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1.  Disorder-to-Order Transition in the CyaA Toxin RTX Domain: Implications for Toxin Secretion 
Toxins  2014;7(1):1-20.
The past decade has seen a fundamental reappraisal of the protein structure-to-function paradigm because it became evident that a significant fraction of polypeptides are lacking ordered structures under physiological conditions. Ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered regions. This trait is exhibited by RTX (Repeat in ToXin) motifs found in more than 250 virulence factors secreted by Gram-negative pathogenic bacteria. We have investigated several RTX-containing polypeptides of different lengths, all derived from the Bordetella pertussis adenylate cyclase toxin, CyaA. Using a combination of experimental approaches, we showed that the RTX proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium. This intrinsic disorder mainly results from internal electrostatic repulsions between negatively charged residues of the RTX motifs. Calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of the RTX proteins. We propose that the intrinsically disordered character of the RTX proteins may facilitate the uptake and secretion of virulence factors through the bacterial secretion machinery. These results support the hypothesis that the folding reaction is achieved upon protein secretion and, in the case of proteins containing RTX motifs, could be finely regulated by the calcium gradient across bacterial cell wall.
PMCID: PMC4303809  PMID: 25559101
adenylate cyclase CyaA toxin; intrinsically disordered proteins (IDP); natively unfolded proteins; repeat in toxin (RTX); calcium-binding proteins; calcium-induced protein folding
2.  Characterization of 27 Mycotoxin Binders and the Relation with in Vitro Zearalenone Adsorption at a Single Concentration 
Toxins  2015;7(1):21-33.
The aim of this study was to characterize 27 feed additives marketed as mycotoxin binders and to screen them for their in vitro zearalenone (ZEN) adsorption. Firstly, 27 mycotoxin binders, commercially available in Belgium and The Netherlands, were selected and characterized. Characterization was comprised of X-ray diffraction (XRD) profiling of the mineral content and d-spacing, determination of the cation exchange capacity (CEC) and the exchangeable base cations, acidity, mineral fraction, relative humidity (RH) and swelling volume. Secondly, an in vitro screening experiment was performed to evaluate the adsorption of a single concentration of ZEN in a ZEN:binder ratio of 1:20,000. The free concentration of ZEN was measured after 4 h of incubation with each of the 27 mycotoxin binders at a pH of 2.5, 6.5 and 8.0. A significant correlation between the free concentration of ZEN and both the d-spacing and mineral fraction of the mycotoxin binders was seen at the three pH levels. A low free concentration of ZEN was demonstrated using binders containing mixed-layered smectites and binders containing humic acids.
PMCID: PMC4303810  PMID: 25568976
mycotoxin; binders; characterization; zearalenone; adsorption screening
3.  Plectasin, First Animal Toxin-Like Fungal Defensin Blocking Potassium Channels through Recognizing Channel Pore Region 
Toxins  2015;7(1):34-42.
The potassium channels were recently found to be inhibited by animal toxin-like human β-defensin 2 (hBD2), the first defensin blocker of potassium channels. Whether there are other defensin blockers from different organisms remains an open question. Here, we reported the potassium channel-blocking plectasin, the first defensin blocker from a fungus. Based on the similar cysteine-stabilized alpha-beta (CSαβ) structure between plectasin and scorpion toxins acting on potassium channels, we found that plectasin could dose-dependently block Kv1.3 channel currents through electrophysiological experiments. Besides Kv1.3 channel, plectasin could less inhibit Kv1.1, Kv1.2, IKCa, SKCa3, hERG and KCNQ channels at the concentration of 1 μΜ. Using mutagenesis and channel activation experiments, we found that outer pore region of Kv1.3 channel was the binding site of plectasin, which is similar to the interacting site of Kv1.3 channel recognized by animal toxin blockers. Together, these findings not only highlight the novel function of plectasin as a potassium channel inhibitor, but also imply that defensins from different organisms functionally evolve to be a novel kind of potassium channel inhibitors.
