Toxins

33 pages, 13540 KiB

Open AccessReview

Structure, Biology, and Therapeutic Application of Toxin–Antitoxin Systems in Pathogenic Bacteria

by Ki-Young Lee and Bong-Jin Lee^*

Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea

Toxins 2016, 8(10), 305; https://doi.org/10.3390/toxins8100305 - 22 Oct 2016

Cited by 96 | Viewed by 19084

Bacterial toxin–antitoxin (TA) systems have received increasing attention for their diverse identities, structures, and functional implications in cell cycle arrest and survival against environmental stresses such as nutrient deficiency, antibiotic treatments, and immune system attacks. In this review, we describe the biological functions [...] Read more.

Bacterial toxin–antitoxin (TA) systems have received increasing attention for their diverse identities, structures, and functional implications in cell cycle arrest and survival against environmental stresses such as nutrient deficiency, antibiotic treatments, and immune system attacks. In this review, we describe the biological functions and the auto-regulatory mechanisms of six different types of TA systems, among which the type II TA system has been most extensively studied. The functions of type II toxins include mRNA/tRNA cleavage, gyrase/ribosome poison, and protein phosphorylation, which can be neutralized by their cognate antitoxins. We mainly explore the similar but divergent structures of type II TA proteins from 12 important pathogenic bacteria, including various aspects of protein–protein interactions. Accumulating knowledge about the structure–function correlation of TA systems from pathogenic bacteria has facilitated a novel strategy to develop antibiotic drugs that target specific pathogens. These molecules could increase the intrinsic activity of the toxin by artificially interfering with the intermolecular network of the TA systems. Full article

(This article belongs to the Special Issue Toxin-Antitoxin System in Bacteria)

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21 pages, 4706 KiB

Open AccessReview

A Comprehensive View of the Structural and Functional Alterations of Extracellular Matrix by Snake Venom Metalloproteinases (SVMPs): Novel Perspectives on the Pathophysiology of Envenoming

by José María Gutiérrez^1,*

, Teresa Escalante¹, Alexandra Rucavado¹, Cristina Herrera^1,2 and Jay W. Fox^3,*

¹ Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José 11501-2060, Costa Rica

² Facultad de Farmacia, Universidad de Costa Rica, San José 11501-2060, Costa Rica

³ School of Medicine, University of Virginia, Charlottesville, VA 22959, USA

Toxins 2016, 8(10), 304; https://doi.org/10.3390/toxins8100304 - 22 Oct 2016

Cited by 88 | Viewed by 10913

Abstract

Snake venom metalloproteinases (SVMPs) affect the extracellular matrix (ECM) in multiple and complex ways. Previously, the combination of various methodological platforms, including electron microscopy, histochemistry, immunohistochemistry, and Western blot, has allowed a partial understanding of such complex pathology. In recent years, the proteomics [...] Read more.

Snake venom metalloproteinases (SVMPs) affect the extracellular matrix (ECM) in multiple and complex ways. Previously, the combination of various methodological platforms, including electron microscopy, histochemistry, immunohistochemistry, and Western blot, has allowed a partial understanding of such complex pathology. In recent years, the proteomics analysis of exudates collected in the vicinity of tissues affected by SVMPs has provided novel and exciting information on SVMP-induced ECM alterations. The presence of fragments of an array of ECM proteins, including those of the basement membrane, has revealed a complex pathological scenario caused by the direct action of SVMPs. In addition, the time-course analysis of these changes has underscored that degradation of some fibrillar collagens is likely to depend on the action of endogenous proteinases, such as matrix metalloproteinases (MMPs), synthesized as a consequence of the inflammatory process. The action of SVMPs on the ECM also results in the release of ECM-derived biologically-active peptides that exert diverse actions in the tissue, some of which might be associated with reparative events or with further tissue damage. The study of the effects of SVMP on the ECM is an open field of research which may bring a renewed understanding of snake venom-induced pathology. Full article

(This article belongs to the Special Issue Snake Venom Metalloproteinases)

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21 pages, 2115 KiB

Open AccessArticle

The Snake with the Scorpion’s Sting: Novel Three-Finger Toxin Sodium Channel Activators from the Venom of the Long-Glanded Blue Coral Snake (Calliophis bivirgatus)

by Daryl C. Yang^1,2,†, Jennifer R. Deuis^3,†, Daniel Dashevsky^2,†, James Dobson^2,†, Timothy N. W. Jackson², Andreas Brust³, Bing Xie⁴

, Ivan Koludarov², Jordan Debono², Iwan Hendrikx², Wayne C. Hodgson¹, Peter Josh⁵, Amanda Nouwens⁵, Gregory J. Baillie³, Timothy J. C. Bruxner³, Paul F. Alewood³, Kelvin Kok Peng Lim⁶, Nathaniel Frank⁷, Irina Vetter^3,8,* and Bryan G. Fry^2,*

¹ Department of Pharmacology, Biomedicine Discovery Institute, Monash University, Clayton 3168, Australia

² Venom Evolution Lab, School of Biological Sciences, University of Queensland, St. Lucia 4072, Australia

³ Institute for Molecular Bioscience, University of Queensland, St. Lucia 4072, Australia

⁴ Bejing Genomics Institute-Shenzhen, Shenzhen 518083, China

⁵ School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia 4072, Australia

⁶ Lee Kong Chian Natural History Museum, National University of Singapore, 2 Conservatory Drive, Singapore 117377, Singapore

⁷ Mtoxins, 1111 Washington ave, Oshkosh, WI 54901, USA

⁸ School of Pharmacy, University of Queensland, Woolloongabba 4102, Australia

^† These authors contributed equally to this work.

Toxins 2016, 8(10), 303; https://doi.org/10.3390/toxins8100303 - 18 Oct 2016

Cited by 53 | Viewed by 42223

Abstract

Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components [...] Read more.

Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake (Calliophis bivirgatus), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake’s body length and nestle within the rib cavity. Despite the snake’s notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (δ-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of Na_V1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (Na_V) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with Na_V function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that Na_V modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of Na_V toxins that will provide significant insights into the pharmacology and physiology of Na_V. The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery. Full article

(This article belongs to the Section Animal Venoms)

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18 pages, 7377 KiB

Open AccessArticle

Cross-Neutralisation of In Vitro Neurotoxicity of Asian and Australian Snake Neurotoxins and Venoms by Different Antivenoms

by Anjana Silva^1,2,*

, Wayne C. Hodgson¹ and Geoffrey K. Isbister^1,3

¹ Monash Venom Group, Department of Pharmacology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia

² Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, Saliyapura 50008, Sri Lanka

³ Clinical Toxicology Research Group, University of Newcastle, Newcastle, NSW 2298, Australia

Toxins 2016, 8(10), 302; https://doi.org/10.3390/toxins8100302 - 18 Oct 2016

Cited by 27 | Viewed by 6509

Abstract

There is limited information on the cross-neutralisation of neurotoxic venoms with antivenoms. Cross-neutralisation of the in vitro neurotoxicity of four Asian and four Australian snake venoms, four post-synaptic neurotoxins (α-bungarotoxin, α-elapitoxin-Nk2a, α-elapitoxin-Ppr1 and α-scutoxin; 100 nM) and one pre-synaptic neurotoxin (taipoxin; 100 nM) [...] Read more.

There is limited information on the cross-neutralisation of neurotoxic venoms with antivenoms. Cross-neutralisation of the in vitro neurotoxicity of four Asian and four Australian snake venoms, four post-synaptic neurotoxins (α-bungarotoxin, α-elapitoxin-Nk2a, α-elapitoxin-Ppr1 and α-scutoxin; 100 nM) and one pre-synaptic neurotoxin (taipoxin; 100 nM) was studied with five antivenoms: Thai cobra antivenom (TCAV), death adder antivenom (DAAV), Thai neuro polyvalent antivenom (TNPAV), Indian Polyvalent antivenom (IPAV) and Australian polyvalent antivenom (APAV). The chick biventer cervicis nerve-muscle preparation was used for this study. Antivenom was added to the organ bath 20 min prior to venom. Pre- and post-synaptic neurotoxicity of Bungarus caeruleus and Bungarus fasciatus venoms was neutralised by all antivenoms except TCAV, which did not neutralise pre-synaptic activity. Post-synaptic neurotoxicity of Ophiophagus hannah was neutralised by all antivenoms, and Naja kaouthia by all antivenoms except IPAV. Pre- and post-synaptic neurotoxicity of Notechis scutatus was neutralised by all antivenoms, except TCAV, which only partially neutralised pre-synaptic activity. Pre- and post-synaptic neurotoxicity of Oxyuranus scutellatus was neutralised by TNPAV and APAV, but TCAV and IPAV only neutralised post-synaptic neurotoxicity. Post-synaptic neurotoxicity of Acanthophis antarcticus was neutralised by all antivenoms except IPAV. Pseudonaja textillis post-synaptic neurotoxicity was only neutralised by APAV. The α-neurotoxins were neutralised by TNPAV and APAV, and taipoxin by all antivenoms except IPAV. Antivenoms raised against venoms with post-synaptic neurotoxic activity (TCAV) cross-neutralised the post-synaptic activity of multiple snake venoms. Antivenoms raised against pre- and post-synaptic neurotoxic venoms (TNPAV, IPAV, APAV) cross-neutralised both activities of Asian and Australian venoms. While acknowledging the limitations of adding antivenom prior to venom in an in vitro preparation, cross-neutralization of neurotoxicity means that antivenoms from one region may be effective in other regions which do not have effective antivenoms. TCAV only neutralized post-synaptic neurotoxicity and is potentially useful in distinguishing pre-synaptic and post-synaptic effects in the chick biventer cervicis preparation. Full article

