MS<sup>1</sup> spectra display the <span class="html-italic">m</span>/<span class="html-italic">z</span> range between 1230 and 1400 comprising Gb4Cer lipoforms with variable fatty acid from C16:0 up to C24:1/C24:0 as assigned in the spectrum. All Gb4Cer variants carry constant sphingosine (d18:1) in their respective ceramide moiety. The two asterisks point to hydroxylated Gb4Cer in the male duodenum (<b>A</b>), whereas no hydroxylated Gb4Cer lipoforms were detectable in the male colon (<b>B</b>).</p> Full article ">Figure 6
<p>Overview mass spectra of Gb4Cer lipoforms derived from: cortex (<b>A</b>); medulla (<b>B</b>); and pelvis (<b>C</b>) of the male piglet. The MS<sup>1</sup> spectra depict the <span class="html-italic">m</span>/<span class="html-italic">z</span> range from 1230 to 1400 encompassing Gb4Cer variants with variable fatty acid from C16:0 up to C24:1/C24:0 as marked in the spectrum. All Gb4Cer variants harbor constant sphingosine (d18:1) in their respective ceramide moiety. The single asterisk in each spectrum indicates hydroxylated Gb4Cer in the male: cortex (<b>A</b>); medulla (<b>B</b>); and pelvis (<b>C</b>).</p> Full article ">Figure 7
<p>Detection of receptor GSLs with Stx2e in tissues and organs of the male piglet. Aliquots of the GSL extracts of 25 tissues and organs (see <a href="#toxins-08-00357-t001" class="html-table">Table 1</a>) were applied to TLC separation, followed by TLC overlay assays with Stx2e. A reference GSL mixture (R) containing equimolar quantities of Gb3Cer, Gb4Cer and Forssman GSL served as positive control (0.1 µg).</p> Full article ">Figure 8
<p>Detection of Stx2e receptors with anti-Gb3Cer antibody in tissues and organs of the female piglet. Aliquots of the GSL extracts of 25 tissues and organs (see <a href="#toxins-08-00357-t001" class="html-table">Table 1</a>) were applied to TLC separation, followed by TLC overlay assays with polyclonal anti-Gb3Cer antibody. A reference GSL mixture (R) containing equimolar quantities of Gb3Cer, Gb4Cer and Forssman GSL served as positive control. Left-hand and right-hand references correspond to 0.1 µg and 0.02 µg of the GSL mixture, respectively.</p> Full article ">Figure 9
<p>Detection of Stx2e receptors with anti-Gb4Cer antibody in tissues and organs of the female piglet. Aliquots of the GSL extracts of 25 tissues and organs (see <a href="#toxins-08-00357-t001" class="html-table">Table 1</a>) were applied to TLC separation, followed by TLC overlay assays with polyclonal anti-Gb4Cer antibody. A reference GSL mixture (R) containing equimolar quantities of Gb3Cer, Gb4Cer and Forssman GSL was used as positive control. References on the left and the right correspond to 0.1 µg and 0.02 µg of the GSL mixture, respectively.</p> Full article ">Figure 10
<p>Overview mass spectra of Gb4Cer lipoforms derived from: duodenum (<b>A</b>); and colon (<b>B</b>) of the female piglet. The MS<sup>1</sup> spectra display the <span class="html-italic">m</span>/<span class="html-italic">z</span> range between 1230 and 1400 comprising Gb4Cer lipoforms with variable fatty acid from C16:0 up to C24:1/C24:0 as assigned in the spectrum. All Gb4Cer variants carry constant sphingosine (d18:1) in their respective ceramide moiety. The asterisks indicate hydroxylated Gb4Cer in the female: duodenum (<b>A</b>); and colon (<b>B</b>).</p> Full article ">Figure 11