PMCID: PMC4303811  PMID: 25568977
plectasin; defensin; potassium channels; Kv1.3 channel; molecular mechanism; functional evolution
4.  Acknowledgement to Reviewers of Toxins in 2014 
Toxins  2015;7(1):43-48.
PMCID: PMC4303812
5.  Ricin Trafficking in Cells 
Toxins  2015;7(1):49-65.
The heterodimeric plant toxin ricin binds exposed galactosyls at the cell surface of target mammalian cells, and, following endocytosis, is transported in vesicular carriers to the endoplasmic reticulum (ER). Subsequently, the cell-binding B chain (RTB) and the catalytic A chain (RTA) are separated reductively, RTA embeds in the ER membrane and then retrotranslocates (or dislocates) across this membrane. The protein conducting channels used by RTA are usually regarded as part of the ER-associated protein degradation system (ERAD) that removes misfolded proteins from the ER for destruction by the cytosolic proteasomes. However, unlike ERAD substrates, cytosolic RTA avoids destruction and folds into a catalytic conformation that inactivates its target ribosomes. Protein synthesis ceases, and subsequently the cells die apoptotically. This raises questions about how this protein avoids the pathways that are normally sanctioned for ER-dislocating substrates. In this review we focus on the molecular events that occur with non-tagged ricin and its isolated subunits at the ER–cytosol interface. This focus reveals that intra-membrane interactions of RTA may control its fate, an area that warrants further investigation.
PMCID: PMC4303813  PMID: 25584427
ricin; ER-cytosol retrotranslocation; p24 proteins; ERP2; HRD1; proteasome; RPT; chaperone
6.  Effects of Natural Flavonoids on Photosynthetic Activity and Cell Integrity in Microcystis aeruginosa 
Toxins  2015;7(1):66-80.
Flavonoids are natural polyphenolic compounds produced by many aquatic plants and released in their environments. In this study, the effects of several aquatic flavonoids on cyanobacterial Microcystis aeruginosa, especially in relation to the cell growth, photosynthetic activity, cell morphology, and cell membrane integrity, were investigated. Significant growth inhibition was observed when the cyanobacteria were exposed to three flavonoids, namely, 5,4'-dihydroxyflavone (DHF), apigenin, and luteolin. Luteolin reduced the effective quantum yield, photosynthetic efficiency, and maximal electron transport rate by 70%, 59% and 44%, respectively, whereas 5,4'-DHF and apigenin slightly affected these parameters, which implies that luteolin disrupts the photosynthetic system. Moreover, 5,4'-DHF and apigenin compromised the membrane integrity, and induced membrane depolarization in 52% and 38%, and permeabilization in 30% and 44% of the cells, respectively. The 5,4'-DHF and apigenin showed more pronounced effects on M. aeruginosa morphology and membrane integrity, compared to the luteolin. These results suggest that flavonoids could have significant effects on growth and physiological functions in cyanobacterial species.
PMCID: PMC4303814  PMID: 25584428
flavonoid; cyanobacteria; pulse-amplitude modulation; flow cytometry; photosynthesis
7.  Triggering Apoptotic Death of Human Epidermal Keratinocytes by Malic Acid: Involvement of Endoplasmic Reticulum Stress- and Mitochondria-Dependent Signaling Pathways 
Toxins  2015;7(1):81-96.
Malic acid (MA) has been commonly used in cosmetic products, but the safety reports in skin are sparse. To investigate the biological effects of MA in human skin keratinocytes, we investigated the potential cytotoxicity and apoptotic effects of MA in human keratinocyte cell lines (HaCaT). The data showed that MA induced apoptosis based on the observations of DAPI staining, DNA fragmentation, and sub-G1 phase in HaCaT cells and normal human epidermal keratinocytes (NHEKs). Flow cytometric assays also showed that MA increased the production of mitochondrial superoxide (mito-SOX) but decreased the mitochondrial membrane potential. Analysis of bioenergetics function with the XF 24 analyzer Seahorse extracellular flux analyzer demonstrated that oxygen consumption rate (OCR) was significantly decreased whereas extracellular acidification rate (ECAR) was increased in MA-treated keratinocytes. The occurrence of apoptosis was proved by the increased expressions of FasL, Fas, Bax, Bid, caspases-3, -8, -9, cytochrome c, and the declined expressions of Bcl-2, PARP. MA also induced endoplasmic reticulum stress associated protein expression such as GRP78, GADD153, and ATF6α. We demonstrated that MA had anti-proliferative effect in HaCaT cell through the inhibition of cell cycle progression at G0/G1, and the induction of programmed cell death through endoplasmic reticulum stress- and mitochondria-dependent pathways.
