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Toxins, Volume 5, Issue 5 (May 2013) – 13 articles , Pages 865-1050

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625 KiB  
Article
Venomous Secretions from Marine Snails of the Terebridae Family Target Acetylcholine Receptors
by Yvonne Kendel, Christian Melaun, Alexander Kurz, Annette Nicke, Steve Peigneur, Jan Tytgat, Cora Wunder, Dietrich Mebs and Silke Kauferstein
Toxins 2013, 5(5), 1043-1050; https://doi.org/10.3390/toxins5051043 - 21 May 2013
Cited by 14 | Viewed by 9240
Abstract
Venoms from cone snails (Conidae) have been extensively studied during the last decades, but those from other members of the suborder Toxoglossa, such as of Terebridae and Turridae superfamilies attracted less interest so far. Here, we report the effects of venom and gland [...] Read more.
Venoms from cone snails (Conidae) have been extensively studied during the last decades, but those from other members of the suborder Toxoglossa, such as of Terebridae and Turridae superfamilies attracted less interest so far. Here, we report the effects of venom and gland extracts from three species of the superfamily Terebridae. By 2-electrode voltage-clamp technique the gland extracts were tested on Xenopus oocytes expressing nicotinic acetylcholine receptors (nAChRs) of rat neuronal (α3β2, α3β4, α4β2, α4β4, α7) and muscle subtypes (α1β1γδ), and expressing potassium (Kv1.2 and Kv1.3) and sodium channels (Nav1.2, 1.3, 1.4, 1.6). The extracts were shown to exhibit remarkably high inhibitory activities on almost all nAChRs tested, in particular on the α7 subtype suggesting the presence of peptides of the A-superfamily from the venom of Conus species. In contrast, no effects on the potassium and sodium channels tested were observed. The venoms of terebrid snails may offer an additional source of novel biologically active peptides. Full article
(This article belongs to the Special Issue Toxins from Aquatic Organisms)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>SEM-pictures of radula teeth from two Terebridae species, <span class="html-italic">Terebra consobrina</span> and <span class="html-italic">T. argus</span>.</p> Full article ">Figure 2
<p>Effects of <span class="html-italic">Terebra consobrina</span> venom gland extract (12.9 μg) on six nicotinic acetylcholine receptor (nAChR) subtypes. 100 µM ACh or nicotine (in the case of the α<sub>7</sub>) were applied for 2 s in 4 min intervals. Two current responses before application of gland extracts, one response directly after extract application (horizontal bar, 3 min incubation) and two subsequent responses after washout of the extracts are shown for each subtype.</p> Full article ">
285 KiB  
Communication
Estimated Dietary Exposure to Mycotoxins after Taking into Account the Cooking of Staple Foods in Japan
by Hisako Sakuma, Yasushi Watanabe, Hiroko Furusawa, Tomoya Yoshinari, Hajime Akashi, Hiroshi Kawakami, Shiro Saito and Yoshiko Sugita-Konishi
Toxins 2013, 5(5), 1032-1042; https://doi.org/10.3390/toxins5051032 - 21 May 2013
Cited by 22 | Viewed by 6129
Abstract
Mycotoxins are commonly present in cereal grains and are not completely destroyed during their cooking and processing. When mycotoxins contaminate staple foods, the risk for exposure becomes serious. In East Asia, including Japan, rice is consumed as a staple food, and with the [...] Read more.
Mycotoxins are commonly present in cereal grains and are not completely destroyed during their cooking and processing. When mycotoxins contaminate staple foods, the risk for exposure becomes serious. In East Asia, including Japan, rice is consumed as a staple food, and with the increasingly Westernized lifestyle, the consumption of wheat has increased. The mycotoxins commonly associated with rice and wheat are total aflatoxin (AFL) and ochratoxin A (OTA), respectively. This study examined the retention of AFL and OTA during the cooking of rice and pasta. AFL was retained at 83%–89% the initial level after the cooking of steamed rice. In pasta noodles, more than 60% of the OTA was retained. These results show that AFL and OTA are relatively stable during the cooking process, suggesting that a major reduction in the exposure to these mycotoxins cannot be expected to occur by cooking rice and pasta. The estimated exposure assessment at the high consumer level (95th percentile) and the mycotoxin contamination level determined by taking into account these reductions in the present study should be useful for the establishment of practical regulations for mycotoxins in staple foods. Full article
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Figure 1
<p>The residual ratio (%) of AFB1, AFB2, AFG1 and AFG2 in rice after cooking. The naturally contaminated rice and the artificially contaminated rice (spiked 1 and 2) were cooked as described in the Materials and Methods section. Spike 1 means that the polished rice was spiked with 2.5 ng/mL of AFB1, 1.875 ng/mL of AFG1 and 0.3125 ng/mL of AFB2 and AFG2. Spike 2 means that the polished rice was spiked with 5.0 ng/mL of AFB1, 3.75 ng/mL of AFG1 and 0.625 ng/mL of AFB2 and AFG2.</p> Full article ">Figure 2
<p>The residual ratio ofochratoxin A (OTA) in pasta after cooking. The pasta was made from durum wheat spiked with 0, 5 or 10 ng/g of OTA.</p> Full article ">
774 KiB  
Review
Sialorrhea: Anatomy, Pathophysiology and Treatment with Emphasis on the Role of Botulinum Toxins
by Amanda Amrita Lakraj, Narges Moghimi and Bahman Jabbari
Toxins 2013, 5(5), 1010-1031; https://doi.org/10.3390/toxins5051010 - 21 May 2013
Cited by 103 | Viewed by 24418
Abstract
Sialorrhea or excessive drooling is a major issue in children with cerebral palsy and adults with neurodegenerative disorders. In this review, we describe the clinical features, anatomy and physiology of sialorrhea, as well as a review of the world literature on medical treatment [...] Read more.
