Optimization of Surface-Enhanced Raman Spectroscopy Detection Conditions for Interaction between Gonyautoxin and Its Aptamer.

This study aimed to optimize the detection conditions for surface-enhanced Raman spectroscopy (SERS) of single-stranded DNA (ssDNA) in four different buffers and explore the interaction between gonyautoxin (GTX1/4) and its aptamer, GO18. The influence of the silver colloid solution and MgSO4 concentration (0.01 M) added under four different buffered conditions on DNA SERS detection was studied to determine the optimum detection conditions. We explored the interaction between GTX1/4 and GO18 under the same conditions as those in the systematic evolution of ligands by exponential enrichment technique, using Tris-HCl as the buffer. The characteristic peaks of GO18 and its G-quadruplex were detected in four different buffer solutions. The change in peak intensity at 1656 cm-1 confirmed that the binding site between GTX1/4 and GO18 was in the G-quadruplex plane. The relative intensity of the peak at 1656 cm-1 was selected for the GTX1/4-GO18 complex (I1656/I1099) to plot the ratio of GTX1/4 in the Tris-HCl buffer condition (including 30 µL of silver colloid solution and 2 µL of MgSO4), and a linear relationship was obtained as follows: Y = 0.1867X + 1.2205 (R2 = 0.9239). This study provides a basis for subsequent application of SERS in the detection of ssDNA, as well as the binding of small toxins and aptamers.

Using Budding Yeast to Identify Molecules That Block Cancer Cell 'Mitotic Slippage' Only in the Presence of Mitotic Poisons.

Research on the budding yeast Saccharomyces cerevisiae has yielded fundamental discoveries on highly conserved biological pathways and yeast remains the best-studied eukaryotic cell in the world. Studies on the mitotic cell cycle and the discovery of cell cycle checkpoints in budding yeast has led to a detailed, although incomplete, understanding of eukaryotic cell cycle progression. In multicellular eukaryotic organisms, uncontrolled aberrant cell division is the defining feature of cancer. Some of the most successful classes of anti-cancer chemotherapeutic agents are mitotic poisons. Mitotic poisons are thought to function by inducing a mitotic spindle checkpoint-dependent cell cycle arrest, via the assembly of the highly conserved mitotic checkpoint complex (MCC), leading to apoptosis. Even in the presence of mitotic poisons, some cancer cells continue cell division via 'mitotic slippage', which may correlate with a cancer becoming refractory to mitotic poison chemotherapeutic treatments. In this review, knowledge about budding yeast cell cycle control is explored to suggest novel potential drug targets, namely, specific regions in the highly conserved anaphase-promoting complex/cyclosome (APC/C) subunits Apc1 and/or Apc5, and in a specific N-terminal region in the APC/C co-factor cell division cycle 20 (Cdc20), which may yield molecules which block 'mitotic slippage' only in the presence of mitotic poisons.

Identifying, naming and documenting of test and tool compound stocks.

Handling of chemicals is an often-neglected area of test descriptions. Some important aspects are highlighted here, using methyl-phenyl-tetrahydropyridine (MPTP), ferrous sulfate (FeSO4·xH2O) and ciguatoxin as example compounds. These are used to provide some background on aspects of acid-base equilibria, redox state, crystal water, natural compound mixtures, and chemical naming systems. Also, solvents and impurities are addressed, for instance concerning their often high (millimolar range) concentrations in assay buffers and cell culture media. The discussion of these aspects calls for a more standardized preparation of test solutions and a more extensive disclosure of the procedure in publications; it also suggests more flexibility in data mining, as compounds with clearly different identifiers may have been used to produce highly similar or fully identical test conditions. While this short overview is not intended as definitive guidance, it does demand more active involvement of all test developers and performers with these issues, and it calls for more transparent information disclosure concerning the preparation and use of test and control chemical solutions.

Evaluation of the Individual and Combined Toxicity of Fumonisin Mycotoxins in Human Gastric Epithelial Cells.