<p>Overview mass spectra of Gb4Cer lipoforms derived from: cortex (<b>A</b>); medulla (<b>B</b>); and pelvis (<b>C</b>) of the female piglet. The MS<sup>1</sup> spectra depict the <span class="html-italic">m/z</span> range from 1230 to 1400 encompassing Gb4Cer variants with variable fatty acid from C16:0 up to C24:1/C24:0 as marked in the spectrum. All Gb4Cer variants harbor constant sphingosine (d18:1) in their respective ceramide moiety. The single asterisk in each spectrum indicates hydroxylated Gb4Cer in the female: cortex (<b>A</b>); medulla (<b>B</b>); and pelvis (<b>C</b>).</p> Full article ">Figure 12
<p>Detection of receptor GSLs with Stx2e in tissues and organs of the female piglet. Aliquots of the GSL extracts of 25 tissues and organs (see <a href="#toxins-08-00357-t001" class="html-table">Table 1</a>), were applied to TLC separation, followed by TLC overlay assays with Stx2e. A reference GSL mixture (R) containing equimolar quantities of Gb3Cer, Gb4Cer and Forssman GSL served as positive control (0.1 µg).</p> Full article ">Figure 13
<p>Relative content of antibody-detected Stx2e receptor Gb3Cer in tissues and organs of male and female piglet. Gb3Cer distribution to the various tissues and organs of the male and the female piglet is depicted as a bar chart (<b>A</b>) in the order of the sample numbers from left to right according to <a href="#toxins-08-00357-f001" class="html-fig">Figure 1</a> (male piglet) and <a href="#toxins-08-00357-f008" class="html-fig">Figure 8</a> (female piglet) and as a schematic topographical atlas in the form of a cartoon map of pig tissues/organs (<b>B</b>). The size of the circle diameters corresponds to the relative Gb3Cer amount and the scale of the “pie slices” indicates the relative Gb3Cer content of the male (black) and the female animal (red).</p> Full article ">Figure 14
<p>Relative content of antibody-detected Stx2e receptor Gb4Cer in tissues and organs of male and female piglet. Gb4Cer distribution to the various tissues and organs of the male and the female piglet is depicted as a bar chart (<b>A</b>) in the order of the sample numbers from left to right according to <a href="#toxins-08-00357-f004" class="html-fig">Figure 4</a>A (male piglet) and <a href="#toxins-08-00357-f009" class="html-fig">Figure 9</a> (female piglet) and as a schematic topographical atlas in the form of a cartoon map of pig tissues/organs (<b>B</b>). The size of the circle diameters corresponds to the relative Gb4Cer amount and the scale of the “pie slices” indicates the relative Gb4Cer content of the male (black) and the female animal (red).</p> Full article ">
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Article
Contrasting Roles of Deoxynivalenol and Nivalenol in Host-Mediated Interactions between Fusarium graminearum and Sitobion avenae
by Jassy Drakulic, Mohd Haziq Kahar, Olubukola Ajigboye, Toby Bruce and Rumiana V. Ray
Toxins 2016, 8(12), 353; https://doi.org/10.3390/toxins8120353 - 30 Nov 2016
Cited by 13 | Viewed by 6589
Abstract
Fusarium graminearum is the predominant causal species of Fusarium head blight in Europe and North America. Different chemotypes of the species exist, each producing a plethora of mycotoxins. Isolates of differing chemotypes produce nivalenol (NIV) and deoxynivalenol (DON), which differ in toxicity to [...] Read more.