Sialorrhea or excessive drooling is a major issue in children with cerebral palsy and adults with neurodegenerative disorders. In this review, we describe the clinical features, anatomy and physiology of sialorrhea, as well as a review of the world literature on medical treatment using Yale University’s search engine; including but not limited to Medline and Erasmus. Level of drug efficacy is defined according to the guidelines of American Academy of Neurology. Current medical management is unsatisfactory. Topical agents (scopolamine and tropicamide) and oral agents (glyccopyrolate) combined render a level B evidence (probably effective); however, this treatment is associated with troublesome side effects. Double-blind and placebo-controlled studies of botulinum toxin (BoNT) provide a level A evidence for type B (two class I studies; effective and established) and both overall and individual B level of evidence for OnabotulinumtoxinA (A/Ona) and AbobotulinumtoxinA (A/Abo); these are probably effective. For IncobotulinumtoxinA (A/Inco), the level of evidence is U (insufficient) due to lack of blinded studies. Side effects are uncommon; transient and comparable between the two types of toxin. A clinical note at the end of this review comments on fine clinical points. Administration of BoNTs into salivary glands is currently the most effective way of treating sialorrhea. Full article
(This article belongs to the Special Issue Neurotoxins: Health Threats and Biological Tools)
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Figure 1
<p>Locations for Parotid gland injections. This figure depicts the way in which Lagalla <span class="html-italic">et al</span>. [<a href="#B29-toxins-05-01010" class="html-bibr">29</a>] inject into the parotid gland (the black x’s). Many of thestudies have used the same approach, injecting in only 2 sites on the parotid gland. At our institution, we inject into nine different sites and have modified the figure to portray this by the blue dots. Modified with permission from Springer [<a href="#B29-toxins-05-01010" class="html-bibr">29</a>].</p> Full article ">Figure 2
<p>Facial Nerve location in relation to parotid gland. It is important to note the anatomical location of the facial nerve in relation to the parotid gland in order to avoid injury to this functionally important nerve during injection.</p> Full article ">
375 KiB  
Article
Variations in the Microcystin Content of Different Fish Species Collected from a Eutrophic Lake
by Justine R. Schmidt, Mylynda Shaskus, John F. Estenik, Carl Oesch, Roman Khidekel and Gregory L. Boyer
Toxins 2013, 5(5), 992-1009; https://doi.org/10.3390/toxins5050992 - 15 May 2013
Cited by 54 | Viewed by 13058
Abstract
Microcystins produced from cyanobacteria can accumulate in fish tissues. Liquid chromatography coupled with tandem quadrupole mass spectrometry (LC-MS/MS) is an attractive alternative to immunoassays for the determination of low concentrations of microcystins in tissues. Fish taken from Grand Lake St. Marys, a eutrophic [...] Read more.
Microcystins produced from cyanobacteria can accumulate in fish tissues. Liquid chromatography coupled with tandem quadrupole mass spectrometry (LC-MS/MS) is an attractive alternative to immunoassays for the determination of low concentrations of microcystins in tissues. Fish taken from Grand Lake St. Marys, a eutrophic lake in Ohio, USA, were analyzed for microcystin-LR in their fillets using LC-MS/MS. Of 129 fish tested for microcystins, only black crappie (Pomoxis nigromaculatus) and common carp (Cyprinus carpio) tested positive for microcystin-LR. Less than 10% of Pomoxis and 7% of Cyprinus samples contained measurable levels of microcystin-LR. Statistical analysis yielded a p-value of 0.07 between Pomoxis and the pooled results of the other four fish species. However, this comparison was complicated by the large difference in sample size between species. Further sampling in Grand Lake St. Marys for microcystin-LR would help determine if microcystin-LR exposure occurs through foodweb transfer. Full article
(This article belongs to the Special Issue Cyanotoxins)
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<p>The dates of water and fish collection relative to the microcystin concentration (µg/L) in raw, unfiltered water collected from the Celina public water supply (PWS) water treatment plant over time. The City of Celina PWS is located on the northwestern shore of Grand Lake St. Marys, and the microcystin data is replotted from the public Ohio EPA database. Microcystin concentrations ranged from 0.2 µg/L to over 100 µg/L. Values over 100 µg/L are represented here as at 100 µg/L.</p> Full article ">Figure 2
<p>Microcystin-LR concentration in fish tissue by species. The detection limit for all samples is indicated by the striped bars. The collection date is given for positive <span class="html-italic">Pomoxis</span> samples greater than 10 µg/kg. In total, three positive <span class="html-italic">Pomoxis</span> samples were collected on 6/2/2011, three on 6/6/2012, and one on 7/25/2012. The single positive <span class="html-italic">Cyprinus</span> sample was collected on 8/30/2011.</p> Full article ">Figure 3
<p>LC-MS/MS chromatograms of a positive <span class="html-italic">Pomoxis</span> sample from Grand Lake St. Marys, collected 6/6/2012. The third chromatogram shows the <span class="html-italic">m/z</span> 995 to 135 transition, which was used to quantitate microcystin-LR in the samples. The remaining four chromatograms show transitions used for the confirmation of microcystin-LR in a sample.</p> Full article ">
214 KiB  
Article
Feasibility of Video Clip Analysis on Effect of Botulinum Toxin-A Injection for Post-Stroke Upper Limb Spasticity
by Woo-Jin Kim, Witsanu Kumthornthip, Byung Mo Oh, Eun Joo Yang and Nam-Jong Paik
Toxins 2013, 5(5), 983-991; https://doi.org/10.3390/toxins5050983 - 10 May 2013
Cited by 3 | Viewed by 6635
Abstract
Existing functional evaluation tools do not accurately reveal the improved function following botulinum toxin A (BTX-A) injection for post-stroke upper limb spasticity. With the aim of developing an alternate method of measuring functional improvement following BTX-A injection, this study tested the feasibility, validity [...] Read more.