Fumonisin contaminates food and feed extensively throughout the world, causing chronic and acute toxicity in human and animals. Currently, studies on the toxicology of fumonisins mainly focus on fumonisin B1 (FB1). Considering that FB1, fumonisin B2 (FB2) and fumonisin B3 (FB3) could coexist in food and feed, a study regarding a single toxin, FB1, may not completely reflect the toxicity of fumonisin. The gastrointestinal tract is usually exposed to these dietary toxins. In our study, the human gastric epithelial cell line (GES-1) was used as in vitro model to evaluate the toxicity of fumonisin. Firstly, we found that they could cause a decrease in cell viability, and increase in membrane leakage, cell death and the induction of expression of markers for endoplasmic reticulum (ER) stress. Their toxicity potency rank is FB1 > FB2 >> FB3. The results also showed that the synergistic effect appeared in the combinations of FB1 + FB2 and FB1 + FB3. Nevertheless, the combinations of FB2 + FB3 and FB1 + FB2 + FB3 showed a synergistic effect at low concentration and an antagonistic effect at high concentration. We also found that myriocin (ISP-1) could alleviate the cytotoxicity induced by fumonisin in GES-1 cells. Finally, this study may help to determine or optimize the legal limits and risk assessment method of mycotoxins in food and feed and provide a potential method to block the fumonisin toxicity.

Substrate Promiscuity of a Paralytic Shellfish Toxin Amidinotransferase.

Secondary metabolites are assembled by enzymes that often perform reactions with high selectivity and specificity. Many of these enzymes also tolerate variations in substrate structure, exhibiting promiscuity that enables various applications of a given biocatalyst. However, initial enzyme characterization studies frequently do not explore beyond the native substrates. This limited assessment of substrate scope contributes to the difficulty of identifying appropriate enzymes for specific synthetic applications. Here, we report the natural function of cyanobacterial SxtG, an amidinotransferase involved in the biosynthesis of paralytic shellfish toxins, and demonstrate its ability to modify a breadth of non-native substrates. In addition, we report the first X-ray crystal structure of SxtG, which provides rationale for this enzyme's substrate scope. Taken together, these data confirm the function of SxtG and exemplify its potential utility in biocatalytic synthesis.

Binding characteristics of aflatoxin B1 with free DNA in vitro.

In this paper, the binding characteristics of aflatoxin B1 (AFB1) with the herring sperm deoxyribonucleic acid (DNA) in vitro were investigated through different analytical methods. The ultraviolet-visible spectroscopy (UV-vis), fluorescence, and circular dichroism (CD) spectra results showed that a new AFB1-DNA complex was formed. All the results suggested that AFB1 interacted with free DNA in vitro in an intercalating binding mode. The results of the DNA melting experiments also showed that the melting temperature of DNA increased by about 12.1 °C due to the addition of AFB1, which was supposed to be closely related to the intercalation of AFB1 into DNA. The agar gel electrophoresis experiments further confirmed that the binding mode of AFB1 and free DNA in vitro was indeed intercalation. In addition, the fluorescence quenching induced by adding AFB1 to the ethidium bromide-DNA (EB-DNA) mixture indicated the presence of competitive non-covalent intercalating binding interaction with a competitive binding constant of 5.58 L/mol between AFB1, EB, and DNA. The thermodynamic data demonstrated that the main driving forces of the binding reaction were van der Waals forces and hydrogen bond. The resonance light scattering (RLS) assay results showed that the DNA binding saturation values of AFB1, EB, psoralen (PSO), and angelicin (ANG) were 2.14, 15.59, 0.74, and 0.74, respectively. These results indicated that the DNA binding capacity of AFB1 was weaker than that of EB, but stronger than those of PSO and ANG.

Venom peptides in association with standard drugs: a novel strategy for combating antibiotic resistance - an overview

Development of antibiotic resistance that leads to resurgence of bacterial infections poses a threat to disease-free existence for humankind and is a challenge for the welfare of the society at large. Despite research efforts directed towards treatment of pathogens, antibiotics within new improved classes have not emerged for years, a fact largely attributable to the pharmacological necessities compelling drug development. Recent reversion to the use of natural products alone or in combination with standard drugs has opened up new vistas for alternative therapeutics. The success of this strategy is evident in the sudden interest in plant extracts as additives/synergists for treatment of maladies caused by drug-resistant bacterial strains. Animal venoms have long fascinated scientists as sources of pharmacologically active components that can be exploited for the treatment of specific ailments and should be promoted further to clinical trials. In the present review, we outline the scope and possible methods for the applications of animal venoms in combination with commercial antibiotics to offer a better treatment approach against antibiotic-resistant infections.(AU)

[Citrate anticoagulation hemoperfusion in the treatment of 273 patients].