(This article belongs to the Section Animal Venoms)

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13 pages, 958 KiB

Open AccessArticle

Higher Fusarium Toxin Accumulation in Grain of Winter Triticale Lines Inoculated with Fusarium culmorum as Compared with Wheat

by Tomasz Góral^1,*

, Halina Wiśniewska², Piotr Ochodzki¹ and Dorota Walentyn-Góral¹

¹ Department of Plant Pathology, Plant Breeding and Acclimatization Institute-National Research Institute, Radzików, 05-870 Blonie, Poland

² Institute of Plant Genetics, Polish Academy of Sciences, 34 Strzeszynska str., 60-479 Poznan, Poland

Toxins 2016, 8(10), 301; https://doi.org/10.3390/toxins8100301 - 18 Oct 2016

Cited by 18 | Viewed by 6531

Abstract

Resistance to Fusarium head blight in 32 winter triticale and 34 winter wheat accessions was evaluated. Triticale and wheat were sown in field experiments in two locations. At the time of flowering, heads were inoculated with three Fusarium culmorum isolates. Fusarium head blight [...] Read more.

Resistance to Fusarium head blight in 32 winter triticale and 34 winter wheat accessions was evaluated. Triticale and wheat were sown in field experiments in two locations. At the time of flowering, heads were inoculated with three Fusarium culmorum isolates. Fusarium head blight index was scored and after the harvest percentage of Fusarium damaged kernels was assessed. Grain was analysed for type B trichothecenes (deoxynivalenol and derivatives, nivalenol) and zearalenone (ZEN) content. The average Fusarium head blight indexes were 28.0% for wheat and 19.2% for triticale accessions. The percentage of Fusarium damaged kernels was also higher for wheat and came to 55.6%, while for triticale this figure was 40.2%. The average content of deoxynivalenol (DON) for wheat amounted to 11.65 mg/kg and was lower than the result for triticale which was 14.12 mg/kg. The average contents of nivalenol were similar in both cereals: 4.13 mg/kg and 5.19 mg/kg for wheat and triticale respectively. Considerable amounts of DON derivatives in the cereals were also detected. The ZEN content in the grain was 0.60 mg/kg for wheat and 0.66 mg/kg for triticale. Relationships between Fusarium head blight index, Fusarium damaged kernels and mycotoxin contents were statistically significant for wheat and mostly insignificant for triticale. Triticale proved to have less infected heads and kernels than wheat. However, the content of type B trichothecenes was higher in triticale grain than in wheat grain. Full article

(This article belongs to the Collection Understanding Mycotoxin Occurrence in Food and Feed Chains)

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14 pages, 945 KiB

Open AccessArticle

Proteomic Characterization and Comparison of Malaysian Tropidolaemus wagleri and Cryptelytrops purpureomaculatus Venom Using Shotgun-Proteomics

by Syafiq Asnawi Zainal Abidin¹

, Pathmanathan Rajadurai^1,2, Md Ezharul Hoque Chowdhury¹, Muhamad Rusdi Ahmad Rusmili³, Iekhsan Othman^1,*

and Rakesh Naidu¹

¹ Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan 47500, Malaysia

² Ramsay Sime Darby Healthcare, Sime Darby Medical Centre, No. 1, Jalan SS12/1A, Subang Jaya, Selangor Darul Ehsan 47500, Malaysia

³ Kuliyyah of Pharmacy, International Islamic University Malaysia, Kuantan Campus, Bandar Indera Mahkota, Kuantan, Pahang Darul Makmur 25200, Malaysia

Toxins 2016, 8(10), 299; https://doi.org/10.3390/toxins8100299 - 18 Oct 2016

Cited by 30 | Viewed by 8264

Abstract

Tropidolaemus wagleri and Cryptelytrops purpureomaculatus are venomous pit viper species commonly found in Malaysia. Tandem mass spectrometry analysis of the crude venoms has detected different proteins in T. wagleri and C. purpureomaculatus. They were classified into 13 venom protein families consisting of [...] Read more.

Tropidolaemus wagleri and Cryptelytrops purpureomaculatus are venomous pit viper species commonly found in Malaysia. Tandem mass spectrometry analysis of the crude venoms has detected different proteins in T. wagleri and C. purpureomaculatus. They were classified into 13 venom protein families consisting of enzymatic and nonenzymatic proteins. Enzymatic families detected in T. wagleri and C. purpureomaculatus venom were snake venom metalloproteinase, phospholipase A₂, ʟ-amino acid oxidase, serine proteases, 5′-nucleotidase, phosphodiesterase, and phospholipase B. In addition, glutaminyl cyclotransferase was detected in C. purpureomaculatus. C-type lectin-like proteins were common nonenzymatic components in both species. Waglerin was present and unique to T. wagleri—it was not in C. purpureomaculatus venom. In contrast, cysteine-rich secretory protein, bradykinin-potentiating peptide, and C-type natriuretic peptide were present in C. purpureomaculatus venom. Composition of the venom proteome of T. wagleri and C. purpureomaculatus provides useful information to guide production of effective antivenom and identification of proteins with potential therapeutic applications. Full article

(This article belongs to the Special Issue Venomics, Venom Proteomics and Venom Transcriptomics)

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11 pages, 1817 KiB

Open AccessArticle

Effect of Degradation of Zearalenone-Contaminated Feed by Bacillus licheniformis CK1 on Postweaning Female Piglets

by Guanhua Fu¹, Junfei Ma¹, Lihong Wang¹, Xin Yang¹, Jeruei Liu² and Xin Zhao^1,3,*

¹ College of Animal Science and Technology, Northwest A & F University, Yangling 712100, Shaanxi, China

² Institute of Biotechnology and Department of Animal Science and Technology, National Taiwan University, Taipei 10617, Taiwan

³ Department of Animal Science, McGill University, Montreal, QC H9X 3V9, Canada

Toxins 2016, 8(10), 300; https://doi.org/10.3390/toxins8100300 - 17 Oct 2016

Cited by 39 | Viewed by 7616

Abstract

Zearalenone (ZEA), an estrogenic mycotoxin, is mainly produced by Fusarium fungi. In this study, Bacillus licheniformis CK1 isolated from soil with the capability of degrading ZEA was evaluated for its efficacy in reducing the adverse effects of ZEA in piglets. The gilts were [...] Read more.

Zearalenone (ZEA), an estrogenic mycotoxin, is mainly produced by Fusarium fungi. In this study, Bacillus licheniformis CK1 isolated from soil with the capability of degrading ZEA was evaluated for its efficacy in reducing the adverse effects of ZEA in piglets. The gilts were fed one of the following three diets for 14 days: a basic diet for the control group; the basic diet supplemented with ZEA-contaminated basic diet for the treatment 1 (T1) group; and the basic diet supplemented with fermented ZEA-contaminated basic diet by CK1 for the treatment 2 (T2) group. The actual ZEA contents (analyzed) were 0, 1.20 ± 0.11, 0.47 ± 0.22 mg/kg for the control, T1, and T2 diets, respectively. The results showed that the T1 group had significantly increased the size of vulva and the relative weight of reproductive organs compared to the control group at the end of the trial. The T1 group significantly decreased the concentration of the luteinizing hormone (LH) compared with the control and T2 groups. Expression of ERβ was significantly up-regulated in the T2 group compared with the control. In addition, expression of ERβ was not different between the control and the T1 group. In summary, our results suggest that Bacillus licheniformis CK1 could detoxify ZEA in feed and reduce the adverse effects of ZEA in the gilts. Full article

(This article belongs to the Special Issue Promising Detoxification Strategies to Mitigate Mycotoxins in Food and Feed)

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10 pages, 1780 KiB

Open AccessArticle

Improvement of the Pharmacological Properties of Maize RIP by Cysteine-Specific PEGylation

by Ka-Yee Au¹, Wei-Wei Shi¹, Shuai Qian², Zhong Zuo² and Pang-Chui Shaw^1,*

¹ Centre for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China

² School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China

Toxins 2016, 8(10), 298; https://doi.org/10.3390/toxins8100298 - 17 Oct 2016

Cited by 3 | Viewed by 4630

Abstract

To improve the pharmacological properties of maize ribosome-inactivating protein (maize RIP) for targeting HIV-infected cells, the previously engineered TAT-fused active form of maize RIP (MOD) was further engineered for cysteine-directed PEGylation. In this work, two potential antigenic sites, namely Lys-78 and Lys-264, were [...] Read more.