Existing functional evaluation tools do not accurately reveal the improved function following botulinum toxin A (BTX-A) injection for post-stroke upper limb spasticity. With the aim of developing an alternate method of measuring functional improvement following BTX-A injection, this study tested the feasibility, validity and reliability of video clip analysis performed by the clinicians. Seventy-nine patients administered BTX-A due to post-stroke upper limb spasticity, were retrospectively evaluated using video clip analysis. Pre- and post-injection video clips recorded at 1-month intervals were randomly allocated and sent to three blinded physician evaluators who were asked to choose the one that seemed more improved in terms of hand motion and associated upper limb reaction during gait. The three physicians chose the post-injection video clip as depicting improved hand motion (82.3%, 79.7%, and 72.2%) and associated upper limb reaction during gait (73.4%, 70.9%, and 70.9%). Kappa and intraclass correlation coefficient as a measure of interrater reliability among the three physicians was 0.86 and 0.79 for the hand, and 0.92 and 0.92 for associated upper limb reaction during gait, respectively. The percent overall agreement of the physicians was 78.1% and 71.7% for hand function and associated upper limb reaction, respectively. Retrospective pre- and post-BTX-A injection video clip analyses is a clinically feasible alternative method to evaluate the improvement following BTX-A injection for post-stroke upper limb spasticity, especially in busy clinical practice setting. Full article
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Figure 1
<p>Pre and post-operative captured images of video-clip, demonstrating improvement of the cylindrical grasp and release on the right.</p> Full article ">
335 KiB  
Article
BiP Negatively Affects Ricin Transport
by Tone F. Gregers, Sigrid S. Skånland, Sébastien Wälchli, Oddmund Bakke and Kirsten Sandvig
Toxins 2013, 5(5), 969-982; https://doi.org/10.3390/toxins5050969 - 10 May 2013
Cited by 9 | Viewed by 8620
Abstract
The AB plant toxin ricin binds both glycoproteins and glycolipids at the cell surface via its B subunit. After binding, ricin is endocytosed and then transported retrogradely through the Golgi to the endoplasmic reticulum (ER). In the ER, the A subunit is retrotranslocated [...] Read more.
The AB plant toxin ricin binds both glycoproteins and glycolipids at the cell surface via its B subunit. After binding, ricin is endocytosed and then transported retrogradely through the Golgi to the endoplasmic reticulum (ER). In the ER, the A subunit is retrotranslocated to the cytosol in a chaperone-dependent process, which is not fully explored. Recently two separate siRNA screens have demonstrated that ER chaperones have implications for ricin toxicity. ER associated degradation (ERAD) involves translocation of misfolded proteins from ER to cytosol and it is conceivable that protein toxins exploit this pathway. The ER chaperone BiP is an important ER regulator and has been implicated in toxicity mediated by cholera and Shiga toxin. In this study, we have investigated the role of BiP in ricin translocation to the cytosol. We first show that overexpression of BiP inhibited ricin translocation and protected cells against the toxin. Furthermore, shRNA-mediated depletion of BiP enhanced toxin translocation resulting in increased cytotoxicity. BiP-dependent inhibition of ricin toxicity was independent of ER stress. Our findings suggest that in contrast to what was shown with the Shiga toxin, the presence of BiP does not facilitate, but rather inhibits the entry of ricin into the cytosol. Full article
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>(<b>A</b>) HEK293 cells were transfected with BiP or an empty vector (ctrl). Lysates were run on SDS-PAGE, transferred to a PVDF membrane and examined for BiP expression using an anti-myc antibody or anti-BiP antibody. Anti-actin was used for loading control. (<b>B</b>) Cells were transfected as indicated and incubated with increasing amounts of ricin in leucine-free medium for 3 h, then washed and incubated with 1 µCi/mL [<sup>3</sup>H]leucine for 20 min. The incorporation of [<sup>3</sup>H]leucine was measured and data presented relative to the control (CTRL). This experiment was repeated three times with similar results in duplicates. (<b>C</b>) Cells transfected as indicated were pre-incubated with 0.2 mCi/mL <sup>35</sup>SO<sub>4</sub><sup>2-</sup> for 3 h and then with ricin sulf-1 for an additional 3 h. The cells were lysed and ricin was immunoprecipitated and subjected to SDS-PAGE under non-reducing conditions. The amount of ricin A-chain reduced from the holotoxin in each sample was quantified and compared to total sulfated ricin in the samples. (<b>D</b>) Cells were transfected with myc-tagged BiP constructs as indicated (wild type, WT or substrate binding mutant, P495L) before lysis and incubated with 1 μg His-tagged ricin A-chain for 1 h. The toxin was pulled down using a Ni-NTA column. The beads were separated in a SDS-PAGE and analyzed by Western blot. BiP proteins were detected using anti-myc antibodies and ricin with anti-His antibodies. Whole cell lysates (WCL, left panels) and pull down (+His-ricin A NTA, right panels) are shown.</p> Full article ">Figure 2
<p>(<b>A</b>) Cells were transfected with a control shRNA vector or two different BiP shRNA constructs either alone or in combination, for three days. Cell lysates were analysed by Western blot using anti-BiP and anti-actin antibodies. (<b>B</b>) Cells transfected with the indicated shRNA were incubated with increasing amounts of ricin in leucine-free medium for 3 h, then washed and incubated with 1 µCi/mL [<sup>3</sup>H]leucine for 20 min. The incorporation of [<sup>3</sup>H]leucine was measured and data presented relative to the control (CTRL). Left panel: a representative experiment performed with parallels. Right panel: average IC<sub>50</sub> values of 3–4 experiments. Error bars represent standard error of the mean (<span class="html-italic">p</span> &lt; 0.001 for shRNA1 (<span class="html-italic">n</span> = 4) and shRNA1+2 (<span class="html-italic">n</span> = 3) and <span class="html-italic">p</span> = 0.047 for shRNA2 (<span class="html-italic">n</span> = 3)). (<b>C</b>) Cells transfected with the indicated shRNA constructs were incubated with 0.2 mCi/mL<sup>35</sup>SO<sub>4</sub><sup>2-</sup> for 3 h and then with ricin sulf-1 for an additional 3 h. The cells were lysed and ricin was immunoprecipitated and subjected to SDS-PAGE under non-reducing conditions. The amount of ricin A-chain in each sample was quantified and compared to total sulfated ricin in the samples.</p> Full article ">Figure 3
<p>(<b>A</b> and <b>B</b>) Cells transfected as indicated were incubated with 0.2 mCi/mL <sup>35</sup>SO<sub>4</sub><sup>2-</sup> for 3 h and then with ricin sulf-1 for an additional 3 h. The cells were then permeabilized in 3 µg/mL digitonin before lysis and separation of the cytosolic/membrane fractions. Ricin was then immunoprecipitated from each fraction and subjected to SDS-PAGE under reducing conditions. The amount of ricin A-chain in the cytosol was quantified and compared to total sulfated ricin in the samples.</p> Full article ">Figure 4