Early hemoperfusion in poisoned patients can remove poisons rapidly and effectively, which plays an important role in improving the prognosis of patients. The key of hemoperfusion therapy is the safe and effective anticoagulation. The local citrate anticoagulation effect acid is good, it also has little effect on the systemic coagulation mechanism and internal environment of patients, so it is worthy of promotion. We retrospectively analyzed the clinical data and treatment of 273 patients who were poisoned by citrate anticoagulant in the emergency intensive care unit of the Second Affiliated Hospital of Shandong First Medical University, aiming at perfusion of citrate anticoagulant in patients with poisoning. Provide a certain clinical reference.

Retrospective and Prospective Look at Aflatoxin Research and Development from a Practical Standpoint.

Among the array of structurally and toxicologically diverse mycotoxins, aflatoxins have attracted the most interest of scientific research due to their high toxicity and incidence in foods and feeds. Despite the undeniable progress made in various aspects related to aflatoxins, the ultimate goal consisting of reducing the associated public health risks worldwide is far from being reached due to multiplicity of social, political, economic, geographic, climatic, and development factors. However, a reasonable degree of health protection is attained in industrialized countries owing to their scientific, administrative, and financial capacities allowing them to use high-tech agricultural management systems. Less fortunate situations exist in equatorial and sub-equatorial developing countries mainly practicing traditional agriculture managed by smallholders for subsistence, and where the climate is suitable for mould growth and aflatoxin production. This situation worsens due to climatic change producing conditions increasingly suitable for aflatoxigenic mould growth and toxin production. Accordingly, it is difficult to harmonize the regulatory standards of aflatoxins worldwide, which prevents agri-foods of developing countries from accessing the markets of industrialized countries. To tackle the multi-faceted aflatoxin problem, actions should be taken collectively by the international community involving scientific research, technological and social development, environment protection, awareness promotion, etc. International cooperation should foster technology transfer and exchange of pertinent technical information. This review presents the main historical discoveries leading to our present knowledge on aflatoxins and the challenges that should be addressed presently and in the future at various levels to ensure higher health protection for everybody. In short, it aims to elucidate where we come from and where we should go in terms of aflatoxin research/development.

Curare alkaloids from Matis Dart Poison: Comparison with d-tubocurarine in interactions with nicotinic, 5-HT3 serotonin and GABAA receptors.

Several novel bisbenzylisoquinoline alkaloids (BBIQAs) have recently been isolated from a Matis tribe arrow poison and shown by two-electrode voltage-clamp to inhibit mouse muscle nicotinic acetylcholine receptors (nAChR). Here, using radioligand assay with Aplysia californica AChBP and radioiodinated α-bungarotoxin ([125I]-αBgt), we show that BBIQA1, BBIQA2, and d-tubocurarine (d-TC) have similar affinities to nAChR orthosteric site. However, a competition with [125I]-αBgt for binding to the Torpedo californica muscle-type nAChR revealed that BBIQAs1, 2, and 3 are less potent (IC50s = 26.3, 8.75, and 17.0 µM) than d-TC (IC50 = 0.39 µM), while with α7 nAChR in GH4C1 cells, BBIQA1 was less potent that d-TC (IC50s = 162 µM and 7.77 µM, respectively), but BBIQA2 was similar (IC50 = 5.52 µM). In inhibiting the Ca2+ responses induced by acetylcholine in Neuro2a cells expressing the mouse adult α1ß1εδ nAChR or human α7 nAChR, BBIQAs1 and 2 had similar potencies to d-TC (IC50s in the range 0.75-3.08 µM). Our data suggest that BBIQA1 and BBIQA2 can inhibit adult muscle α1ß1εδ nAChR by both competitive and noncompetitive mechanisms. Further experiments on neuronal α3ß2, α4ß2, and α9α10 nAChRs, expressed in Xenopus laevis oocytes, showed that similar potencies for BBIQAs1, 2, and d-TC. With α3ß2γ2 GABAAR currents were almost completely inhibited by d-TC at a high (100 µM) concentration, but BBIQAs1 and 2 were less potent (only 40-50% inhibition), whereas in competition with Alexa Fluor 546-α-cobratoxin for binding to α1ß3γ2 GABAAR in Neuro2a cells, d-TC and these analogs had comparable affinities. Especially interesting effects of BBIQAs1 and 2 in comparison with d-TC were observed for 5-HT3AR: BBIQA1 and BBIQA2 were 5- and 87-fold less potent than d-TC (IC50 = 22.63 nM). Thus, our results reveal that these BBIQAs differ from d-TC in their potencies towards certain Cys-loop receptors, and we suggest that understanding the reasons behind this might be useful for future drug design.