Fusarium graminearum is the predominant causal species of Fusarium head blight in Europe and North America. Different chemotypes of the species exist, each producing a plethora of mycotoxins. Isolates of differing chemotypes produce nivalenol (NIV) and deoxynivalenol (DON), which differ in toxicity to mammals and plants. However, the effect of each mycotoxin on volatile emissions of plant hosts is not known. Host volatiles are interpreted by insect herbivores such as Sitobion avenae, the English grain aphid, during host selection. Previous work has shown that grain aphids are repelled by wheat infected with DON-producing F. graminearum, and this study seeks to determine the influence of pathogen mycotoxins to host volatile chemistry. Volatile collections from infected hosts and olfactometer bioassays with alate aphids were performed. Infections with isolates that produced DON and NIV were compared, as well as a trichothecene deficient transformant derived from the NIV-producing isolate. This work confirmed the repellent nature of infected hosts with DON accumulation. NIV accumulation produced volatiles that were attractive to aphids. Attraction did not occur when NIV was absent and was, therefore, a direct consequence of NIV production. Full article
(This article belongs to the Collection Understanding Mycotoxin Occurrence in Food and Feed Chains)
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<p>Severity at 7 days after inoculation (DAI) of (<b>a</b>) disease (<span class="html-italic">p</span> &lt; 0.001) and (<b>b</b>) bleaching (<span class="html-italic">p</span> &lt; 0.001); and severity at 14 DAI of (<b>c</b>) disease (<span class="html-italic">p</span> = 0.005) and (<b>d</b>) bleaching (<span class="html-italic">p</span> &lt; 0.001). Disease severity shows the percentage of spikelets with lesions and/or bleaching; bleaching severity shows the percentage of spikelets with bleaching. All data were angularly transformed. DON-wt: deoxynivalenol (DON)-producer; NIV-wt: nivalenol (NIV)-producer; NIV-ko: <span class="html-italic">tri5</span> knock out transformant of NIV-wt. Data marked with different letters are significantly different (lsd; <span class="html-italic">p</span> = 0.05). NIV-ko data for bleaching severity at 7 DAI was zero and, therefore, excluded from analysis.</p> Full article ">Figure 2
<p>Fungal biomass quantified using Real-time QPCR with primers specific for (<b>a</b>) <span class="html-italic">F. graminearum</span> (<span class="html-italic">p</span> = 0.06); (<b>b</b>) NIV-chemotype isolates, based on <span class="html-italic">Tri12</span> (<span class="html-italic">p</span> = 0.096); and (<b>c</b>) 15ADON-chemotype isolates, based on <span class="html-italic">Tri12</span> (<span class="html-italic">p</span> = 0.003). Data log<sub>10</sub> transformed. DON-wt: DON-producer; NIV-wt: NIV-producer; NIV-ko: <span class="html-italic">tri5</span> knock out transformant of NIV-wt. Data marked with different letters are significantly different (least significant difference (lsd); <span class="html-italic">p</span> = 0.05). DON-wt data were zero for NIV DNA so were excluded from analysis.</p> Full article ">Figure 3
<p>Concentration of DON (log<sub>10</sub> transformed) in ears treated with <span class="html-italic">F. graminearum</span> of different chemotypes (<span class="html-italic">p</span> &lt; 0.001). DON-wt: DON-producer; NIV-wt: NIV-producer; NIV-ko: <span class="html-italic">tri5</span> knock out transformant of NIV-wt. Data marked with different letters are significantly different (lsd; <span class="html-italic">p</span> = 0.05).</p> Full article ">Figure 4
<p>Behavioural responses of <span class="html-italic">Sitobion avenae</span> to volatile chemicals sampled from <span class="html-italic">Fusarium</span>-infected hosts. DON-wt: DON-producer; NIV-wt: NIV-producer; NIV-ko: <span class="html-italic">tri5</span> knock out transformant of NIV-wt; NTC: non-treated control. *** <span class="html-italic">p</span> &lt; 0.001, * 0.01 ≤ <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 5
<p>Mean abundances of volatile chemicals, log<sub>10</sub> transformed, across all time points. (<b>a</b>) Nonanal (<span class="html-italic">p</span> = 0.004); (<b>b</b>) ocimene (ns); (<b>c</b>) 2-heptanone (ns); (<b>d</b>) α-cedrene (<span class="html-italic">p</span> = 0.015); (<b>e</b>) 2-pentadecanone (<span class="html-italic">p</span> &lt; 0.001); (<b>f</b>) 2-undecanone (<span class="html-italic">p</span> &lt; 0.001). DON-wt: DON-producer; NIV-wt: NIV-producer; NIV-ko: <span class="html-italic">tri5</span> knock out transformant of NIV-wt; NTC: non-treated control. Data marked with different letters are significantly different (lsd; <span class="html-italic">p</span> = 0.05).</p> Full article ">Figure 6
<p>Change in abundance of 2-pentadecanone over time. Treatment was significant (<span class="html-italic">p</span> = 0.019) as was the interaction between treatment and time (<span class="html-italic">p</span> &lt; 0.001). DON-wt: DON-producer; NIV-wt: NIV-producer; NIV-ko: <span class="html-italic">tri5</span> knock out transformant of NIV-wt; NTC: non-treated control. Greenhouse-Geisser ε = 0.68.</p> Full article ">
3762 KiB  
Review
Dehydropyrrolizidine Alkaloid Toxicity, Cytotoxicity, and Carcinogenicity
by Bryan L. Stegelmeier, Steven M. Colegate and Ammon W. Brown
Toxins 2016, 8(12), 356; https://doi.org/10.3390/toxins8120356 - 29 Nov 2016
Cited by 41 | Viewed by 8566
Abstract
Dehydropyrrolizidine alkaloid (DHPA)-producing plants have a worldwide distribution amongst flowering plants and commonly cause poisoning of livestock, wildlife, and humans. Previous work has produced considerable understanding of DHPA metabolism, toxicity, species susceptibility, conditions, and routes of exposure, and pathogenesis of acute poisoning. Intoxication [...] Read more.