To improve the pharmacological properties of maize ribosome-inactivating protein (maize RIP) for targeting HIV-infected cells, the previously engineered TAT-fused active form of maize RIP (MOD) was further engineered for cysteine-directed PEGylation. In this work, two potential antigenic sites, namely Lys-78 and Lys-264, were identified. They were mutated to cysteine residue and conjugated with PEG_5k or PEG_20k. The resultant PEG derivatives of MOD variants were examined for ribosome-inactivating activity, circulating half-life and immunogenicity. Our results showed that MOD-PEG conjugates had two- to five-fold lower biological activity compared to the wild-type. Mutation of the two sites respectively did not decrease the anti-MOD IgG and IgE level in mice, but the conjugation of PEG did dramatically reduce the antigenicity. Furthermore, pharmacokinetics studies demonstrated that attachment of PEG_20k prolonged the plasma half-life by five-fold for MOD-K78C and 17-fold for MOD-K264C, respectively. The site-specific mutation together with PEGylation therefore generated MOD derivatives with improved pharmacological properties. Full article

(This article belongs to the Special Issue Ribosome Inactivating Toxins)

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38 pages, 8079 KiB

Open AccessArticle

Toxicity and Growth Assessments of Three Thermophilic Benthic Dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima and Coolia monotis) Developing in the Southern Mediterranean Basin

by Hela Ben-Gharbia^1,*, Ons Kéfi-Daly Yahia¹, Zouher Amzil²

, Nicolas Chomérat³, Eric Abadie⁴, Estelle Masseret⁴, Manoella Sibat²

, Habiba Zmerli Triki¹, Habiba Nouri⁵ and Mohamed Laabir⁴

¹ Research Group on Oceanography and Plankton Ecology, Tunisian National Institute of Agronomy (INAT), IRESA—Carthage University, 43 Avenue Charles Nicolle, Tunis 1082, Tunisia

² Phycotoxins Laboratory, Institut Français de Recherche pour l’Exploitation de la Mer (IFREMER), Rue De l’Ile d’Yeu, BP 21105, Nantes Cedex 3 F-44311, France

³ Laboratoire Environnement Ressource de Bretagne Occidentale (LER-BO), Institut Français de Recherche pour l’Exploitation de la Mer (IFREMER), Station de Biologie Marine, Place de la Croix, BP 40537, Concarneau F-29185, France

⁴ Center for Marine Biodiversity, Exploitation and Conservation (MARBEC), Montpellier University, Centre National de la Recherche Scientifique (CNRS), Institut de Recherche pour le Développement (IRD), Institut Français de Recherche pour l’Exploitation de la Mer (IFREMER), Place Eugène Bataillon, CC093, Montpellier Cedex 5 F-34095, France

⁵ Institut de Recherche pour le Développement (IRD), 2 Rue Des Sports-El Menzah 1, BP 434, Tunis 1004, Tunisia

Toxins 2016, 8(10), 297; https://doi.org/10.3390/toxins8100297 - 15 Oct 2016

Cited by 67 | Viewed by 10307

Abstract

Harmful benthic dinoflagellates, usually developing in tropical areas, are expanding to temperate ecosystems facing water warming. Reports on harmful benthic species are particularly scarce in the Southern Mediterranean Sea. For the first time, three thermophilic benthic dinoflagellates (Ostreopsis cf. ovata, Prorocentrum [...] Read more.

Harmful benthic dinoflagellates, usually developing in tropical areas, are expanding to temperate ecosystems facing water warming. Reports on harmful benthic species are particularly scarce in the Southern Mediterranean Sea. For the first time, three thermophilic benthic dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima and Coolia monotis) were isolated from Bizerte Bay (Tunisia, Mediterranean) and monoclonal cultures established. The ribotyping confirmed the morphological identification of the three species. Maximum growth rates were 0.59 ± 0.08 d⁻¹ for O. cf. ovata, 0.35 ± 0.01 d⁻¹ for C. monotis and 0.33 ± 0.04 d⁻¹ for P. lima. Toxin analyses revealed the presence of ovatoxin-a and ovatoxin-b in O. cf. ovata cells. Okadaic acid and dinophysistoxin-1 were detected in P. lima cultures. For C. monotis, a chromatographic peak at 5.6 min with a mass m/z = 1061.768 was observed, but did not correspond to a mono-sulfated analogue of the yessotoxin. A comparison of the toxicity and growth characteristics of these dinoflagellates, distributed worldwide, is proposed. Full article

(This article belongs to the Section Marine and Freshwater Toxins)

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14 pages, 981 KiB

Open AccessArticle

Pentahydroxyscirpene—Producing Strains, Formation In Planta, and Natural Occurrence

by Elisabeth Varga^1,†

, Gerlinde Wiesenberger^2,*,†

, Philipp Fruhmann³

, Alexandra Malachová¹, Thomas Svoboda²

, Marc Lemmens⁴, Gerhard Adam²

and Franz Berthiller¹

¹ Center for Analytical Chemistry and Christian Doppler Laboratory for Mycotoxin Metabolism, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad-Lorenz-Straße 20, Tulln 3430, Austria

² Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad-Lorenz-Straße 24, Tulln 3430, Austria

³ Institute of Applied Synthetic Chemistry, Vienna University of Technology (VUT), Getreidemarkt 9/163, Vienna 1060, Austria

⁴ Institute for Biotechnology in Plant Production, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad-Lorenz-Straße 20, Tulln 3430, Austria

^† These authors contributed equally to this work.

Toxins 2016, 8(10), 295; https://doi.org/10.3390/toxins8100295 - 14 Oct 2016

Cited by 1 | Viewed by 4825

Abstract

Trichothecenes are a class of structurally diverse mycotoxins with more than 200 naturally occurring compounds. Previously, a new compound, pentahydroxyscirpene (PHS), was reported as a byproduct of a nivalenol producing Fusarium strain, IFA189. PHS contains a hydroxy group at C-8 instead of the [...] Read more.

Trichothecenes are a class of structurally diverse mycotoxins with more than 200 naturally occurring compounds. Previously, a new compound, pentahydroxyscirpene (PHS), was reported as a byproduct of a nivalenol producing Fusarium strain, IFA189. PHS contains a hydroxy group at C-8 instead of the keto group of type B trichothecenes. In this work, we demonstrate that IFA189 belongs to the species Fusarium kyushuense using molecular tools. Production of PHS in vitro was also observed for several isolates of other Fusarium species producing nivalenol. Furthermore, we report the formation of 4-acetyl-PHS by F. kyushuense on inoculated rice. Wheat ears of the variety Remus were infected with IFA189 and the in planta production of PHS was confirmed. Natural occurrence of PHS was verified in barley samples from the Czech Republic using a liquid chromatographic-tandem mass spectrometric method validated for this purpose. Toxicity of PHS to wheat ribosomes was evaluated with a coupled in vitro transcription and translation assay, which showed that PHS inhibits protein biosynthesis slightly less than nivalenol and deoxynivalenol. Full article

(This article belongs to the Collection Fusarium Toxins – Relevance for Human and Animal Health)

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12 pages, 3680 KiB

Open AccessArticle

Crystal Structure of Ribosome-Inactivating Protein Ricin A Chain in Complex with the C-Terminal Peptide of the Ribosomal Stalk Protein P2

by Wei-Wei Shi^†, Yun-Sang Tang^†, See-Yuen Sze, Zhen-Ning Zhu, Kam-Bo Wong and Pang-Chui Shaw^*

¹ Centre for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China

^† These authors contributed equally to this work.

Toxins 2016, 8(10), 296; https://doi.org/10.3390/toxins8100296 - 13 Oct 2016

Cited by 32 | Viewed by 8302

Abstract

Ricin is a type 2 ribosome-inactivating protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors [...] Read more.