<p>(<b>A</b>) Cells were transfected with control vector or BiP shRNA constructs together with an ATF6-luciferase reporter construct and <span class="html-italic">Renilla</span> luciferase. 2 μg/mL tunicamycin was used as positive control. Cells were analyzed for luciferase activity 3 days post transfection and the relative activity was normalized to the <span class="html-italic">Renilla</span> luciferase internal control. (<b>B</b>) Lysate from cells transfected with the shRNA constructs were analyzed for the level of Grp94 by Western blot using rat anti-Grp94. The intensity of each band was quantified by ImageQuant 5.0 software. (<b>C</b>) Non transfected cells were incubated with leucine-free medium with or without 2 µg/mL tunicamycin (TUN) for 21 h before different concentrations of ricin were added for subsequently 3 h, then washed in leucine-free medium and incubated with 1 µCi/mL [<sup>3</sup>H]leucine for 20 min. The amount of radioactive protein was finally measured. (<b>D</b>) Cells were treated with 2 µg/mL tunicamycin (TUN) for 0, 8 and 24 h in parallels before lysis. Lysates were subjected to SDS-PAGE and Western blot analysis. Anti-BiP was used to detect BiP and anti-actin was used as loading control. The intensity of each band was quantified by ImageQuant 5.0 software where endogenous level of BiP at time point 0 was used as the reference level.</p> Full article ">
1001 KiB  
Brief Report
Insights into Diphthamide, Key Diphtheria Toxin Effector
by Wael Abdel-Fattah, Viktor Scheidt, Shanow Uthman, Michael J. R. Stark and Raffael Schaffrath
Toxins 2013, 5(5), 958-968; https://doi.org/10.3390/toxins5050958 - 3 May 2013
Cited by 21 | Viewed by 13571
Abstract
Diphtheria toxin (DT) inhibits eukaryotic translation elongation factor 2 (eEF2) by ADP-ribosylation in a fashion that requires diphthamide, a modified histidine residue on eEF2. In budding yeast, diphthamide formation involves seven genes, DPH1-DPH7. In an effort to further study diphthamide synthesis and [...] Read more.
Diphtheria toxin (DT) inhibits eukaryotic translation elongation factor 2 (eEF2) by ADP-ribosylation in a fashion that requires diphthamide, a modified histidine residue on eEF2. In budding yeast, diphthamide formation involves seven genes, DPH1-DPH7. In an effort to further study diphthamide synthesis and interrelation among the Dph proteins, we found, by expression in E. coli and co-immune precipitation in yeast, that Dph1 and Dph2 interact and that they form a complex with Dph3. Protein-protein interaction mapping shows that Dph1-Dph3 complex formation can be dissected by progressive DPH1 gene truncations. This identifies N- and C-terminal domains on Dph1 that are crucial for diphthamide synthesis, DT action and cytotoxicity of sordarin, another microbial eEF2 inhibitor. Intriguingly, dph1 truncation mutants are sensitive to overexpression of DPH5, the gene necessary to synthesize diphthine from the first diphthamide pathway intermediate produced by Dph1-Dph3. This is in stark contrast to dph6 mutants, which also lack the ability to form diphthamide but are resistant to growth inhibition by excess Dph5 levels. As judged from site-specific mutagenesis, the amidation reaction itself relies on a conserved ATP binding domain in Dph6 that, when altered, blocks diphthamide formation and confers resistance to eEF2 inhibition by sordarin. Full article
(This article belongs to the Special Issue Diphtheria Toxin)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Diphthamide synthesis on yeast translation elongation factor 2 (eEF2) and ADP-ribosylation by diphtheria toxin (DT). For details, see text.</p> Full article ">Figure 2
<p>(His)<sub>6</sub>-tagged Dph1 and Dph2 expressed from <span class="html-italic">E. coli</span> interact with each other <span class="html-italic">in vitro</span>. (<b>A</b>) Anti-(His)<sub>6</sub> Western blot following denaturing conditions (12% SDS-PAGE). (<b>B</b>) Western blot under native conditions (10% native PAGE, 0.5 × TBE).</p> Full article ">Figure 3
<p>Use of DT and sordarin as diagnostic tools to map Dph1 regions crucial for Dph2 and Dph3 interaction. (<b>A</b>) Diagram illustrating the N- and C-terminal Dph1 truncation sets (<a href="#toxins-05-00958-s001" class="html-supplementary-material">Figure S1</a>) used to study Dph1 function and interaction profiles. (<b>B</b>) DT and sordarin sensitivity assays. Serial cell dilutions of wild-type (wt), <span class="html-italic">DPH1</span> deletant (<span class="html-italic">dph1Δ</span>) and the strains indicated in panel A were grown in the absence (control) or presence of DT or sordarin. ‘S’ and ‘R’ denote sensitive and resistant traits, respectively. (<b>C</b>, <b>D</b>) Anti-c-Myc co-immune precipitation (IP) assays to study Dph1-Dph2 and Dph1-Dph3 protein-protein interactions. The presence of c-Myc-tagged Dph2 (panel C), Dph3 (panel D), the HA-tagged full-length Dph1 (N, C) and the N- and C-terminal truncation variants of Dph1 in the IPs were monitored by anti-c-Myc and anti-HA Western blots. In addition, the content of full-length and truncated forms of HA-tagged Dph1 was checked by immune blots in the inputs (pre-IP). The positions of Dph2, Dph3 as well as full-length and truncation forms of Dph1 are indicated by arrows.</p> Full article ">Figure 4
<p>Overexpression of <span class="html-italic">DPH5</span> is growth inhibitory to <span class="html-italic">dph1</span> truncation and deletion mutants. Strains with the indicated genetic backgrounds (see <a href="#toxins-05-00958-f003" class="html-fig">Figure 3</a>) and maintaining plasmid p<span class="html-italic">GAL-DPH5</span> for galactose inducible overexpression of Dph5 were serially diluted and spotted onto glucose (2% glc) and galactose (2% gal) media to assay their response to <span class="html-italic">DPH5</span> overexpression. Unaltered tolerance (T) and sensitive (S) responses are indicated.</p> Full article ">Figure 5
<p><span class="html-italic">DPH6</span> mutagenesis identifies domains in Dph6 that are essential for its function in sordarin sensitivity and dipthamide synthesis. (<b>A</b>) Diagram showing the <span class="html-italic">DPH6</span> wild-type and mutant constructs tested in (<b>B</b>), indicating the Alpha_ANH_like_IV (ANH_IV: red) and YjgF-YER057c-UK114 (UK114: blue) domains and the position of point mutations. (<b>B</b>) Ten-fold serial cell dilutions of a <span class="html-italic">dph6Δ</span> deletion strain carrying the constructs shown in (<b>A</b>) or the corresponding empty vector pSU6 were grown onto plates with or without sordarin. ‘S’ and ‘R’ denote sensitive and resistant traits, respectively.</p> Full article ">
439 KiB  
Article
Sedimentation Patterns of Toxin-Producing Microcystis Morphospecies in Freshwater Reservoirs
by Samuel Cirés, Lars Wörmer, David Carrasco and Antonio Quesada
Toxins 2013, 5(5), 939-957; https://doi.org/10.3390/toxins5050939 - 3 May 2013
Cited by 25 | Viewed by 6859
Abstract
Understanding the annual cycle of Microcystis is essential for managing the blooms of this toxic cyanobacterium. The current work investigated the sedimentation of microcystin-producing Microcystis spp. in three reservoirs from Central Spain during the summer and autumn of 2006 and 2007. We confirmed [...] Read more.