Dehydropyrrolizidine alkaloid (DHPA)-producing plants have a worldwide distribution amongst flowering plants and commonly cause poisoning of livestock, wildlife, and humans. Previous work has produced considerable understanding of DHPA metabolism, toxicity, species susceptibility, conditions, and routes of exposure, and pathogenesis of acute poisoning. Intoxication is generally caused by contaminated grains, feed, flour, and breads that result in acute, high-dose, short-duration poisoning. Acute poisoning produces hepatic necrosis that is usually confirmed histologically, epidemiologically, and chemically. Less is known about chronic poisoning that may result when plant populations are sporadic, used as tisanes or herbal preparations, or when DHPAs contaminate milk, honey, pollen, or other animal-derived products. Such subclinical exposures may contribute to the development of chronic disease in humans or may be cumulative and probably slowly progress until liver failure. Recent work using rodent models suggest increased neoplastic incidence even with very low DHPA doses of short durations. These concerns have moved some governments to prohibit or limit human exposure to DHPAs. The purpose of this review is to summarize some recent DHPA research, including in vitro and in vivo DHPA toxicity and carcinogenicity reports, and the implications of these findings with respect to diagnosis and prognosis for human and animal health. Full article
(This article belongs to the Collection Toxicity of Natural Alkaloids)
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<p>The top row contains the general structure of a non-toxic pyrrolizidine alkaloid. The middle two structures are toxic dehydropyrrolizidine alkaloids, heliotridine and retronecine, with a 1,2 unsaturation that is characteristic of toxic alkaloids. The top right structure is, a dehydroxypyrrolizidine alkaloid <span class="html-italic">N</span>-oxide. The second line contains dehydroxypyrrolizidine alkaloid, riddelliine, which is a macrocyclic diester retronecine base alkaloid. The center structure is a “pyrrolic” riddelliine, dehydroriddelliine, or didehydroxypyrrolizidine alkaloid. The last structure on the right is a “pyrrolic” metabolite or macromolecule adduct.</p> Full article ">Figure 2
<p>California White chick dosed with riddelliine at 0.26 mMol/kg BW/day for 10 days. Notice the extensive ascites (<b>a</b>) with mild fibrinous pleuritis (<b>p</b>) and the extremely small and firm liver (<b>l</b>).</p> Full article ">Figure 3
<p>Photomicrograph of the liver from a California White chick dosed with riddelliine at 0.26 mMol/kg BW/day for 10 days. Notice the massive hepatocellular necrosis (<b>n</b>) with the collapse of hepatic cords with hemorrhage (<b>h</b>). There is minimal periportal inflammation, edema, and early proliferation of ovalocytes (<b>arrow</b>).</p> Full article ">Figure 4
<p>Photomicrograph of the liver of a California White chick dosed with riddelliine at 0.4 mMol/kg BW/ for 10 days. Notice the enlarged hepatocytes (<b>arrow</b>) with large nuclei and abundant heterochromatin (most likely developing megalocytes). There is also periportal inflammation and fibrosis (<b>*</b>) with oval cell and biliary epithelial proliferation (<b>bh</b>).</p> Full article ">Figure 5
<p>Structures of dehydropyrrolizidine alkaloids that were ranked in this review. Lasiocarpine (heliotridine diester), seneciphylline and senecionine (retronecine macrocyclic diester), heliotrine (heliotridine monoester), riddelliine, monocrotaline, riddelliine <span class="html-italic">N-</span>oxide (retronecine macrocyclic diesters), intermedine, lycopsamine, lycopsamine <span class="html-italic">N-</span>oxide (heliotridine monoester), and senecionine <span class="html-italic">N-</span>oxide (retronecine macrocyclic diester).</p> Full article ">
1242 KiB  
Communication
Fast Screening of Antibacterial Compounds from Fusaria
by Teis Esben Sondergaard, Marlene Fredborg, Ann-Maria Oppenhagen Christensen, Sofie K. Damsgaard, Nikoline F. Kramer, Henriette Giese and Jens Laurids Sørensen
Toxins 2016, 8(12), 355; https://doi.org/10.3390/toxins8120355 - 29 Nov 2016
Cited by 32 | Viewed by 6759
Abstract
Bio-guided screening is an important method to identify bioactive compounds from fungi. In this study we applied a fast digital time-lapse microscopic method for assessment of the antibacterial properties of secondary metabolites from the fungal genus Fusarium. Here antibacterial effects could be [...] Read more.