Ricin is a type 2 ribosome-inactivating protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation center of the ribosome. Here, we present the 1.6 Å crystal structure of Ricin A chain (RTA) complexed to the C-terminal peptide of the ribosomal stalk protein P2, which plays a crucial role in specific recognition of elongation factors and recruitment of eukaryote-specific RIPs to the ribosomes. Our structure reveals that the C-terminal GFGLFD motif of P2 peptide is inserted into a hydrophobic pocket of RTA, while the interaction assays demonstrate the structurally untraced SDDDM motif of P2 peptide contributes to the interaction with RTA. This interaction mode of RTA and P protein is in contrast to that with trichosanthin (TCS), Shiga-toxin (Stx) and the active form of maize RIP (MOD), implying the flexibility of the P2 peptide-RIP interaction, for the latter to gain access to ribosome. Full article

(This article belongs to the Special Issue Ribosome Inactivating Toxins)

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9 pages, 795 KiB

Open AccessCommunication

Quantitative Nuclear Magnetic Resonance Spectroscopy Based on PULCON Methodology: Application to Quantification of Invaluable Marine Toxin, Okadaic Acid

by Ryuichi Watanabe¹, Chika Sugai², Taichi Yamazaki³, Ryoji Matsushima¹, Hajime Uchida¹, Masahiro Matsumiya², Akiko Takatsu³ and Toshiyuki Suzuki^1,*

¹ National Research Institute of Fisheries Science, 2-12-4 Fukuura, Kanazawa, Yokohama 236-8648, Japan

² Department of Marine Science and Resource, Nihon University, Kameino, Fujisawa 252-0880, Japan

³ National Institute of Advanced Industrial Science and Technology, Tsukuba Chuo 3, 1-1-1 Umezono, Tsukuba 305-8563, Japan

Toxins 2016, 8(10), 294; https://doi.org/10.3390/toxins8100294 - 13 Oct 2016

Cited by 54 | Viewed by 8005

Abstract

ERETIC2 (Electronic Reference To access In vivo Concentrations 2) based on PULCON (Pulse Length–based Concentration determination) methodology is a quantitative NMR (qNMR) using an external standard. The performance of the PULCON method was assessed using maleic acid (MA). Quantification of the diarrhetic shellfish [...] Read more.

ERETIC2 (Electronic Reference To access In vivo Concentrations 2) based on PULCON (Pulse Length–based Concentration determination) methodology is a quantitative NMR (qNMR) using an external standard. The performance of the PULCON method was assessed using maleic acid (MA). Quantification of the diarrhetic shellfish toxin and okadaic acid by PULCON was successfully consistent with that obtained by a conventional internal standard method, demonstrating that the PULCON method is useful for the quantification of invaluable marine toxins without any contaminations by an internal standard. Full article

(This article belongs to the Collection Marine and Freshwater Toxins)

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16 pages, 1636 KiB

Open AccessArticle

Pertussis Toxin Exploits Host Cell Signaling Pathways Induced by Meningitis-Causing E. coli K1-RS218 and Enhances Adherence of Monocytic THP-1 Cells to Human Cerebral Endothelial Cells

by Laura Julia Starost^1,†, Sascha Karassek^1,†,‡, Yasuteru Sano², Takashi Kanda², Kwang Sik Kim³, Ulrich Dobrindt⁴, Christian Rüter¹ and Marcus Alexander Schmidt^1,*

¹ Institute of Infectiology, Center of Molecular Biology of Inflammation, Westfälische Wilhelms-Universität Münster, Münster D-48149, Germany

² Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, Yamaguchi, Ube City 755-8505, Japan

³ Pediatric Infectious Diseases Division, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA

⁴ Institute of Hygiene, Microbial Genome Plasticity—Molecular Infection Biology, Westfälische Wilhelms-Universität Münster, Münster D-48149, Germany

^† These authors contributed equally to this study.

^‡ Present address: Oncology Business Unit, WuXi AppTec Inc., Shanghai 200131, China.

Toxins 2016, 8(10), 291; https://doi.org/10.3390/toxins8100291 - 13 Oct 2016

Cited by 3 | Viewed by 6689

Abstract

Pertussis toxin (PTx), the major virulence factor of the whooping cough-causing bacterial pathogen Bordetella pertussis, permeabilizes the blood–brain barrier (BBB) in vitro and in vivo. Breaking barriers might promote translocation of meningitis-causing bacteria across the BBB, thereby facilitating infection. PTx activates several [...] Read more.

Pertussis toxin (PTx), the major virulence factor of the whooping cough-causing bacterial pathogen Bordetella pertussis, permeabilizes the blood–brain barrier (BBB) in vitro and in vivo. Breaking barriers might promote translocation of meningitis-causing bacteria across the BBB, thereby facilitating infection. PTx activates several host cell signaling pathways exploited by the neonatal meningitis-causing Escherichia coli K1-RS218 for invasion and translocation across the BBB. Here, we investigated whether PTx and E. coli K1-RS218 exert similar effects on MAPK p38, NF-κB activation and transcription of downstream targets in human cerebral endothelial TY10 cells using qRT-PCR, Western blotting, and ELISA in combination with specific inhibitors. PTx and E. coli K1-RS218 activate MAPK p38, but only E. coli K1-RS218 activates the NF-κB pathway. mRNA and protein levels of p38 and NF-κB downstream targets including IL-6, IL-8, CxCL-1, CxCL-2 and ICAM-1 were increased. The p38 specific inhibitor SB203590 blocked PTx-enhanced activity, whereas E. coli K1-RS218’s effects were inhibited by the NF-κB inhibitor Bay 11-7082. Further, we found that PTx enhances the adherence of human monocytic THP-1 cells to human cerebral endothelial TY10 cells, thereby contributing to enhanced translocation. These modulations of host cell signaling pathways by PTx and meningitis-causing E. coli support their contributions to pathogen and monocytic THP-1 cells translocation across the BBB. Full article

(This article belongs to the Section Bacterial Toxins)

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17 pages, 2617 KiB

Open AccessArticle

Microcystin-Bound Protein Patterns in Different Cultures of Microcystis aeruginosa and Field Samples

by Nian Wei^1,2, Lili Hu^1,2, Lirong Song^1,* and Nanqin Gan^1,*

¹ State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China

² University of Chinese Academy of Sciences, Beijing 100049, China

Toxins 2016, 8(10), 293; https://doi.org/10.3390/toxins8100293 - 12 Oct 2016

Cited by 33 | Viewed by 5590

Abstract

Micocystin (MC) exists in Microcystis cells in two different forms, free and protein-bound. We examined the dynamic change in extracellular free MCs, intracellular free MCs and protein-bound MCs in both batch cultures and semi-continuous cultures, using high performance liquid chromatography and Western blot. [...] Read more.

Micocystin (MC) exists in Microcystis cells in two different forms, free and protein-bound. We examined the dynamic change in extracellular free MCs, intracellular free MCs and protein-bound MCs in both batch cultures and semi-continuous cultures, using high performance liquid chromatography and Western blot. The results showed that the free MC per cell remained constant, while the quantity of protein-bound MCs increased with the growth of Microcystis cells in both kinds of culture. Significant changes in the dominant MC-bound proteins occurred in the late exponential growth phase of batch cultures, while the dominant MC-bound proteins in semi-continuous cultures remained the same. In field samples collected at different months in Lake Taihu, the dominant MC-bound proteins were shown to be similar, but the amount of protein-bound MC varied and correlated with the intracellular MC content. We identified MC-bound proteins by two-dimensional electrophoresis immunoblots and mass spectrometry. The 60 kDa chaperonin GroEL was a prominent MC-bound protein. Three essential glycolytic enzymes and ATP synthase alpha subunit were also major targets of MC-binding, which might contribute to sustained growth in semi-continuous culture. Our results indicate that protein-bound MC may be important for sustaining growth and adaptation of Microcystis sp. Full article

(This article belongs to the Collection Marine and Freshwater Toxins)

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18 pages, 2635 KiB

Open AccessArticle

Novel Catalytically-Inactive PII Metalloproteinases from a Viperid Snake Venom with Substitutions in the Canonical Zinc-Binding Motif

by Erika Camacho¹, Libia Sanz², Teresa Escalante¹, Alicia Pérez², Fabián Villalta¹, Bruno Lomonte¹

, Ana Gisele C. Neves-Ferreira³, Andrés Feoli¹, Juan J. Calvete^2,4

, José María Gutiérrez¹

and Alexandra Rucavado^1,*

¹ Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José 11501, Costa Rica

² Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas, Valencia 46010, Spain

³ Laboratório de Toxinologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro 21040-900, Brazil

⁴ Departamento de Biotecnología, Universidad Politécnica de Valencia, Valencia 46022, Spain

Toxins 2016, 8(10), 292; https://doi.org/10.3390/toxins8100292 - 12 Oct 2016

Cited by 11 | Viewed by 5958

Abstract

Snake venom metalloproteinases (SVMPs) play key biological roles in prey immobilization and digestion. The majority of these activities depend on the hydrolysis of relevant protein substrates in the tissues. Hereby, we describe several isoforms and a cDNA clone sequence, corresponding to PII SVMP [...] Read more.