Understanding the annual cycle of Microcystis is essential for managing the blooms of this toxic cyanobacterium. The current work investigated the sedimentation of microcystin-producing Microcystis spp. in three reservoirs from Central Spain during the summer and autumn of 2006 and 2007. We confirmed remarkable settling fluxes during and after blooms ranging 106–109 cells m−2 d−1, which might represent 0.1%–7.6% of the organic matter settled. A comprehensive analysis of the Valmayor reservoir showed average Microcystis settling rates (0.04 d−1) and velocities (0.7 m d−1) that resembled toxin settling in the same reservoir and were above most reported elsewhere. M. aeruginosa settling rate was significantly higher than that of M. novacekii and M. flos-aquae. Despite the fact that colony sizes did not differ significantly in their average settling rates, we observed extremely high and low rates in large colonies (>5000 cells) and a greater influence of a drop in temperature on small colonies (<1000 cells). We found a 4–14 fold decrease in microcystin cell quota in settling Microcystis of the Cogotas and Valmayor reservoirs compared with pelagic populations, and the hypothetical causes of this are discussed. Our study provides novel data on Microcystis settling patterns in Mediterranean Europe and highlights the need for including morphological, chemotypical and physiological criteria to address the sedimentation of complex Microcystis populations. Full article
(This article belongs to the Special Issue Cyanotoxins)
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Figure 1
<p>Bloom dynamics and MCs in the Cogotas and Valmayor reservoirs. Cyanobacteria are represented in the top graphs by area plots: <span class="html-italic">Microcystis aeruginosa</span> (dark grey); <span class="html-italic">Microcystis flos-aquae</span> (light grey); <span class="html-italic">Microcystis novacekii</span> (black); others (white). MC concentration (sum of MC-LR, MC-RR and MC-YR) is represented by black circles and a solid line. Estimated MC cell quota (sum of MC-LR, MC-RR and MC-YR) in <span class="html-italic">Microcystis</span> is represented in the bottom graphs by white triangles and a dashed line.</p> Full article ">Figure 2
<p>Colony sizes of <span class="html-italic">Microcystis</span> morphospecies in subsurface water at the Valmayor reservoir (box-plots). Dots represent the 5th and 95th percentiles.</p> Full article ">Figure 3
<p>Box-plots representing the number of single <span class="html-italic">Microcystis</span> cells among the total number of <span class="html-italic">Microcystis</span> cells settled in two depths of the Valmayor reservoir.</p> Full article ">
1621 KiB  
Article
Effects of Clostridium difficile Toxin A and B on Human T Lymphocyte Migration
by Dan Wu, Antony George Joyee, Saravanan Nandagopal, Marianela Lopez, Xiuli Ma, Jody Berry and Francis Lin
Toxins 2013, 5(5), 926-938; https://doi.org/10.3390/toxins5050926 - 3 May 2013
Cited by 13 | Viewed by 7651
Abstract
Bacterial products such as toxins can interfere with a variety of cellular processes, leading to severe human diseases. Clostridium difficile toxins, TcdA and TcdB are the primary contributing factors to the pathogenesis of C. difficile-associated diseases (CDAD). While the mechanisms for TcdA [...] Read more.