PMCID: PMC4303815  PMID: 25584429
malic acid (MA); HaCaT cells; seahorse XF 24 analyzer; apoptosis
8.  Nephroprotective Role of Resveratrol and Ursolic Acid in Aristolochic Acid Intoxicated Zebrafish 
Toxins  2015;7(1):97-109.
The nephrotoxicity of aristolochic acid (AA) is well known, but information regarding the attenuation of AA-induced toxicity is limited. The aim of the present study was to study the nephroprotective effects of resveratrol (Resv) and ursolic acid (UA) in a zebrafish model. We used two transgenic lines, Tg(wt1b:EGFP) and Tg(gata1:DsRed), to evaluate the nephroprotective effects of Resv and UA by recording subtle changes in the kidney and red blood cell circulation. Our results demonstrated that both Resv and UA treatment can attenuate AA-induced kidney malformations and improve blood circulation. Glomerular filtration rate assays revealed that both Resv and UA treatment can restore renal function (100% for Mock; 56.1% ± 17.3% for AA-treated; 80.2% ± 11.3% for Resv+AA; and 83.1% ± 8.1% for UA+AA, n = 15). Furthermore, real-time RT-PCR experiments showed that pre-treatment with either Resv or UA suppresses expression of pro-inflammatory genes. In conclusion, our findings reveal that AA-induced nephrotoxicities can be attenuated by pre-treatment with either Resv or UA. Therefore, we believe that zebrafish represent an efficient model for screening AA-protective natural compounds.
PMCID: PMC4303816  PMID: 25590276
aristolochic acid; kidney; nephrotoxicity; resveratrol; ursolic acid; zebrafish
9.  The Cytolytic Activity of Vaginolysin Strictly Depends on Cholesterol and Is Potentiated by Human CD59 
Toxins  2015;7(1):110-128.
Gardnerella vaginalis produces cytolysin vaginolysin (VLY), which has been suggested to be a contributor to bacterial vaginosis pathogenesis. VLY along with intermedilysin (ILY) from Streptococcus intermedius have been attributed to a group of cholesterol-dependent cytolysins (CDCs) whose pore-forming activity depends on human CD59 (hCD59). Here, we show that different types of cells lacking hCD59 are susceptible to VLY-mediated lysis, albeit to different extents. We analyze the effects of both hCD59 and cholesterol on VLY cytolytic activity. We show that VLY binds to cholesterol-rich membranes of non-human cells, while VLY with an impaired cholesterol recognition site retains binding to the hCD59-containing cells. We further demonstrate that cholesterol binding by VLY is sufficient to trigger the formation of oligomeric complexes on cholesterol rich-liposomes lacking hCD59. Thus, VLY may induce cell lysis following two alternative pathways. One requires only cholesterol and does not depend on hCD59. The second pathway involves hCD59 contribution similarly to ILY. Apparently, under physiological conditions VLY acts in the most effective way by accepting the assistance of hCD59.
PMCID: PMC4303817  PMID: 25590277
cytolysin; vaginolysin; hCD59; cholesterol; liposomes; pore
10.  Increased Circulating Levels of Vitamin D Binding Protein in MS Patients 
Toxins  2015;7(1):129-137.