Snake venom metalloproteinases (SVMPs) play key biological roles in prey immobilization and digestion. The majority of these activities depend on the hydrolysis of relevant protein substrates in the tissues. Hereby, we describe several isoforms and a cDNA clone sequence, corresponding to PII SVMP homologues from the venom of the Central American pit viper Bothriechis lateralis, which have modifications in the residues of the canonical sequence of the zinc-binding motif HEXXHXXGXXH. As a consequence, the proteolytic activity of the isolated proteins was undetectable when tested on azocasein and gelatin. These PII isoforms comprise metalloproteinase and disintegrin domains in the mature protein, thus belonging to the subclass PIIb of SVMPs. PII SVMP homologues were devoid of hemorrhagic and in vitro coagulant activities, effects attributed to the enzymatic activity of SVMPs, but induced a mild edema. One of the isoforms presents the characteristic RGD sequence in the disintegrin domain and inhibits ADP- and collagen-induced platelet aggregation. Catalytically-inactive SVMP homologues may have been hitherto missed in the characterization of snake venoms. The presence of such enzymatically-inactive homologues in snake venoms and their possible toxic and adaptive roles deserve further investigation. Full article

(This article belongs to the Special Issue Snake Venom Metalloproteinases)

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11 pages, 537 KiB

Open AccessArticle

Effects of Milk Yield, Feed Composition, and Feed Contamination with Aflatoxin B1 on the Aflatoxin M1 Concentration in Dairy Cows’ Milk Investigated Using Monte Carlo Simulation Modelling

by H. J. Van der Fels-Klerx^* and Louise Camenzuli

RIKILT Wageningen University & Research, P.O. Box 230, Wageningen 6700 AE, The Netherlands

Toxins 2016, 8(10), 290; https://doi.org/10.3390/toxins8100290 - 9 Oct 2016

Cited by 26 | Viewed by 6980

Abstract

This study investigated the presence of aflatoxin M1 (AfM1) in dairy cows’ milk, given predefined scenarios for milk production, compound feed (CF) contamination with aflatoxin B1 (AfB1), and inclusion rates of ingredients, using Monte Carlo simulation modelling. The model simulated a typical dairy [...] Read more.

This study investigated the presence of aflatoxin M1 (AfM1) in dairy cows’ milk, given predefined scenarios for milk production, compound feed (CF) contamination with aflatoxin B1 (AfB1), and inclusion rates of ingredients, using Monte Carlo simulation modelling. The model simulated a typical dairy farm in the Netherlands. Six different scenarios were considered, based on two lactation and three CF composition scenarios. AfB1 contamination of the CF was based on results from the Dutch national monitoring programme for AfB1 in feed materials from 2000 until 2010. Monitoring data from feed materials used in CF production for dairy cattle in the Netherlands were used. Additionally, AfB1 contamination data from an incident in maize in 2013 were used. In each scenario, five different transfer equations of AfB1 from feed to AfM1 in the milk were used, and 1000 iterations were run for each scenario. The results showed that under these six scenarios, the weekly farm concentration of AfM1 in milk was above the EC threshold in less than 1% of the iterations, with all five transfer equations considered. However, this increased substantially in weeks when concentrations from the contaminated maize batch were included, and up to 28.5% of the iterations exceeded the EC threshold. It was also observed that an increase in the milk production had a minimal effect on the exceedance of the AfM1 threshold due to an apparent dilution effect. Feeding regimes, including the composition of CF and feeding roughages of dairy cows, should be carefully considered based on the potential AfM1 contamination of the farm’s milk. Full article

(This article belongs to the Special Issue Selected Papers from the 5th International Symposium on Mycotoxins and Toxigenic Moulds: Challenges and Perspectives)

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17 pages, 3077 KiB

Open AccessArticle

The Possible Mechanisms Involved in Degradation of Patulin by Pichia caribbica

by Xiangfeng Zheng, Qiya Yang, Hongyin Zhang^*, Jing Cao, Xiaoyun Zhang and Maurice Tibiru Apaliya

School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, Jiangsu, China

Toxins 2016, 8(10), 289; https://doi.org/10.3390/toxins8100289 - 9 Oct 2016

Cited by 51 | Viewed by 7926

Abstract

In this work, we examined the mechanisms involved in the degradation of patulin by Pichia caribbica. Our results indicate that cell-free filtrate of P. caribbica reduced patutlin content. The heat-killed cells could not degrade patulin. However, the live cells significantly reduced the [...] Read more.

In this work, we examined the mechanisms involved in the degradation of patulin by Pichia caribbica. Our results indicate that cell-free filtrate of P. caribbica reduced patutlin content. The heat-killed cells could not degrade patulin. However, the live cells significantly reduced the concentration of the patulin. In furtherance to this, it was observed that patulin was not detected in the broken yeast cells and cell wall. The addition of cycloheximide to the P. caribbica cells decreased the capacity of degradation of patulin. Proteomics analyses revealed that patulin treatment resulted in an upregulated protein which was involved in metabolism and stress response processes. Our results suggested that the mechanism of degradation of patulin by P. caribbica was not absorption; the presence of patulin can induce P. caribbica to produce associated intracellular and extracellular enzymes, both of which have the ability to degrade patulin. The result provides a new possible method that used the enzymes produced by yeast to detoxify patulin in food and feed. Full article

(This article belongs to the Special Issue Promising Detoxification Strategies to Mitigate Mycotoxins in Food and Feed)

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14 pages, 3562 KiB

Open AccessArticle

A Combinational Strategy upon RNA Sequencing and Peptidomics Unravels a Set of Novel Toxin Peptides in Scorpion Mesobuthus martensii

by Ning Luan^1,†, Wang Shen^1,†, Jie Liu^2,3,†, Bo Wen^2,3, Zhilong Lin^2,3, Shilong Yang^4,5,6, Ren Lai^1,4,5,6,*, Siqi Liu^2,3,* and Mingqiang Rong^4,5,6,*

¹ Life Sciences College of Nanjing Agricultural University, Nanjing 210095, Jiangsu, China

² BGI-Shenzhen, Shenzhen 518083, China

³ China National GeneBank-Shenzhen, BGI-Shenzhen, Shenzhen, Guangdong 518083, China

⁴ Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming 650223, Yunnan, China

⁵ United Laboratory of Natural Peptide of University of Science and Technology of China & Kunming Institute of Zoology, Chinese Academy of Science, Kunming 650223, Yunnan, China

⁶ Sino-African Joint Research Center, Chinese Academy of Science, Wuhan 430074, Hubei, China

^† These authors contributed equally to this paper.

Toxins 2016, 8(10), 286; https://doi.org/10.3390/toxins8100286 - 5 Oct 2016

Cited by 10 | Viewed by 6030

Abstract

Scorpion venom is deemed to contain many toxic peptides as an important source of natural compounds. Out of the two hundred proteins identiﬁed in Mesobuthus martensii (M. martensii), only a few peptide toxins have been found so far. Herein, a combinational approach [...] Read more.

Scorpion venom is deemed to contain many toxic peptides as an important source of natural compounds. Out of the two hundred proteins identiﬁed in Mesobuthus martensii (M. martensii), only a few peptide toxins have been found so far. Herein, a combinational approach based upon RNA sequencing and Liquid chromatography-mass spectrometry/mass spectrometry (LC MS/MS) was employed to explore the venom peptides in M. martensii. A total of 153 proteins were identiﬁed from the scorpion venom, 26 previously known and 127 newly identiﬁed. Of the novel toxins, 97 proteins exhibited sequence similarities to known toxins, and 30 were never reported. Combining peptidomic and transcriptomic analyses, the peptide sequence of BmKKx1 was reannotated and four disulﬁde bridges were conﬁrmed within it. In light of the comparison of conservation and variety of toxin amino acid sequences, highly conserved and variable regions were perceived in 24 toxins that were parts of two sodium channel and two potassium channel toxins families. Taking all of this evidences together, the peptidomic analysis on M. martensii indeed identiﬁed numerous novel scorpion peptides, expanded our knowledge towards the venom diversity, and afforded a set of pharmaceutical candidates. Full article

(This article belongs to the Section Animal Venoms)

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14 pages, 3738 KiB

Open AccessArticle

Structural and Functional Elucidation of Peptide Ts11 Shows Evidence of a Novel Subfamily of Scorpion Venom Toxins

by Caroline M. Cremonez^1,†

, Mohitosh Maiti^2,†, Steve Peigneur³

, Juliana Silva Cassoli⁴, Alexandre A. A. Dutra⁴, Etienne Waelkens⁵, Eveline Lescrinier²

, Piet Herdewijn², Maria Elena De Lima⁴, Adriano M. C. Pimenta⁴, Eliane C. Arantes¹

and Jan Tytgat^3,*

¹ Laboratório de Toxinas Animais, Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto 14040-903, São Paulo, Brasil

² Laboratory for Medicinal Chemistry, Rega Institute for Medical Research, University of Leuven (KU Leuven), P.O. Box 922, Leuven 3000, Belgium

³ Toxicology & Pharmacology, University of Leuven (KU Leuven), Campus Gasthuisberg O&N2, P.O. Box 922, Leuven 3000, Belgium

⁴ Laboratório de Venenos e Toxinas Animais, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte 31270-901, Brasil

⁵ Laboratory of Protein Phosphorylation and Proteomics, University of Leuven (KU Leuven), P.O. Box 922, Leuven 3000, Belgium

^† These authors contributed equally to this work.