Bacterial products such as toxins can interfere with a variety of cellular processes, leading to severe human diseases. Clostridium difficile toxins, TcdA and TcdB are the primary contributing factors to the pathogenesis of C. difficile-associated diseases (CDAD). While the mechanisms for TcdA and TcdB mediated cellular responses are complex, it has been shown that these toxins can alter chemotactic responses of neutrophils and intestinal epithelial cells leading to innate immune responses and tissue damages. The effects of C. difficile toxins on the migration and trafficking of other leukocyte subsets, such as T lymphocytes, are not clear and may have potential implications for adaptive immunity. We investigated here the direct and indirect effects of TcdA and TcdB on the migration of human blood T cells using conventional cell migration assays and microfluidic devices. It has been found that, although both toxins decrease T cell motility, only TcdA but not TcdB decreases T cell chemotaxis. Similar effects are observed in T cell migration toward the TcdA- or TcdB-treated human epithelial cells. Our study demonstrated the primary role of TcdA (compared to TcdB) in altering T cell migration and chemotaxis, suggesting possible implications for C. difficile toxin mediated adaptive immune responses in CDAD. Full article
(This article belongs to the Special Issue Novel Properties of Well-Characterized Toxins)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Effects of TcdA and TcdB on T cell viability. Human blood T cells were treated with TcdA (<b>A</b>) or TcdB (<b>B</b>) for 3 h and the cell viability was determined by trypan blue staining using a hemocytometer (at least 3 independent experiments for each condition). The circled toxin concentrations (<span class="html-italic">i.e.</span>, TcdA: 50 ng/mL; TcdB: 25 ng/mL) were selected for all subsequent experiments. (<span class="html-italic">i.e.</span>, the qualitative selection criteria for TcdA and TcdB concentration are (1) comparable viability level ~80%; (2) the concentration before the viability level drops relatively fast.).</p> Full article ">Figure 2
<p>Effects of TcdA and TcdB on T cell motility and chemotaxis in transwell assays. (<b>A</b>) Migration of TcdA- or TcdB pre-treated T cells or untreated cells to either medium alone or medium containing CCL19 (100 nM), is presented as the percentage of input cells that migrated to the bottom well of the transwell assay; (<b>B</b>) Fold change of cell migration to 100 nM CCL19 comparing to medium alone; (<b>C</b>) Flowcytometric analysis of CCR7 expression on T cells with or without TcdA or TcdB treatment. T cells were incubated with 50 ng/mL TcdA or 25 ng/mL TcdB for 3 h before CCR7 antibody staining (anti-human CCR7-APC); (<b>D</b>) T cell migration to the medium control, 50 ng/mL TcdA or 25 ng/mL TcdB. Data are normalized to the percentage of T cells migrated to medium alone. All migration experiments (at least 3 independent experiments for each condition) were performed in RPMI containing 0.4% BSA for 90 min. The <span class="html-italic">p</span> values for each comparison from the 2-sample <span class="html-italic">t</span> test are shown: <b>**</b> <span class="html-italic">p &lt;</span> 0.01.</p> Full article ">Figure 3
<p>Effects of TcdA and TcdB on T cell chemotaxis to a CCL19 gradient in microfluidic devices. (<b>A</b>) Chemotactic Index (C.I.) and speed of T cells with or without toxin pre-treatment (TcdA: 50 ng/mL; TcdB: 25 ng/mL; 3 h treatment before the cell migration experiments) over a 35 min cell migration experiment in a 100 nM CCL19 gradient. Results are presented as average ± S.E.M. The percentage of cells migrating towards the CCL19 gradient is shown on the top of the C.I; (<b>B</b>–<b>D</b>) Images of T cells without toxin treatment (<b>B</b>), treated by TcdA (<b>C</b>) or TcdB (<b>D</b>) in microfluidic devices. The <span class="html-italic">p</span> values for each comparison from the 2-sample <span class="html-italic">t</span> test are shown. <b>*</b> <span class="html-italic">p</span> &lt; 0.05; <b>**</b> <span class="html-italic">p</span> &lt; 0.01. Three independent experiments were performed for each condition with similar results and one representative experiment for each condition is presented.</p> Full article ">Figure 4
<p>Effects of TcdA or TcdB on T cell migration to HT-29 cell culture. (<b>A</b>) T cell migration to HT-29 cell culture treated with TcdA or TcdB for 24 h or to untreated HT-29 cell culture. Data are normalized to the percentage of T cells migrated to the untreated HT-29 cell culture; (<b>B</b>–<b>D</b>) Morphological changes of HT-29 cells without (<b>B</b>) or with TcdA (<b>C</b>) or TcdB (<b>D</b>) treatment after 24 h. All cell migration experiment were performed in RPMI with 10% FBS for 90 min. The <span class="html-italic">p</span> values for each comparison from the 2-sample <span class="html-italic">t</span> test are shown. <b>*</b> <span class="html-italic">p</span> &lt; 0.05; <b>**</b> <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">
216 KiB  
Review
The Toxicological Impacts of the Fusarium Mycotoxin, Deoxynivalenol, in Poultry Flocks with Special Reference to Immunotoxicity
by Wageha Awad, Khaled Ghareeb, Josef Böhm and Jürgen Zentek
Toxins 2013, 5(5), 912-925; https://doi.org/10.3390/toxins5050912 - 29 Apr 2013
Cited by 77 | Viewed by 10150
Abstract
Deoxynivalenol (DON) is a common Fusarium toxin in poultry feed. Chickens are more resistant to the adverse impacts of deoxynivalenol (DON) compared to other species. In general, the acute form of DON mycotoxicosis rarely occurs in poultry flocks under normal conditions. However, if [...] Read more.
Deoxynivalenol (DON) is a common Fusarium toxin in poultry feed. Chickens are more resistant to the adverse impacts of deoxynivalenol (DON) compared to other species. In general, the acute form of DON mycotoxicosis rarely occurs in poultry flocks under normal conditions. However, if diets contain low levels of DON (less than 5 mg DON/kg diet), lower productivity, impaired immunity and higher susceptibility to infectious diseases can occur. The molecular mechanism of action of DON has not been completely understood. A significant influence of DON in chickens is the impairment of immunological functions. It was known that low doses of DON elevated the serum IgA levels and affected both cell-mediated and humoral immunity in animals. DON is shown to suppress the antibody response to infectious bronchitis vaccine (IBV) and to Newcastle disease virus (NDV) in broilers (10 mg DON/kg feed) and laying hens (3.5 to 14 mg of DON/kg feed), respectively. Moreover, DON (10 mg DON/kg feed) decreased tumor necrosis factor alpha (TNF-α) in the plasma of broilers. DON can severely affect the immune system and, due to its negative impact on performance and productivity, can eventually result in high economic losses to poultry producers. The present review highlights the impacts of DON intoxication on cell mediated immunity, humoral immunity, gut immunity, immune organs and pro-inflammatory cytokines in chickens. Full article
(This article belongs to the Special Issue Novel Properties of Well-Characterized Toxins)
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<p>Chemical structure of deoxynivalenol.</p> Full article ">
566 KiB  
Review
Structure, Function, and Biology of the Enterococcus faecalis Cytolysin
by Daria Van Tyne, Melissa J. Martin and Michael S. Gilmore
Toxins 2013, 5(5), 895-911; https://doi.org/10.3390/toxins5050895 - 29 Apr 2013
Cited by 140 | Viewed by 25411
Abstract
Enterococcus faecalis is a Gram-positive commensal member of the gut microbiota of a wide range of organisms. With the advent of antibiotic therapy, it has emerged as a multidrug resistant, hospital-acquired pathogen. Highly virulent strains of E. faecalis express a pore-forming exotoxin, called [...] Read more.