An engineered lipocalin that tightly complexes the plant poison colchicine for use as antidote and in bioanalytical applications.

Colchicine is a toxic alkaloid prevalent in autumn crocus (Colchicum autumnale) that binds to tubulin and inhibits polymerization of microtubules. Using combinatorial and rational protein design, we have developed an artificial binding protein based on the human lipocalin 2 that binds colchicine with a dissociation constant of 120 pm, i.e. 10000-fold stronger than tubulin. Crystallographic analysis of the engineered lipocalin, dubbed Colchicalin, revealed major structural changes in the flexible loop region that forms the ligand pocket at the open end of the eight-stranded ß-barrel, resulting in a lid-like structure over the deeply buried colchicine. A cis-peptide bond between residues Phe71 and Pro72 in loop #2 constitutes a peculiar feature and allows intimate contact with the tricyclic ligand. Using directed evolution, we achieved an extraordinary dissociation half-life of more than 9 h for the Colchicalin-colchicine complex. Together with the chemical robustness of colchicine and availability of activated derivatives, this also opens applications as a general-purpose affinity reagent, including facile quantification of colchicine in biological samples. Given that engineered lipocalins, also known as Anticalin® proteins, represent a class of clinically validated biopharmaceuticals, Colchicalin may offer a therapeutic antidote to scavenge colchicine and reverse its poisoning effect in situations of acute intoxication.

A salamander's toxic arsenal: review of skin poison diversity and function in true salamanders, genus Salamandra.

Terrestrial salamanders of the genus Salamandra represent one of the most prominent groups of amphibians. They are mainly distributed across Europe but also reach Northern Africa and the Near East. Members of the six currently accepted species have long been known to be poisonous; however, work on their toxins was mostly published in German language, and therefore, many nuances of these studies have remained hidden from the majority of herpetologists and toxinologists. Several Salamandra species are called fire salamanders due to their highly contrasted, black-yellow colouration which probably serves to deter predators, although thorough evidence for aposematism in Salamandra is still lacking. Salamandra skin toxins do not only represent a potent antipredator defence but may also have antimicrobial effects. A better understanding of this dual function of Salamandra skin secretions is of utmost importance in the face of the emergence of a fungal disease causing catastrophic declines of fire salamanders in Central Europe, caused by the fungus Batrachochytrium salamandrivorans. In this review, we summarize the knowledge on Salamandra toxins, providing a list of the compounds so far isolated from their secretion and focusing on the bioactivity of the major compounds in Salamandra secretions, the steroidal alkaloids. We identify priorities for future research, including a screening of co-occurrence of steroidal alkaloids and tetrodotoxins in salamandrids, chemical characterization of already identified novel steroidal compounds, elucidation of the presence and role of peptides and proteins in the secretion, and experimental in vitro and in vivo study of the interactions between bioactive compounds in Salamandra skin secretions and cutaneous fungal and bacterial pathogens.

Application of Metabolomic Tools for Studying Low Molecular-Weight Fraction of Animal Venoms and Poisons.

Both venoms and poisonous secretions are complex mixtures that assist in defense, predation, communication, and competition in the animal world. They consist of variable bioactive molecules, such as proteins, peptides, salts and also metabolites. Metabolomics opens up new perspectives for the study of venoms and poisons as it gives an opportunity to investigate their previously unexplored low molecular-weight components. The aim of this article is to summarize the available literature where metabolomic technologies were used for examining the composition of animal venoms and poisons. The paper discusses only the low molecular-weight components of venoms and poisons collected from snakes, spiders, scorpions, toads, frogs, and ants. An overview is given of the analytical strategies used in the analysis of the metabolic content of the samples. We paid special attention to the classes of compounds identified in various venoms and poisons and potential applications of the small molecules (especially bufadienolides) discovered. The issues that should be more effectively addressed in the studies of animal venoms and poisons include challenges related to sample collection and preparation, species-related chemical diversity of compounds building the metabolome and a need of an online database that would enhance identification of small molecule components of these secretions.