Toxins 2016, 8(10), 288; https://doi.org/10.3390/toxins8100288 - 30 Sep 2016

Cited by 28 | Viewed by 6850

Abstract

To date, several families of peptide toxins specifically interacting with ion channels in scorpion venom have been described. One of these families comprise peptide toxins (called KTxs), known to modulate potassium channels. Thus far, 202 KTxs have been reported, belonging to several subfamilies [...] Read more.

To date, several families of peptide toxins specifically interacting with ion channels in scorpion venom have been described. One of these families comprise peptide toxins (called KTxs), known to modulate potassium channels. Thus far, 202 KTxs have been reported, belonging to several subfamilies of KTxs (called α, β, γ, κ, δ, and λ-KTxs). Here we report on a previously described orphan toxin from Tityus serrulatus venom, named Ts11. We carried out an in-depth structure-function analysis combining 3D structure elucidation of Ts11 and electrophysiological characterization of the toxin. The Ts11 structure is highlighted by an Inhibitor Cystine Knot (ICK) type scaffold, completely devoid of the classical secondary structure elements (α-helix and/or β-strand). This has, to the best of our knowledge, never been described before for scorpion toxins and therefore represents a novel, 6th type of structural fold for these scorpion peptides. On the basis of their preferred interaction with voltage-gated K channels, as compared to all the other targets tested, it can be postulated that Ts11 is the first member of a new subfamily, designated as ε-KTx. Full article

(This article belongs to the Section Animal Venoms)

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9 pages, 1104 KiB

Open AccessArticle

Transcript Abundance of Photorhabdus Insect-Related (Pir) Toxin in Manduca sexta and Galleria mellonella Infections

by Anaïs Castagnola¹, Geraldine Mulley², Nathaniel Davis³, Nicholas Waterfield⁴

and S. Patricia Stock^1,5,*

¹ Center for Insect Science, University of Arizona, Tucson, AZ 85721, USA

² School of Biological Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, UK

³ Pima Community College, Tucson, AZ 85721, USA

⁴ Division of Biomedical Sciences, Warwick Medical School, Warwick University, Coventry CV4 7AL, UK

⁵ Department of Entomology, University of Arizona, Tucson, AZ 85721, USA

Toxins 2016, 8(10), 287; https://doi.org/10.3390/toxins8100287 - 29 Sep 2016

Cited by 5 | Viewed by 7342

Abstract

In this study, we assessed pirAB toxin transcription in Photorhabdus luminescens laumondii (strain TT01) (Enterobacteriaceae) by comparing mRNA abundance under in vivo and in vitro conditions. In vivo assays considered both natural and forced infections with two lepidopteran hosts: Galleria mellonella and Manduca [...] Read more.

In this study, we assessed pirAB toxin transcription in Photorhabdus luminescens laumondii (strain TT01) (Enterobacteriaceae) by comparing mRNA abundance under in vivo and in vitro conditions. In vivo assays considered both natural and forced infections with two lepidopteran hosts: Galleria mellonella and Manduca sexta. Three portals of entry were utilized for the forced infection assays: (a) integument; (b) the digestive route (via mouth and anus); and (c) the tracheal route (via spiracles). We also assessed plu4093-2 transcription during the course of a natural infection; this is when the bacteria are delivered by Heterorhabditis bacteriophora nematodes. Transcript abundance in G. mellonella was higher than in M. sexta at two of the observed time points: 15 and 18 h. Expression of pirAB plu4093-2 reached above endogenous control levels at 22 h in G. mellonella but not in M. sexta. Overall, pirAB plu4093-2 transcripts were not as highly expressed in M. sexta as in G. mellonella, from 15 to 22 h. This is the first study to directly compare pirAB plu4093-2 toxin transcript production considering different portals of entry. Full article

(This article belongs to the Section Bacterial Toxins)

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27 pages, 4501 KiB

Open AccessReview

Metalloproteases Affecting Blood Coagulation, Fibrinolysis and Platelet Aggregation from Snake Venoms: Definition and Nomenclature of Interaction Sites

by R. Manjunatha Kini^*

and Cho Yeow Koh

Protein Science Laboratory, Department of Biological Sciences, Faculty of Science, 14 Science Drive 4, National University of Singapore, Singapore 117543, Singapore

Toxins 2016, 8(10), 284; https://doi.org/10.3390/toxins8100284 - 29 Sep 2016

Cited by 129 | Viewed by 16912

Abstract

Snake venom metalloproteases, in addition to their contribution to the digestion of the prey, affect various physiological functions by cleaving specific proteins. They exhibit their activities through activation of zymogens of coagulation factors, and precursors of integrins or receptors. Based on their structure–function [...] Read more.

Snake venom metalloproteases, in addition to their contribution to the digestion of the prey, affect various physiological functions by cleaving specific proteins. They exhibit their activities through activation of zymogens of coagulation factors, and precursors of integrins or receptors. Based on their structure–function relationships and mechanism of action, we have defined classification and nomenclature of functional sites of proteases. These metalloproteases are useful as research tools and in diagnosis and treatment of various thrombotic and hemostatic conditions. They also contribute to our understanding of molecular details in the activation of specific factors involved in coagulation, platelet aggregation and matrix biology. This review provides a ready reference for metalloproteases that interfere in blood coagulation, fibrinolysis and platelet aggregation. Full article

(This article belongs to the Special Issue Snake Venom Metalloproteinases)

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10 pages, 590 KiB

Open AccessArticle

Effects of Citric and Lactic Acid on the Reduction of Deoxynivalenol and Its Derivatives in Feeds

by Elke Humer¹, Annegret Lucke¹, Hauke Harder¹, Barbara U. Metzler-Zebeli², Josef Böhm¹ and Qendrim Zebeli^1,*

¹ Institute of Animal Nutrition and Functional Plant Compounds, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna; Veterinaerplatz 1, Vienna 1210, Austria

² University Clinic for Swine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Veterinaerplatz 1, Vienna 1210, Austria

Toxins 2016, 8(10), 285; https://doi.org/10.3390/toxins8100285 - 28 Sep 2016

Cited by 28 | Viewed by 6115

Abstract

Exposure to mycotoxin-contaminated feeds represents a serious health risk. This has necessitated the need for the establishment of practical methods for mycotoxin decontamination. This study investigated the effects of citric acid (CA) and lactic acid (LA) on common trichothecene mycotoxins in feeds contaminated [...] Read more.

Exposure to mycotoxin-contaminated feeds represents a serious health risk. This has necessitated the need for the establishment of practical methods for mycotoxin decontamination. This study investigated the effects of citric acid (CA) and lactic acid (LA) on common trichothecene mycotoxins in feeds contaminated with Fusarium mycotoxins. Contaminated feed samples were processed either with 5% CA or 5% LA solutions in a ratio of 1:1.2 (w/v) for 5, 24, or 48 h, and analyzed for multiple mycotoxin metabolites using a liquid chromatography–tandem mass spectrometric method. The analyses showed that treating the feed with CA and LA lowered the concentration of deoxynivalenol (DON), whereby 5% LA lowered the original DON concentration in the contaminated feed samples by half, irrespective of the processing time. Similar lowering effects were observed for the concentrations of 15Ac-DON, 5-hydroxyculmorin, and sambucinol. The concentration of nivalenol was only lowered by the LA treatment. In contrast, CA and LA treatments showed no or only small effects on the concentration of several mycotoxins and their derivatives, including zearalenone, fumonisins, and culmorin. In conclusion, the present results indicate that the use of 5% solutions of LA and CA might reduce the concentration of common trichothecene mycotoxins, especially DON and its derivate 15Ac-DON. However, further research is required to determine the effect on overall toxicity and to identify the underlying mechanisms. Full article

(This article belongs to the Section Mycotoxins)

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14 pages, 1936 KiB

Open AccessReview

Structure, Evolution, and Functions of Bacterial Type III Toxin-Antitoxin Systems

by Nathalie Goeders, Ray Chai, Bihe Chen, Andrew Day and George P. C. Salmond^*

Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK

Toxins 2016, 8(10), 282; https://doi.org/10.3390/toxins8100282 - 28 Sep 2016

Cited by 43 | Viewed by 8496

Abstract

Toxin-antitoxin (TA) systems are small genetic modules that encode a toxin (that targets an essential cellular process) and an antitoxin that neutralises or suppresses the deleterious effect of the toxin. Based on the molecular nature of the toxin and antitoxin components, TA systems [...] Read more.