Bio-guided screening is an important method to identify bioactive compounds from fungi. In this study we applied a fast digital time-lapse microscopic method for assessment of the antibacterial properties of secondary metabolites from the fungal genus Fusarium. Here antibacterial effects could be detected for antibiotic Y, aurofusarin, beauvericin, enniatins and fusaric acid after six hours of cultivation. The system was then used in a bio-guided screen of extracts from 14 different Fusarium species, which had been fractionated by HPLC. In this screen, fractions containing the red pigments aurofusarin and bikaverin showed effects against strains of Lactobacillus and Bifidobacterium. The IC50 for aurofusarin against Lactobacillus acidophilus was 8 µM, and against Bifidobacterium breve it was 64 µM. Aurofusarin only showed an effect on probiotic bacteria, leading to the speculation that only health-promoting bacteria with a positive effect in the gut system are affected. Full article
(This article belongs to the Collection Fusarium Toxins – Relevance for Human and Animal Health)
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<p>The inhibition effects of aurofusarin on <span class="html-italic">Lactobacillus</span> spp. and <span class="html-italic">Bifidobacterium</span> spp. Aurofusarin was tested in concentrations between 2 and 128 µM. The IC<sub>50</sub> is marked with a red dotted line. The values are normalized mean values from three independent experiments. Ethanol 1% is used as control.</p> Full article ">Figure 2
<p>Isolated fractions with antibacterial effect. Secondary metabolites from each fungus were fractionized by preparative HPLC. Fractions in red showed antibacterial effect against <span class="html-italic">Staph. aureus.</span> Fractions in yellow showed antibacterial effect against <span class="html-italic">L. acidophilus</span>. <span class="html-italic">F. avenaceum</span> was grown on YMA, <span class="html-italic">F. graminearum</span> on PDA, <span class="html-italic">F. poae</span> and <span class="html-italic">F. pseudograminearum</span> on YES and <span class="html-italic">F. oxysporum</span> on rice. AUR: Aurofusarin. Enn: Enniatins. ZEA: Zearalenone. RUB: Rubrofusarin. BEA: Beauvericin. Fus C: Fusarin C. BIK: Bikaverin. ?: unknown.</p> Full article ">
2412 KiB  
Article
Growth-Phase Sterigmatocystin Formation on Lactose Is Mediated via Low Specific Growth Rates in Aspergillus nidulans
by Zoltán Németh, Ákos P. Molnár, Balázs Fejes, Levente Novák, Levente Karaffa, Nancy P. Keller and Erzsébet Fekete
Toxins 2016, 8(12), 354; https://doi.org/10.3390/toxins8120354 - 28 Nov 2016
Cited by 15 | Viewed by 5077
Abstract
Seed contamination with polyketide mycotoxins such as sterigmatocystin (ST) produced by Aspergilli is a worldwide issue. The ST biosynthetic pathway is well-characterized in A. nidulans, but regulatory aspects related to the carbon source are still enigmatic. This is particularly true for lactose, [...] Read more.