Absolute Configuration and Pharmacology of the Poison Frog Alkaloid Phantasmidine.

Phantasmidine, a rigid congener of the well-known nicotinic acetylcholine receptor agonist epibatidine, is found in the same species of poison frog ( Epipedobates anthonyi). Natural phantasmidine was found to be a 4:1 scalemic mixture, enriched in the (2a R,4a S,9a S) enantiomer by chiral-phase LC-MS comparison to the synthetic enantiomers whose absolute configurations were previously established by Mosher's amide analysis. The major enantiomer has the opposite S configuration at the benzylic carbon to natural epibatidine, whose benzylic carbon is R. Pharmacological characterization of the synthetic racemate and separated enantiomers established that phantasmidine is ∼10-fold less potent than epibatidine, but ∼100-fold more potent than nicotine in most receptors tested. Unlike epibatidine, phantasmidine is sharply enantioselective in its activity and the major natural enantiomer whose benzylic carbon has the 4a S configuration is more active. The stereoselective pharmacology of phantasmidine is ascribed to its rigid and asymmetric shape as compared to the nearly symmetric conformations previously suggested for epibatidine enantiomers. While phantasmidine itself is too toxic for direct therapeutic use, we believe it is a useful platform for the development of potent and selective nicotinic agonists, which may have value as pharmacological tools.

Synthesis of carbon quantum dots-doped dummy molecularly imprinted polymer monolithic column for selective enrichment and analysis of aflatoxin B1 in peanut.

In this study, a novel method was proposed to sensitively determine aflatoxin B1 (AFB1) in peanut sample by using a carbon quantum dots-coated dummy molecularly imprinted polymer (CDs-DMIP) monolithic column for pretreatment coupled with high performance liquid chromatography-fluorescence detection (HPLC-FLD). The CDs-DMIP monolithic column was prepared by in-situ polymerization in a water bath using 5,7-dimethoxycoumarin as dummy template molecule. The CDs-DMIP monolithic column was applied to determine AFB1 by HPLC-FLD. Satisfactory linearity was obtained over 0.5-2000ngmL-1, with a high correlation coefficient of 0.9999. The recoveries of AFB1 in peanut sample ranged from 79.5% to 91.2%, and the intraday and interday relative standard deviation ranged from 1.2% to 4.9%. Limit of detection (S/N=3) and limit of quantitation (S/N=10) were 0.118ngmL-1 and 0.393ngmL-1, respectively. Under the optimized conditions, the enrichment factor was over 71-fold. AFB1 in peanut sample and even some other samples could be sensitively determined by CDs-DMIP-HPLC-FLD method.

Zinc oxide nanoparticles provide anti-cholera activity by disrupting the interaction of cholera toxin with the human GM1 receptor.

Vibrio cholerae causes cholera and is the leading cause of diarrhea in developing countries, highlighting the need for the development of new treatment strategies to combat this disease agent. While exploring the possibility of using zinc oxide (ZnO) nanoparticles (NPs) in cholera treatment, we previously found that ZnO NPs reduce fluid accumulation in mouse ileum induced by the cholera toxin (CT) protein. To uncover the mechanism of action of ZnO NPs on CT activity, here we used classical (O395) and El Tor (C6706) V. cholerae biotypes in growth and biochemical assays. We found that a ZnO NP concentration of 10 µg/ml did not affect the growth rates of these two strains, nor did we observe that ZnO NPs reduce the expression levels of CT mRNA and protein. It was observed that ZnO NPs form a complex with CT, appear to disrupt the CT secondary structure, and block its interaction with the GM1 ganglioside receptor in the outer leaflet of the plasma membrane in intestinal (HT-29) cells and thereby reduce CT uptake into the cells. In the range of 2.5-10 µg/ml, ZnO NPs exhibited no cytotoxicity on kidney (HEK293) and HT-29 cells. We conclude that ZnO NPs prevent the first step in the translocation of cholera toxin into intestinal epithelial cells without exerting measurable toxic effects on HEK293 and HT-29 cells.