Toxin-antitoxin (TA) systems are small genetic modules that encode a toxin (that targets an essential cellular process) and an antitoxin that neutralises or suppresses the deleterious effect of the toxin. Based on the molecular nature of the toxin and antitoxin components, TA systems are categorised into different types. Type III TA systems, the focus of this review, are composed of a toxic endoribonuclease neutralised by a non-coding RNA antitoxin in a pseudoknotted configuration. Bioinformatic analysis shows that the Type III systems can be classified into subtypes. These TA systems were originally discovered through a phage resistance phenotype arising due to a process akin to an altruistic suicide; the phenomenon of abortive infection. Some Type III TA systems are bifunctional and can stabilise plasmids during vegetative growth and sporulation. Features particular to Type III systems are explored here, emphasising some of the characteristics of the RNA antitoxin and how these may affect the co-evolutionary relationship between toxins and cognate antitoxins in their quaternary structures. Finally, an updated analysis of the distribution and diversity of these systems are presented and discussed. Full article

(This article belongs to the Special Issue Toxin-Antitoxin System in Bacteria)

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15 pages, 4697 KiB

Open AccessArticle

Ex Vivo Smooth Muscle Pharmacological Effects of a Novel Bradykinin-Related Peptide, and Its Analogue, from Chinese Large Odorous Frog, Odorrana livida Skin Secretions

by Jie Xiang^1,†

, Hui Wang^2,†, Chengbang Ma¹, Mei Zhou¹

, Yuxin Wu^1,*

, Lei Wang^1,*, Shaodong Guo³, Tianbao Chen¹

and Chris Shaw¹

¹ Natural Drug Discovery Group, School of Pharmacy, Queen’s University Belfast, Belfast BT9 7BL, Northern Ireland, UK

² School of Pharmaceutical Sciences, China Medical University, Shenyang 110001, China

³ Department of Nutrition and Food Science, College of Agriculture and Life Sciences, Texas A&M University, 123A Cater Mattil Hall, 2253 TAMU, College Station, TX 77843, USA

Toxins 2016, 8(10), 283; https://doi.org/10.3390/toxins8100283 - 27 Sep 2016

Cited by 8 | Viewed by 6066

Abstract

Bradykinin-related peptides (BRPs) are one of the most extensively studied frog secretions-derived peptide families identified from many amphibian species. The diverse primary structures of BRPs have been proven essential for providing valuable information in understanding basic mechanisms associated with drug modification. Here, we [...] Read more.

Bradykinin-related peptides (BRPs) are one of the most extensively studied frog secretions-derived peptide families identified from many amphibian species. The diverse primary structures of BRPs have been proven essential for providing valuable information in understanding basic mechanisms associated with drug modification. Here, we isolated, identified and characterized a dodeca-BRP (RAP-L1, T6-BK), with primary structure RAPLPPGFTPFR, from the skin secretions of Chinese large odorous frogs, Odorrana livida. This novel peptide exhibited a dose-dependent contractile property on rat bladder and rat ileum, and increased the contraction frequency on rat uterus ex vivo smooth muscle preparations; it also showed vasorelaxant activity on rat tail artery smooth muscle. In addition, the analogue RAP-L1, T6, L8-BK completely abolished these effects on selected rat smooth muscle tissues, whilst it showed inhibition effect on bradykinin-induced rat tail artery relaxation. By using canonical antagonist for bradykinin B1 or B2 type receptors, we found that RAP-L1, T6-BK -induced relaxation of the arterial smooth muscle was very likely to be modulated by B2 receptors. The analogue RAP-L1, T6, L8-BK further enhanced the bradykinin inhibitory activity only under the condition of co-administration with HOE140 on rat tail artery, suggesting a synergistic inhibition mechanism by which targeting B2 type receptors. Full article

(This article belongs to the Section Animal Venoms)

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16 pages, 3178 KiB

Open AccessArticle

BmP02 Atypically Delays Kv4.2 Inactivation: Implication for a Unique Interaction between Scorpion Toxin and Potassium Channel

by Bin Wu¹, Yan Zhu¹, Jian Shi^1,2,*, Jie Tao^3,* and Yonghua Ji^1,*

¹ Lab of Neuropharmacology and Neurotoxicology, Shanghai University, Nanchen Road 333, Shanghai 200444, China

² Vascular Biology Research Centre, Institute of Cardiovascular and Cell Sciences, St. George’s, University of London, Cranmer Terrace, London SW17 0RE, UK

³ Central Laboratory, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, 164 Lanxi road, Shanghai 200062, China

Toxins 2016, 8(10), 280; https://doi.org/10.3390/toxins8100280 - 27 Sep 2016

Cited by 5 | Viewed by 4778

Abstract

BmP02, a short-chain peptide with 28 residues from the venom of Chinese scorpion Buthus martensi Karsch, has been reported to inhibit the transient outward potassium currents (I_to) in rat ventricular muscle cells. However, it remains unclear whether BmP02 modulates the Kv4.2 [...] Read more.

BmP02, a short-chain peptide with 28 residues from the venom of Chinese scorpion Buthus martensi Karsch, has been reported to inhibit the transient outward potassium currents (I_to) in rat ventricular muscle cells. However, it remains unclear whether BmP02 modulates the Kv4.2 channel, one of the main contributors to I_to. The present study investigated the effects of BmP02 on Kv4.2 kinetics and its underlying molecular mechanism. The electrophysiological recordings showed that the inactivation of Kv4.2 expressed in HEK293T cells was significantly delayed by BmP02 in a dose-response manner with EC₅₀ of ~850 nM while the peak current, activation and voltage-dependent inactivation of Kv4.2 were not affected. Meanwhile, the recovery from inactivation of Kv4.2 was accelerated and the deactivation was slowed after the application of BmP02. The site-directed mutagenesis combined with computational modelling identified that K347 and K353, located in the turret motif of the Kv4.2, and E4/E5, D20/D21 in BmP02 are key residues to interact with BmP02 through electrostatic force. These findings not only reveal a novel interaction between Kv4.2 channel and its peptidyl modulator, but also provide valuable information for design of highly-selective Kv4.2 modulators. Full article

(This article belongs to the Section Animal Venoms)

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11 pages, 235 KiB

Open AccessReview

Botulinum Toxin A for Controlling Obesity

by Raffaela Pero^1,*, Lorena Coretti¹ and Francesca Lembo²

¹ Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II”, Naples 80131, Italy

² Dipartimento di Farmacia, Università degli Studi di Napoli “Federico II”, via D. Montesano 47, Naples 80131, Italy

Toxins 2016, 8(10), 281; https://doi.org/10.3390/toxins8100281 - 26 Sep 2016

Cited by 15 | Viewed by 5967

Abstract

Rapid growth of the overweight population and the number of obese individuals in recent decades suggests that current strategies based on diet, exercise, and pharmacological knowledge are not sufficient to address this epidemic. Obesity is the result of a high caloric intake and [...] Read more.

Rapid growth of the overweight population and the number of obese individuals in recent decades suggests that current strategies based on diet, exercise, and pharmacological knowledge are not sufficient to address this epidemic. Obesity is the result of a high caloric intake and energy storage, not counterbalanced by an equally important energy expense. Botulinum toxin type A (BoNT-A) use is rapidly expanding to include treatment of a variety of ophthalmological, gastrointestinal, urological, orthopedic, dermatological, secretory, painful, and cosmetic disorders. Many studies evaluating the effect of BoNT-A in gastric antrum e/o fundus for the treatment of obesity have been published. This treatment modality was based on the observation that gastric injection of BoNT-A in laparatomized rats induced a significant reduction of food intake and body weight. These studies have been published yielding debated results. Differences in the selection of patients, the doses of BoNT-A, the method of administration of the toxin, and the instruments of evaluation of some parameters among these studies may be the cause. In this review, it will study the state-of-the-art use of BoNT-A in obesity basic science models and review the clinical evidence on the therapeutic applications of BoNT-A for obesity. Full article

(This article belongs to the Section Bacterial Toxins)

31 pages, 1069 KiB

Open AccessArticle

Polyamines as Snake Toxins and Their Probable Pharmacological Functions in Envenomation

by Steven D. Aird^1,2

, Alejandro Villar Briones³

, Michael C. Roy³ and Alexander S. Mikheyev^2,*

¹ Division of Faculty Affairs, Okinawa Institute of Science and Technology Graduate University, 1919-1 Tancha, Onna-son, Kunigami-gun, Okinawa-ken 904-0495, Japan

² Ecology and Evolution Unit, Okinawa Institute of Science and Technology Graduate University, 1919-1 Tancha, Onna-son, Kunigami-gun, Okinawa-ken 904-0495, Japan

³ Division of Research Support, Okinawa Institute of Science and Technology Graduate University, 1919-1 Tancha, Onna-son, Kunigami-gun, Okinawa-ken 904-0495, Japan

Toxins 2016, 8(10), 279; https://doi.org/10.3390/toxins8100279 - 26 Sep 2016

Cited by 9 | Viewed by 6407

Abstract

While decades of research have focused on snake venom proteins, far less attention has been paid to small organic venom constituents. Using mostly pooled samples, we surveyed 31 venoms (six elapid, six viperid, and 19 crotalid) for spermine, spermidine, putrescine, and cadaverine. Most [...] Read more.