Enterococcus faecalis is a Gram-positive commensal member of the gut microbiota of a wide range of organisms. With the advent of antibiotic therapy, it has emerged as a multidrug resistant, hospital-acquired pathogen. Highly virulent strains of E. faecalis express a pore-forming exotoxin, called cytolysin, which lyses both bacterial and eukaryotic cells in response to quorum signals. Originally described in the 1930s, the cytolysin is a member of a large class of lanthionine-containing bacteriocins produced by Gram-positive bacteria. While the cytolysin shares some core features with other lantibiotics, it possesses unique characteristics as well. The current understanding of cytolysin biosynthesis, structure/function relationships, and contribution to the biology of E. faecalis are reviewed, and opportunities for using emerging technologies to advance this understanding are discussed. Full article
(This article belongs to the Special Issue Pore-Forming Toxins)
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Figure 1
<p><b><span class="html-italic">E. faecalis</span> cytolysin expression.</b> (<b>A</b>) Cytolysin operon in the inactive and active states. In the inactive state, CylR2 binds to the P<sub>Lys</sub> (P<sub>L</sub>) promoter [<a href="#B68-toxins-05-00895" class="html-bibr">68</a>]. Autoinduction via quorum sensing triggers an inferred change in the binding of the cytolysin promoter by the CylR2 protein, resulting in high-level expression of the cytolysin operon [<a href="#B67-toxins-05-00895" class="html-bibr">67</a>]. (<b>B</b>) Cytolysin processing and secretion. Large and small subunits are post-translationally modified by CylM [<a href="#B65-toxins-05-00895" class="html-bibr">65</a>], secreted and trimmed by CylB [<a href="#B41-toxins-05-00895" class="html-bibr">41</a>], and further processed by CylA [<a href="#B64-toxins-05-00895" class="html-bibr">64</a>]. (<b>C</b>) Cytolysin activity, in the absence and presence of target cells. In the absence of target cells the subunits form inactive and insoluble multimeric complexes. In the presence of target cells they coordinate to form a pore in the target cell membrane [<a href="#B71-toxins-05-00895" class="html-bibr">71</a>].</p> Full article ">Figure 2
<p><b>Sequences and structures of the <span class="html-italic">E. faecalis</span> cytolysin subunits.</b> (<b>A</b>) Primary amino acid sequences of the cytolysin subunits CylL<sub>L</sub> and CylL<sub>S</sub>. Arrows indicate sites of proteolytic cleavage by CylB and CylA [<a href="#B69-toxins-05-00895" class="html-bibr">69</a>], and brackets show the positions of lanthionine and methyllanthionine bridges. (<b>B</b>) Structures of the processed mature cytolysin subunits. Image is reproduced with permission from [<a href="#B76-toxins-05-00895" class="html-bibr">76</a>].</p> Full article ">
332 KiB  
Article
Assessment of Aflatoxin Contamination of Maize, Peanut Meal and Poultry Feed Mixtures from Different Agroecological Zones in Cameroon
by Jean Raphaël Kana, Benoit Gbemenou Joselin Gnonlonfin, Jagger Harvey, James Wainaina, Immaculate Wanjuki, Robert A. Skilton and Alexis Teguia
Toxins 2013, 5(5), 884-894; https://doi.org/10.3390/toxins5050884 - 29 Apr 2013
Cited by 58 | Viewed by 11164
Abstract
Mycotoxins affect poultry production by being present in the feed and directly causing a negative impact on bird performance. Carry-over rates of mycotoxins in animal products are, in general, small (except for aflatoxins in milk and eggs) therefore representing a small source of [...] Read more.
Mycotoxins affect poultry production by being present in the feed and directly causing a negative impact on bird performance. Carry-over rates of mycotoxins in animal products are, in general, small (except for aflatoxins in milk and eggs) therefore representing a small source of mycotoxins for humans. Mycotoxins present directly in human food represent a much higher risk. The contamination of poultry feed by aflatoxins was determined as a first assessment of this risk in Cameroon. A total of 201 samples of maize, peanut meal, broiler and layer feeds were collected directly at poultry farms, poultry production sites and poultry feed dealers in three agroecological zones (AEZs) of Cameroon and analyzed for moisture content and aflatoxin levels. The results indicate that the mean of the moisture content of maize (14.1%) was significantly (P < 0.05) higher than all other commodities (10.0%–12.7%). Approximately 9% of maize samples were positive for aflatoxin, with concentrations overall ranging from <2 to 42 µg/kg. Most of the samples of peanut meal (100%), broiler (93.3%) and layer feeds (83.0%) were positive with concentrations of positive samples ranging from 39 to 950 µg/kg for peanut meal, 2 to 52 µg/kg for broiler feed and 2 to 23 µg/kg for layer feed. The aflatoxin content of layer feed did not vary by AEZ, while the highest (16.8 µg/kg) and the lowest (8.2 µg/kg) aflatoxin content of broiler feed were respectively recorded in Western High Plateau and in Rainforest agroecological zones. These results suggest that peanut meal is likely to be a high risk feed, and further investigation is needed to guide promotion of safe feeds for poultry in Cameroon. Full article
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<p>Sampling sites across different agroecological zones of Cameroon.</p> Full article ">Figure 2
<p>(<b>a</b>). Average aflatoxin content of aflatoxin positive maize samples. (<b>b</b>). Average aflatoxin content of aflatoxin positive peanut meal samples.</p> Full article ">Figure 3
<p>Average aflatoxin content of poultry feed mixtures collected in two agroecological zones in Cameroon.</p> Full article ">
2031 KiB  
Article
An Ultrasensitive Electrochemiluminescent Immunoassay for Aflatoxin M1 in Milk, Based on Extraction by Magnetic Graphene and Detection by Antibody-Labeled CdTe Quantumn Dots-Carbon Nanotubes Nanocomposite
by Ning Gan, Jing Zhou, Ping Xiong, Futao Hu, Yuting Cao, Tianhua Li and Qianli Jiang
Toxins 2013, 5(5), 865-883; https://doi.org/10.3390/toxins5050865 - 29 Apr 2013
Cited by 64 | Viewed by 11460
Abstract
An ultrasensitive electrochemiluminescent immunoassay (ECLIA) for aflatoxins M1 (ATM1) in milk using magnetic Fe3O4-graphene oxides (Fe-GO) as the absorbent and antibody-labeled cadmium telluride quantum dots (CdTe QDs) as the signal tag is presented. Firstly, Fe3O4 nanoparticles [...] Read more.