Oral Supplementation with Bovine Colostrum Decreases Intestinal Permeability and Stool Concentrations of Zonulin in Athletes.

Increased intestinal permeability has been implicated in various pathologies, has various causes, and can develop during vigorous athletic training. Colostrum bovinum is a natural supplement with a wide range of supposed positive health effects, including reduction of intestine permeability. We assessed influence of colostrum supplementation on intestinal permeability related parameters in a group of 16 athletes during peak training for competition. This double-blind placebo-controlled study compared supplementation for 20 days with 500 mg of colostrum bovinum or placebo (whey). Gut permeability status was assayed by differential absorption of lactulose and mannitol (L/M test) and stool zonulin concentration. Baseline L/M tests found that six of the participants (75%) in the colostrum group had increased intestinal permeability. After supplementation, the test values were within the normal range and were significantly lower than at baseline. The colostrum group Δ values produced by comparing the post-intervention and baseline results were also significantly lower than the placebo group Δ values. The differences in stool zonulin concentration were smaller than those in the L/M test, but were significant when the Δ values due to intervention were compared between the colostrum group and the placebo group. Colostrum bovinum supplementation was safe and effective in decreasing of intestinal permeability in this series of athletes at increased risk of its elevation.

Separation of 20 coumarin derivatives using the capillary electrophoresis method optimized by a series of Doehlert experimental designs.

The aim of this study was to develop the first CE-based method enabling separation of 20 structurally similar coumarin derivatives. To facilitate method optimization a series of three consequent Doehlert experimental designs with the response surface methodology was employed, using number of peaks and the adjusted time of analysis as the selected responses. Initially, three variables were examined: buffer pH, ionic strength and temperature (No. 1 Doehlert design). The optimal conditions provided only partial separation, on that account, several buffer additives were examined at the next step: organic cosolvents and cyclodextrin (No. 2 Doehlert design). The optimal cyclodextrin type was also selected experimentally. The most promising results were obtained for the buffers fortified with methanol, acetonitrile and heptakis(2,3,6-tri-O-methyl)-ß-cyclodextrin. Since these additives may potentially affect acid-base equilibrium and ionization state of analytes, the third Doehlert design (No. 3) was used to reconcile concentration of these additives with optimal pH. Ultimately, the total separation of all 20 compounds was achieved using the borate buffer at basic pH 9.5 in the presence of 10mM cyclodextrin, 9% (v/v) acetonitrile and 36% (v/v) methanol. Identity of all compounds was confirmed using the in-lab build UV-VIS spectra library. The developed method succeeded in identification of coumarin derivatives in three real samples. It demonstrates a huge resolving power of CE assisted by addition of cyclodextrins and organic cosolvents. Our unique optimization approach, based on the three Doehlert designs, seems to be prospective for future applications of this technique.

Endocrine activity of mycotoxins and mycotoxin mixtures.

Reporter gene assays incorporating nuclear receptors (estrogen, androgen, thyroid ß and PPARγ2) have been implemented to assess the endocrine activity of 13 mycotoxins and their mixtures. As expected, zearalenone and its metabolites α-zearalenol and ß- zearalenol turned out to have the strongest estrogenic potency (EC50 8,7 10-10 ± 0,8; 3,1 10-11 ± 0,5 and 1,3 10-8 ± 0,3 M respectively). The metabolite of deoxynivalenol, 3-acetyl-deoxynivalenol also had estrogenic activity (EC50 3,8 10-7 ± 1,1 M). Furthermore, most of the mycotoxins (and their mixtures) showed anti-androgenic effects (15-acetyldeoxynivalenol, 3-acetyl-deoxynivalenol and α-zearalenol with potencies within one order of magnitude of that of the reference compound flutamide). In particular, deoxynivalenol and 15-acetyl-deoxynivalenol acted as antagonists for the PPARy2 receptor. When testing mixtures of mycotoxins on the same cell systems, we showed that most of the mixtures reacted as predicted by the concentration addition (CA) theory. Generally, the CA was within the 95% confidence interval of the observed ones, only minor deviations were detected. Although these reporter gene tests cannot be directly extrapolated in vivo, they can be the basis for further research. Especially the additive effects of ZEN and its metabolites are of importance and could have repercussions in vivo.