While decades of research have focused on snake venom proteins, far less attention has been paid to small organic venom constituents. Using mostly pooled samples, we surveyed 31 venoms (six elapid, six viperid, and 19 crotalid) for spermine, spermidine, putrescine, and cadaverine. Most venoms contained all four polyamines, although some in essentially trace quantities. Spermine is a potentially significant component of many viperid and crotalid venoms (≤0.16% by mass, or 7.9 µmol/g); however, it is almost completely absent from elapid venoms assayed. All elapid venoms contained larger molar quantities of putrescine and cadaverine than spermine, but still at levels that are likely to be biologically insignificant. As with venom purines, polyamines impact numerous physiological targets in ways that are consistent with the objectives of prey envenomation, prey immobilization via hypotension and paralysis. Most venoms probably do not contain sufficient quantities of polyamines to induce systemic effects in prey; however, local effects seem probable. A review of the pharmacological literature suggests that spermine could contribute to prey hypotension and paralysis by interacting with N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, nicotinic and muscarinic acetylcholine receptors, γ-Aminobutyric acid (GABA) receptors, blood platelets, ryanodine receptors, and Ca²⁺-ATPase. It also blocks many types of cation-permeable channels by interacting with negatively charged amino acid residues in the channel mouths. The site of envenomation probably determines which physiological targets assume the greatest importance; however, venom-induced liberation of endogenous, intracellular stores of polyamines could potentially have systemic implications and may contribute significantly to envenomation sequelae. Full article

(This article belongs to the Section Animal Venoms)

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12 pages, 265 KiB

Open AccessArticle

Protective Effects of Sporoderm-Broken Spores of Ganderma lucidum on Growth Performance, Antioxidant Capacity and Immune Function of Broiler Chickens Exposed to Low Level of Aflatoxin B₁

by Tao Liu¹

, Qiugang Ma^1,*

, Lihong Zhao¹, Ru Jia², Jianyun Zhang¹, Cheng Ji¹ and Xinyue Wang¹

¹ State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

² College of Life Science, Shanxi University, Taiyuan 030006, Shanxi, China

Toxins 2016, 8(10), 278; https://doi.org/10.3390/toxins8100278 - 24 Sep 2016

Cited by 45 | Viewed by 6716

Abstract

This study was conducted to investigate the toxic effects of aflatoxin B₁ (AFB₁) and evaluate the effects of sporoderm-broken spores of Ganoderma lucidum (SSGL) in relieving aflatoxicosis in broilers. A total of 300 one-day-old male Arbor Acre broiler chickens were [...] Read more.

This study was conducted to investigate the toxic effects of aflatoxin B₁ (AFB₁) and evaluate the effects of sporoderm-broken spores of Ganoderma lucidum (SSGL) in relieving aflatoxicosis in broilers. A total of 300 one-day-old male Arbor Acre broiler chickens were randomly divided into four dietary treatments; the treatment diets were: Control (a basal diet containing normal peanut meal); AFB₁ (the basal diet containing AFB₁-contaminated peanut meal); SSGL (basal diet with 200 mg/kg of SSGL); AFB₁+SSGL (supplementation of 200 mg/kg of SSGL in AFB₁ diet). The contents of AFB₁ in AFB₁ and AFB₁+SSGL diets were 25.0 μg/kg in the starter period and 22.5 μg/kg in the finisher period. The results showed that diet contaminated with a low level of AFB₁ significantly decreased (p < 0.05) the average daily feed intake and average daily gain during the entire experiment and reduced (p < 0.05) serum contents of total protein IgA and IgG. Furthermore, a dietary low level of AFB₁ not only increased (p < 0.05) levels of hydrogen peroxide and lipid peroxidation, but also decreased (p < 0.05) total antioxidant capability, catalase, glutathione peroxidase, and hydroxyl radical scavenger activity in the liver and spleen of broilers. Moreover, the addition of SSGL to AFB₁-contaminated diet counteracted these negative effects, indicating that SSGL has a protective effect against aflatoxicosis. Full article

(This article belongs to the Special Issue Promising Detoxification Strategies to Mitigate Mycotoxins in Food and Feed)

16 pages, 3217 KiB

Open AccessArticle

Aerobic De-Epoxydation of Trichothecene Mycotoxins by a Soil Bacterial Consortium Isolated Using In Situ Soil Enrichment

by Wei-Jie He^1,2, Qing-Song Yuan^1,3, You-Bing Zhang^1,2, Mao-Wei Guo^1,3, An-Dong Gong^1,3, Jing-Bo Zhang^1,3, Ai-Bo Wu⁴

, Tao Huang^1,2, Bo Qu^1,3, He-Ping Li^1,2,* and Yu-Cai Liao^1,3,5,*

¹ Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070, China

² College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

³ College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

⁴ Key Laboratory of Food Safety Research Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

⁵ National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China

Toxins 2016, 8(10), 277; https://doi.org/10.3390/toxins8100277 - 24 Sep 2016

Cited by 43 | Viewed by 6819

Abstract

Globally, the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) are among the most widely distributed mycotoxins that contaminate small grain cereals. In this study, a bacterial consortium, PGC-3, with de-epoxydation activity was isolated from soil by an in situ soil enrichment method. Screening [...] Read more.

Globally, the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) are among the most widely distributed mycotoxins that contaminate small grain cereals. In this study, a bacterial consortium, PGC-3, with de-epoxydation activity was isolated from soil by an in situ soil enrichment method. Screening of 14 soil samples that were sprayed with DON revealed that 4 samples were able to biotransform DON into de-epoxydized DON (dE-DON). Among these, the PGC-3 consortium showed the highest and most stable activity to biotransform DON into dE-DON and NIV into dE-NIV. PGC-3 exhibited de-epoxydation activity at a wide range of pH (5–10) and temperatures (20–37 °C) values under aerobic conditions. Sequential subculturing with a continued exposure to DON substantially reduced the microbial population diversity of this consortium. Analyses of the 16S rDNA sequences indicated that PGC-3 comprised 10 bacterial genera. Among these, one species, Desulfitobacterium, showed a steady increase in relative abundance, from 0.03% to 1.55% (a 52-fold increase), as higher concentrations of DON were used in the subculture media, from 0 to 500 μg/mL. This study establishes the foundation to further develop bioactive agents that can detoxify trichothecene mycotoxins in cereals and enables for the characterization of detoxifying genes and their regulation. Full article

(This article belongs to the Special Issue Selected papers from 1^st China Mycotoxin Conference: Current trends in mycotoxin prevention and control)

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13 pages, 2413 KiB

Open AccessArticle

Development of an Innovative in Vitro Potency Assay for Anti-Botulinum Antitoxins

by Osnat Rosen

, Eyal Ozeri, Ada Barnea, Alon Ben David and Ran Zichel^*

Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona 7410001, Israel

Toxins 2016, 8(10), 276; https://doi.org/10.3390/toxins8100276 - 24 Sep 2016

Cited by 18 | Viewed by 7242

Abstract

Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming, [...] Read more.

Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming, labor-intensive, costly, and raises ethical issues related to the large numbers of laboratory animals needed. Until now, all efforts to develop an alternative in vitro assay have not provided a valid replacement to the mouse potency assay. In the present study, we report the development of an innovative in vitro assay for determining botulinum antitoxin potency, using botulinum type B as a model. The concept of the assay is to mimic two fundamental steps in botulinum intoxication: receptor binding and catalytic activity. By simulating these steps in vitro we were able to accurately determine the potency of antitoxin preparations. The reproducibility of the assay was high with a CV < 13%. Most importantly, the antitoxin potency measured by the in vitro assay highly correlated with that measured by the standard in vivo mouse assay (r = 0.9842, p < 0.0001). Thus, this new in vitro assay has the potential to be considered, after validation, as a replacement to the mouse assay for quantitating neutralizing antibody concentrations in pharmaceutical botulinum antitoxin preparations. Future adoption of this in vitro assay would minimize the use of laboratory animals, speed up the time, and reduce the cost of botulinum antitoxin approval. Full article

(This article belongs to the Special Issue Botulinum Neurotoxins Antibody and Vaccine)

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Toxins, Volume 8, Issue 10 (October 2016) – 36 articles

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