Vitamin D (vitD) low status is currently considered a main environmental factor in multiple sclerosis (MS) etiology and pathogenesis. VitD and its metabolites are highly hydrophobic and circulate mostly bound to the vitamin D binding protein (DBP) and with lower affinity to albumin, while less than 1% are in a free form. The aim of this study was to investigate whether the circulating levels of either of the two vitD plasma carriers and/or their relationship are altered in MS. We measured DBP and albumin plasma levels in 28 MS patients and 24 healthy controls. MS patients were found to have higher DBP levels than healthy subjects. Concomitant interferon beta therapy did not influence DBP concentration, and the difference with the control group was significant in both females and males. No significant correlation between DBP and albumin levels was observed either in healthy controls or in patients. These observations suggest the involvement of DBP in the patho-physiology of MS.
PMCID: PMC4303818  PMID: 25590278
vitamin D binding protein; albumin; relapsing/remitting multiple sclerosis; beta-interferon; immunology; vitamin D
11.  Important Poisonous Plants in Tibetan Ethnomedicine 
Toxins  2015;7(1):138-155.
Tibetan ethnomedicine is famous worldwide, both for its high effectiveness and unique cultural background. Many poisonous plants have been widely used to treat disorders in the Tibetan medicinal system. In the present review article, some representative poisonous plant species are introduced in terms of their significance in traditional Tibetan medicinal practices. They are Aconitum pendulum, Strychnos nux-vomica, Datura stramonium and Anisodus tanguticus, for which the toxic chemical constituents, bioactivities and pharmacological functions are reviewed herein. The most important toxins include aconitine, strychnine, scopolamine, and anisodamine. These toxic plants are still currently in use for pain-reduction and other purposes by Tibetan healers after processing.
PMCID: PMC4303819  PMID: 25594733
poisonous plants; Tibetan ethnomedicine; toxins; aconitine; strychnine; scopolamine; anisodamine
12.  The Recombinant Maize Ribosome-Inactivating Protein Transiently Reduces Viral Load in SHIV89.6 Infected Chinese Rhesus Macaques 
Toxins  2015;7(1):156-169.
Ribosome inactivating proteins (RIPs) inhibit protein synthesis by depurinating the large ribosomal RNA and some are found to possess anti-human immunodeficiency virus (HIV) activity. Maize ribosome inactivating protein (RIP) has an internal inactivation loop which is proteolytically removed for full catalytic activity. Here, we showed that the recombinant active maize RIP protected chimeric simian-human immunodeficiency virus (SHIV) 89.6-infected macaque peripheral blood mononuclear cells from lysis ex vivo and transiently reduced plasma viral load in SHIV89.6-infected rhesus macaque model. No evidence of immune dysregulation and other obvious side-effects was found in the treated macaques. Our work demonstrates the potential development of maize RIP as an anti-HIV agent without impeding systemic immune functions.
PMCID: PMC4303820  PMID: 25606813
maize RIP; anti-HIV; animal model; viral load
13.  Shiga Toxin 2-Induced Endoplasmic Reticulum Stress Is Minimized by Activated Protein C but Does Not Correlate with Lethal Kidney Injury 
Toxins  2015;7(1):170-186.
Enterohemorrhagic Escherichia coli produce ribotoxic Shiga toxins (Stx), which are responsible for kidney injury and development of hemolytic uremic syndrome. The endoplasmic reticulum (ER) stress response is hypothesized to induce apoptosis contributing to organ injury; however, this process has been described only in vitro. ER stress marker transcripts of spliced XBP1 (1.78-fold), HSP40 (4.45-fold) and CHOP (7.69-fold) were up-regulated early in kidneys of Stx2 challenged mice compared to saline controls. Anti-apoptotic Bcl2 decreased (−2.41-fold vs. saline) and pro-apoptotic DR5 increased (6.38-fold vs. saline) at later time points. Cytoprotective activated protein C (APC) reduced early CHOP expression (−3.3-fold vs. untreated), increased later Bcl2 expression (5.8-fold vs. untreated), and had early effects on survival but did not alter DR5 expression. Changes in kidney ER stress and apoptotic marker transcripts were observed in Stx2-producing C. rodentium challenged mice compared to mice infected with a non-toxigenic control strain. CHOP (4.14-fold) and DR5 (2.81-fold) were increased and Bcl2 (−1.65-fold) was decreased. APC reduced CHOP expression and increased Bcl2 expression, but did not alter mortality. These data indicate that Stx2 induces renal ER stress and apoptosis in murine models of Stx2-induced kidney injury, but decreasing these processes alone was not sufficient to alter survival outcome.