Seed contamination with polyketide mycotoxins such as sterigmatocystin (ST) produced by Aspergilli is a worldwide issue. The ST biosynthetic pathway is well-characterized in A. nidulans, but regulatory aspects related to the carbon source are still enigmatic. This is particularly true for lactose, inasmuch as some ST production mutant strains still synthesize ST on lactose but not on other carbon substrates. Here, kinetic data revealed that on d-glucose, ST forms only after the sugar is depleted from the medium, while on lactose, ST appears when most of the carbon source is still available. Biomass-specified ST production on lactose was significantly higher than on d-glucose, suggesting that ST formation may either be mediated by a carbon catabolite regulatory mechanism, or induced by low specific growth rates attainable on lactose. These hypotheses were tested by d-glucose limited chemostat-type continuous fermentations. No ST formed at a high growth rate, while a low growth rate led to the formation of 0.4 mg·L−1 ST. Similar results were obtained with a CreA mutant strain. We concluded that low specific growth rates may be the primary cause of mid-growth ST formation on lactose in A. nidulans, and that carbon utilization rates likely play a general regulatory role during biosynthesis. Full article
(This article belongs to the Collection Aflatoxins)
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<p>Maximal sterigmatocystin (ST) production of the <span class="html-italic">A. nidulans</span> wild-type strain RDIT 9.32 as a function of the carbon source. <span class="html-small-caps">d</span>-glucose and lactose indicates minimal media initially containing 15 g/L sole carbon substrate. Black and white columns indicate liquid and agar-solidified cultures, respectively. Data presented here are means of three independent experiments (biological replicates). The variations among experiments were estimated by standard deviations (SDs), indicated by the error bars. ST extraction and quantification protocols are described in details in <a href="#sec4-toxins-08-00354" class="html-sec">Section 4</a>.</p> Full article ">Figure 2
<p>Maximal biomass-specific sterigmatocystin (ST) production of <span class="html-italic">A. nidulans</span> strains RDIT 9.32 and RDIT 2.3, carrying the <span class="html-italic">veA+</span> (wild-type) allele, as well as of the <span class="html-italic">A. nidulans</span> R21 strain carrying the <span class="html-italic">veA1</span> (mutant) allele in liquid minimal medium initially containing 15 g/L <span class="html-small-caps">d</span>-glucose or lactose as sole carbon sources. For additional information on the strains, see <a href="#toxins-08-00354-t003" class="html-table">Table 3</a>. DCW: dry cell weight.</p> Full article ">Figure 3
<p>Time-profile of residual sterigmatocystin (ST) concentrations in sterile water (●), in sterile minimal growth medium with <span class="html-small-caps">d</span>-glucose (<b>□</b>) and in <span class="html-italic">A. nidulans</span> liquid batch cultures with <span class="html-small-caps">d</span>-glucose as a sole carbon source (▲).</p> Full article ">Figure 4
<p>Time-profile of growth (□), residual carbon source concentrations (●) as well as sterigmatocystin (ST) production (▲) in batch fermentations of an <span class="html-italic">A. nidulans</span> wild-type strain in minimal media initially containing 15 g/L sole carbon substrate. (<b>A</b>) <span class="html-small-caps">d</span>-glucose; (<b>B</b>) lactose. Mycelial inoculum preformed overnight on glycerol and transferred into the bioreactors was used for all fermentations.</p> Full article ">Figure 5
<p>Time profiles of growth (□), residual <span class="html-small-caps">d</span>-glucose concentrations (●) as well as sterigmatocystin (ST) production (▲) in fed-batch fermentations of an <span class="html-italic">A. nidulans</span> wild-type strain in minimal media. Additional <span class="html-small-caps">d</span>-glucose (indicated by plain arrows) was added at 94 h and 140 h. To increase clarity, the plot gives the mean data of three independent fermentations instead of displaying error bars. The mean standard deviation for the <span class="html-small-caps">d</span>-glucose concentration was 5% and for the biomass concentration, 10%; the maximum deviations were 7% and 13%, respectively. Inoculation of the bioreactor occured as described above.</p> Full article ">Figure 6
<p>Time profile of growth (□), residual carbon source concentrations (●) as well as sterigmatocystin (ST) production (▲) in batch fermentations of an <span class="html-italic">A. nidulans</span> carbon catabolite derepressed CreA mutant strain in minimal media initially containing 15 g/L sole carbon substrate. (<b>A</b>) <span class="html-small-caps">d</span>-glucose; (<b>B</b>) lactose. Inoculation of the bioreactor occurred as described above.</p> Full article ">
246 KiB  
Conference Report
Priority Actions and Progress to Substantially and Sustainably Reduce the Mortality, Morbidity and Socioeconomic Burden of Tropical Snakebite
by Robert A. Harrison and José María Gutiérrez
Toxins 2016, 8(12), 351; https://doi.org/10.3390/toxins8120351 - 24 Nov 2016
Cited by 68 | Viewed by 10172
Abstract
The deliberations and conclusions of a Hinxton Retreat convened in September 2015, entitled “Mechanisms to reverse the public health neglect of snakebite victims” are reported. The participants recommended that the following priority actions be included in strategies to reduce the global impact of [...] Read more.