An ultrasensitive electrochemiluminescent immunoassay (ECLIA) for aflatoxins M1 (ATM1) in milk using magnetic Fe3O4-graphene oxides (Fe-GO) as the absorbent and antibody-labeled cadmium telluride quantum dots (CdTe QDs) as the signal tag is presented. Firstly, Fe3O4 nanoparticles were immobilized on GO to fabricate the magnetic nanocomposites, which were used as absorbent to ATM1. Secondly, aflatoxin M1 antibody (primary antibody, ATM1 Ab1), was attached to the surface of the CdTe QDs-carbon nanotubes nanocomposite to form the signal tag (ATM1 Ab1/CdTe-CNT). The above materials were characterized. The optimal experimental conditions were obtained. Thirdly, Fe-GO was employed for extraction of ATM1 in milk. Results indicated that it can adsorb ATM1 efficiently and selectively within a large extent of pH from 3.0 to 8.0. Adsorption processes reached 95% of the equilibrium within 10 min. Lastly, the ATM1 with a serial of concentrations absorbed on Fe-GO was conjugated with ATM1 Ab1/CdTe-CNT signal tag based on sandwich immunoassay. The immunocomplex can emit a strong ECL signal whose intensity depended linearly on the logarithm of ATM1 concentration from 1.0 to 1.0 × 105 pg/mL, with the detection limit (LOD) of 0.3 pg/mL (S/N = 3). The method was more sensitive for ATM1 detection compared to the ELISA method. Finally, ten samples of milk were tested based on the immunoassay. The method is fast and requires very little sample preparation, which was suitable for high-throughput screening of mycotoxins in food. Full article
(This article belongs to the Special Issue Mycotoxins in Food and Feed)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>The (<b>a</b>) XRD; (<b>b</b>) magnetization hysteresis loops spectrum and (<b>c</b>) magnetic separation of Fe-GO before and after adding the magnetic field.</p> Full article ">Figure 2
<p>The TEM of (<b>a</b>) Fe-GO; (<b>b</b>) CNT; (<b>c</b>) CdTe QDs and (<b>d</b>) CdTe-CNT nanocomposite.</p> Full article ">Figure 3
<p>Fluorescence spectrum of (<b>a</b>) free CdTe and (<b>b</b>) CdTe-CNT.</p> Full article ">Figure 4
<p>The effect of adsorption time on the adsorption capacity.</p> Full article ">Figure 5
<p>Adsorption isotherms of ATM1 onto Fe-GO.</p> Full article ">Figure 6
<p>ECL-potential curves of (<b>a</b>) CdTe QDs; (<b>b</b>) CdTe-CNT and (<b>c</b>) ATM1 Ab1/CdTe-CNT bioconjugates modified SPCE electrode at 0.1 mol/L PBS (pH 7.4) containing 0.1 mol/L KCl and 0.1 mol/L K<sub>2</sub>S<sub>2</sub>O<sub>8</sub>. Scan rate: 100 mV/s. The voltage of the PMT was set at 600 V.</p> Full article ">Figure 7
<p>ECL-potential curves of the (<b>a</b>) Fe-GO; (<b>b</b>) Fe-GO/ATM1/ATM1 Ab1/CdTe; (<b>c</b>) Fe-GO/ATM1/ATM1 Ab1/CdTe-CNT sandwich immunocomplex modified SPCE electrode. Other conditions are the same as <a href="#toxins-05-00865-f006" class="html-fig">Figure 6</a>.</p> Full article ">Figure 8
<p>Effect of (<b>a</b>) Fe-GO composite solutions, (<b>b</b>) pH, (<b>c</b>) incubation temperature, and (<b>d</b>) time on the ECL intensity the immunosensor toward 5 pg/mL ATM1. Other conditions are the same as <a href="#toxins-05-00865-f006" class="html-fig">Figure 6</a>.</p> Full article ">Figure 9
<p>ECL emissions from the immunosensor to the final immunocomplex using 5 pg/mL ATM1 solution under continuous potential scanning for nine cycles. Scan rate: 100 mV/s.</p> Full article ">Figure 10
<p>ECL profiles of the immunosensor before (<b>a</b>) and after (<b>b</b>–<b>j</b>) incubating in different concentrations of ATM1 in pH 7.4 PBS containing 0.1 mol/L KCl and 0.1 mol/L K<sub>2</sub>S<sub>2</sub>O<sub>8</sub>. ATM1 concentration (pg/mL): (<b>a</b>) 0; (<b>b</b>) 5; (<b>c</b>) 10; (<b>d</b>) 100; (<b>e</b>) 5.0 × 10<sup>2</sup>; (<b>f</b>) 1.0 × 10<sup>3</sup>; (<b>g</b>) 5.0 × 10<sup>3</sup>; (<b>h</b>) 1.0 × 10<sup>4</sup>; (<b>i</b>) 5.0 × 10<sup>4</sup>;.(<b>j</b>) 1.0 × 10<sup>5</sup>, Inset A: linear plots of ECL intensity <span class="html-italic">vs</span>. log (ATM1) concentrations.</p> Full article ">Figure 11
<p>The standard curve of ECL intensity <span class="html-italic">vs</span>. log (ATM1) concentrations in milk samples.</p> Full article ">Figure 12
<p>The synthesis steps for ATM Ab1/CdTe-CNT signal tag.</p> Full article ">Figure 13
<p>The detection of ATM1 in samples by the sandwich immunoassay.</p> Full article ">Figure 14
<p>The “sandwich” immunoassay for detection of ATM1.</p> Full article ">
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