PMCID: PMC4303821  PMID: 25609181
Shiga toxin; endoplasmic reticulum stress; apoptosis; kidney injury; enterohemorrhagic Escherichia coli; hemolytic uremic syndrome
14.  Low Toxicity of Deoxynivalenol-3-Glucoside in Microbial Cells 
Toxins  2015;7(1):187-200.
Host plants excrete a glucosylation enzyme onto the plant surface that changes mycotoxins derived from fungal secondary metabolites to glucosylated products. Deoxynivalenol-3-glucoside (DON3G) is synthesized by grain uridine diphosphate-glucosyltransferase, and is found worldwide, although information on its toxicity is lacking. Here, we conducted growth tests and DNA microarray analysis to elucidate the characteristics of DON3G. The Saccharomyces cerevisiae PDR5 mutant strain exposed to DON3G demonstrated similar growth to the dimethyl sulfoxide control, and DNA microarray analysis revealed limited differences. Only 10 genes were extracted, and the expression profile of stress response genes was similar to that of DON, in contrast to metabolism genes like SER3, which encodes 3-phosphoglycerate dehydrogenase. Growth tests with Chlamydomonas reinhardtii also showed a similar growth rate to the control sample. These results suggest that DON3G has extremely low toxicity to these cells, and the glucosylation of mycotoxins is a useful protective mechanism not only for host plants, but also for other species.
PMCID: PMC4303822  PMID: 25609182
deoxynivalenol-3-glucoside; DNA microarray; yeast; Chlamydomonas reinhardtii
15.  Diversification of Ergot Alkaloids in Natural and Modified Fungi 
Toxins  2015;7(1):201-218.
Several fungi in two different families––the Clavicipitaceae and the Trichocomaceae––produce different profiles of ergot alkaloids, many of which are important in agriculture and medicine. All ergot alkaloid producers share early steps before their pathways diverge to produce different end products. EasA, an oxidoreductase of the old yellow enzyme class, has alternate activities in different fungi resulting in branching of the pathway. Enzymes beyond the branch point differ among lineages. In the Clavicipitaceae, diversity is generated by the presence or absence and activities of lysergyl peptide synthetases, which interact to make lysergic acid amides and ergopeptines. The range of ergopeptines in a fungus may be controlled by the presence of multiple peptide synthetases as well as by the specificity of individual peptide synthetase domains. In the Trichocomaceae, diversity is generated by the presence or absence of the prenyl transferase encoded by easL (also called fgaPT1). Moreover, relaxed specificity of EasL appears to contribute to ergot alkaloid diversification. The profile of ergot alkaloids observed within a fungus also is affected by a delayed flux of intermediates through the pathway, which results in an accumulation of intermediates or early pathway byproducts to concentrations comparable to that of the pathway end product.
PMCID: PMC4303823  PMID: 25609183
ergot alkaloids; Claviceps; Epichloë, Neosartorya fumigata, old yellow enzyme; peptide synthetase; prenyl transferase
16.  Uniform Orientation of Biotinylated Nanobody as an Affinity Binder for Detection of Bacillus thuringiensis (Bt) Cry1Ac Toxin 
Toxins  2014;6(12):3208-3222.
Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies) sandwich-ELISA (DAS-ELISA) assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac) were selected as capture antibody (Nb61) and detection antibody (Nb44). The capture antibody (Nb61) was biotinylated in vivo for directional immobilization on wells coated with streptavidin matrix. Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 μg·mL−1 and a working range 0.010–1.0 μg·mL−1. The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system.