The deliberations and conclusions of a Hinxton Retreat convened in September 2015, entitled “Mechanisms to reverse the public health neglect of snakebite victims” are reported. The participants recommended that the following priority actions be included in strategies to reduce the global impact of snake envenoming: (a) collection of accurate global snakebite incidence, mortality and morbidity data to underpin advocacy efforts and help design public health campaigns; (b) promotion of (i) public education prevention campaigns; (ii) transport systems to improve access to hospitals and (iii) establishment of regional antivenom-efficacy testing facilities to ensure antivenoms’ effectiveness and safety; (c) exploration of funding models for investment in the production of antivenoms to address deficiencies in some regions; (d) establishment of (i) programs for training in effective first aid, hospital management and post-treatment care of victims; (ii) a clinical network to generate treatment guidelines and (iii) a clinical trials system to improve the clinical management of snakebite; (e) development of (i) novel treatments of the systemic and local tissue-destructive effects of envenoming and (ii) affordable, simple, point-of-care snakebite diagnostic kits to improve the accuracy and rapidity of treatment; (f) devising and implementation of interventions to help the people and communities affected by physical and psychological sequelae of snakebite. Full article
(This article belongs to the Section Animal Venoms)
574 KiB  
Review
Worldwide Mycotoxins Exposure in Pig and Poultry Feed Formulations
by Philippe Guerre
Toxins 2016, 8(12), 350; https://doi.org/10.3390/toxins8120350 - 24 Nov 2016
Cited by 67 | Viewed by 8936
Abstract
The purpose of this review is to present information about raw materials that can be used in pig and poultry diets and the factors responsible for variations in their mycotoxin contents. The levels of mycotoxins in pig and poultry feeds are calculated based [...] Read more.
The purpose of this review is to present information about raw materials that can be used in pig and poultry diets and the factors responsible for variations in their mycotoxin contents. The levels of mycotoxins in pig and poultry feeds are calculated based on mycotoxin contamination levels of the raw materials with different diet formulations, to highlight the important role the stage of production and the raw materials used can have on mycotoxins levels in diets. Our analysis focuses on mycotoxins for which maximum tolerated levels or regulatory guidelines exist, and for which sufficient contamination data are available. Raw materials used in feed formulation vary considerably depending on the species of animal, and the stage of production. Mycotoxins are secondary fungal metabolites whose frequency and levels also vary considerably depending on the raw materials used and on the geographic location where they were produced. Although several reviews of existing data and of the literature on worldwide mycotoxin contamination of food and feed are available, the impact of the different raw materials used on feed formulation has not been widely studied. Full article
(This article belongs to the Special Issue Exposure and Risk Assessment for Mycotoxins)
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<p>Consequences of a difference in sensitivity for descriptive statistics of mycotoxin contents. (<b>A</b>) Method of analysis with a limit of quantitation (LOQ) of 50 µg/kg (old method); (<b>B</b>) method of analysis with an LOQ of 1 µg/kg (new method).</p> Full article ">
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