PMCID: PMC4280530  PMID: 25474492
nanobody; Cry1Ac toxin; streptavidin; DAS-ELISA
17.  Sensitive Quantification of Aflatoxin B1 in Animal Feeds, Corn Feed Grain, and Yellow Corn Meal Using Immunomagnetic Bead-Based Recovery and Real-Time Immunoquantitative-PCR 
Toxins  2014;6(12):3223-3237.
Aflatoxins are considered unavoidable natural mycotoxins encountered in foods, animal feeds, and feed grains. In this study, we demonstrate the application of our recently developed real-time immunoquantitative PCR (RT iq-PCR) assay for sensitive detection and quantification of aflatoxins in poultry feed, two types of dairy feed (1 and 2), horse feed, whole kernel corn feed grains, and retail yellow ground corn meal. Upon testing methanol/water (60:40) extractions of the above samples using competitive direct enzyme linked immunosorbent assay, the aflatoxin content was found to be <20 μg/kg. The RT iq-PCR assay exhibited high antigen hook effect in samples containing aflatoxin levels higher than the quantification limits (0.1–10 μg/kg), addressed by comparing the quantification results of undiluted and diluted extracts. In testing the reliability of the immuno-PCR assay, samples were spiked with 200 μg/kg of aflatoxin B1, but the recovery of spiked aflatoxin was found to be poor. Considering the significance of determining trace levels of aflatoxins and their serious implications for animal and human health, the RT iq-PCR method described in this study can be useful for quantifying low natural aflatoxin levels in complex matrices of food or animal feed samples without the requirement of extra sample cleanup.
PMCID: PMC4280531  PMID: 25474493
aflatoxin B1; immunomagnetic bead capture; real-time immunoquantitative PCR; animal feeds; feed grains
18.  The Dynamics of Microcystis Genotypes and Microcystin Production and Associations with Environmental Factors during Blooms in Lake Chaohu, China 
Toxins  2014;6(12):3238-3257.
Lake Chaohu, which is a large, shallow, hypertrophic freshwater lake in southeastern China, has been experiencing lake-wide toxic Microcystis blooms in recent decades. To illuminate the relationships between microcystin (MC) production, the genotypic composition of the Microcystis community and environmental factors, water samples and associated environmental data were collected from June to October 2012 within Lake Chaohu. The Microcystis genotypes and MC concentrations were quantified using quantitative real-time PCR (qPCR) and HPLC, respectively. The results showed that the abundances of Microcystis genotypes and MC concentrations varied on spatial and temporal scales. Microcystis exists as a mixed population of toxic and non-toxic genotypes, and the proportion of toxic Microcystis genotypes ranged from 9.43% to 87.98%. Both Pearson correlation and stepwise multiple regressions demonstrated that throughout the entire lake, the abundances of total and toxic Microcystis and MC concentrations showed significant positive correlation with the total phosphorus and water temperature, suggesting that increases in temperature together with the phosphorus concentrations may promote more frequent toxic Microcystis blooms and higher concentrations of MC. Whereas, dissolved inorganic carbon (DIC) was negatively correlated with the abundances of total and toxic Microcystis and MC concentrations, indicating that rising DIC concentrations may suppress toxic Microcystis abundance and reduce the MC concentrations in the future. Therefore, our results highlight the fact that future eutrophication and global climate change can affect the dynamics of toxic Microcystis blooms and hence change the MC levels in freshwater.
PMCID: PMC4280532  PMID: 25474494
Microcystis; microcystin; 16S rDNA; mcyD; qPCR; environmental factors; Lake Chaohu
20.  Effects of Hydrogen Peroxide and Ultrasound on Biomass Reduction and Toxin Release in the Cyanobacterium, Microcystis aeruginosa 
Toxins  2014;6(12):3260-3280.
Cyanobacterial blooms are expected to increase, and the toxins they produce threaten human health and impair ecosystem services. The reduction of the nutrient load of surface waters is the preferred way to prevent these blooms; however, this is not always feasible. Quick curative measures are therefore preferred in some cases. Two of these proposed measures, peroxide and ultrasound, were tested for their efficiency in reducing cyanobacterial biomass and potential release of cyanotoxins. Hereto, laboratory assays with a microcystin (MC)-producing cyanobacterium (Microcystis aeruginosa) were conducted. Peroxide effectively reduced M. aeruginosa biomass when dosed at 4 or 8 mg L−1, but not at 1 and 2 mg L−1. Peroxide dosed at 4 or 8 mg L−1 lowered total MC concentrations by 23%, yet led to a significant release of MCs into the water. Dissolved MC concentrations were nine-times (4 mg L−1) and 12-times (8 mg L−1 H2O2) higher than in the control. Cell lysis moreover increased the proportion of the dissolved hydrophobic variants, MC-LW and MC-LF (where L = Leucine, W = tryptophan, F = phenylalanine). Ultrasound treatment with commercial transducers sold for clearing ponds and lakes only caused minimal growth inhibition and some release of MCs into the water. Commercial ultrasound transducers are therefore ineffective at controlling cyanobacteria.
PMCID: PMC4280534  PMID: 25513892
cyanotoxin; eutrophication control; lake restoration; LC-MS/MS; microcystin profile; PCC 7820
21.  Bacillus thuringiensis Toxins: An Overview of Their Biocidal Activity 
Toxins  2014;6(12):3296-3325.
Bacillus thuringiensis (Bt) is a Gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing Cry and Cyt proteins, some of which are toxic against a wide range of insect orders, nematodes and human-cancer cells. These toxins have been successfully used as bioinsecticides against caterpillars, beetles, and flies, including mosquitoes and blackflies. Bt also synthesizes insecticidal proteins during the vegetative growth phase, which are subsequently secreted into the growth medium. These proteins are commonly known as vegetative insecticidal proteins (Vips) and hold insecticidal activity against lepidopteran, coleopteran and some homopteran pests. A less well characterized secretory protein with no amino acid similarity to Vip proteins has shown insecticidal activity against coleopteran pests and is termed Sip (secreted insecticidal protein). Bin-like and ETX_MTX2-family proteins (Pfam PF03318), which share amino acid similarities with mosquitocidal binary (Bin) and Mtx2 toxins, respectively, from Lysinibacillus sphaericus, are also produced by some Bt strains. In addition, vast numbers of Bt isolates naturally present in the soil and the phylloplane also synthesize crystal proteins whose biological activity is still unknown. In this review, we provide an updated overview of the known active Bt toxins to date and discuss their activities.
PMCID: PMC4280536  PMID: 25514092
Bacillus thuringiensis; Bt biopesticides; toxic activity; Cry toxins; Cyt toxins; Vip toxins; Sip toxins; parasporins
22.  Quantitative Analysis of Cereulide Toxin from Bacillus cereus in Rice and Pasta Using Synthetic Cereulide Standard and 13C6-Cereulide Standard—A Short Validation Study 
Toxins  2014;6(12):3326-3335.
A single laboratory validation study of a rapid and sensitive quantitative method for the analysis of cereulide toxin produced by Bacillus cereus using ultra high performance liquid chromatography-electrospray-tandem mass spectrometry is presented. The analysis of this cyclic peptide toxin was validated for pasta and rice samples using a newly presented synthetic cereulide peptide standard, together with 13C6-cereulide that previously have not been commercially available. The use of cereulide standard was also compared to the most frequently used surrogate standard, the antibiotic valinomycin. The performance of the method was evaluated by analyzing spiked sample pools from different types of rice and pasta, as well as 21 individual rice and pasta samples from differently prepared meals. Inoculation of samples with three cereulide toxin-producing strains of Bacillus cereus was finally used to mimic naturally contaminated foods. The quantification range of the method was 1–500 ng/g (R2 = 0.999) and the limits of detection and quantification were 0.1 and 1 ng/g, respectively. The precision varied from 3% to 7% relative standard deviation and the trueness from −2% to +6% relative bias at different concentration levels in cooked rice and pasta.
PMCID: PMC4280537  PMID: 25514093
cereulide; toxin; Bacillus cereus; validation; foods; UPLC-MS/MS
23.  Protective Effect of Two Yeast Based Feed Additives on Pigs Chronically Exposed to Deoxynivalenol and Zearalenone 
Toxins  2014;6(12):3336-3353.
To evaluate the effects of the mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) on pigs and the benefits of two mycotoxin mitigation strategies, gilts (n = 84, 9.1 ± 0.1 kg) were allotted to four treatments: CON (control); MT (4.8 mg/kg feed DON and 0.3 mg/kg feed ZEA); MT-YC (MT + 2 g/kg of yeast cell wall product); and MT-YF (MT + 2 g/kg of yeast fermentation product). After 42 days of feeding, pigs fed MT had reduced (p < 0.05) growth performance compared with pigs fed CON. Pigs fed MT-YF had greater (p < 0.05) average daily gain and tended to have greater (p = 0.080) average daily feed intake than MT, whereas pigs fed MT-YC did not differ from MT. Oxidative DNA damage increased (p < 0.05) in MT, whereas pigs fed MT-YF tended to have lower (p = 0.067) oxidative stress. Liver hydropic degeneration was increased (p < 0.05) in MT in contrast to CON and MT-YF, and tended to be greater (p = 0.079) than MT-YC. Collectively, feeding diets contaminated with mycotoxins significantly reduced growth performance and impacted pig health. The yeast additives had varied ability to reduce mycotoxin effects on pig growth and health, but may still play a beneficial role in reducing the overall impacts of a mycotoxin challenge on pigs.
PMCID: PMC4280538  PMID: 25533517
deoxynivalenol; feed additives; pigs; yeast; zearalenone
24.  The Fate of Microcystins in the Environment and Challenges for Monitoring 
Toxins  2014;6(12):3354-3387.
Microcystins are secondary metabolites produced by cyanobacteria that act as hepatotoxins in higher organisms. These toxins can be altered through abiotic processes, such as photodegradation and adsorption, as well as through biological processes via metabolism and bacterial degradation. Some species of bacteria can degrade microcystins, and many other organisms metabolize microcystins into a series of conjugated products. There are toxicokinetic models used to examine microcystin uptake and elimination, which can be difficult to compare due to differences in compartmentalization and speciation. Metabolites of microcystins are formed as a detoxification mechanism, and little is known about how quickly these metabolites are formed. In summary, microcystins can undergo abiotic and biotic processes that alter the toxicity and structure of the microcystin molecule. The environmental impact and toxicity of these alterations and the metabolism of microcystins remains uncertain, making it difficult to establish guidelines for human health. Here, we present the current state of knowledge regarding the alterations microcystins can undergo in the environment.
PMCID: PMC4280539  PMID: 25514094
microcystins; food web; microbial degradation; metabolism; glutathione metabolic pathway; toxicokinetics
25.  Omics Meets Biology: Application to the Design and Preclinical Assessment of Antivenoms 
Toxins  2014;6(12):3388-3405.
Snakebite envenoming represents a neglected tropical disease that has a heavy public health impact worldwide, mostly affecting poor people involved in agricultural activities in Africa, Asia, Latin America and Oceania. A key issue that complicates the treatment of snakebite envenomings is the poor availability of the only validated treatment for this disease, antivenoms. Antivenoms can be an efficacious treatment for snakebite envenoming, provided they are safe, effective, affordable, accessible and administered appropriately. The shortage of antivenoms in various regions, particularly in Sub-Saharan Africa and some parts of Asia, can be significantly alleviated by optimizing the use of current antivenoms and by the generation of novel polyspecific antivenoms having a wide spectrum of efficacy. Complementing preclinical testing of antivenom efficacy using in vivo and in vitro functional neutralization assays, developments in venomics and antivenomics are likely to revolutionize the design and preclinical assessment of antivenoms by being able to test new antivenom preparations and to predict their paraspecific neutralization to the level of species-specific toxins.
PMCID: PMC4280540  PMID: 25517863
snake venom; snakebite envenoming; antivenom; preclinical venom neutralization assays; venomics; antivenomics

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