Studies in the use of magnetic microspheres for immunoaffinity extraction of paralytic shellfish poisoning toxins from shellfish

Paralytic shellfish poisoning (PSP) is a potentially fatal human health condition caused by the consumption of shellfish containing high levels of PSP toxins. Toxin extraction from shellfish and from algal cultures for use as standards and analysis by alternative analytical monitoring methods to the mouse bioassay is extensive and laborious. This study investigated whether a selected MAb antibody could be coupled to a novel form of magnetic microsphere (hollow glass magnetic microspheres, brand name Ferrospheres-N) and whether these coated microspheres could be utilized in the extraction of low concentrations of the PSP toxin, STX, from potential extraction buffers and spiked mussel extracts. The feasibility of utilizing a mass of 25 mg of Ferrospheres-N, as a simple extraction procedure for STX from spiked sodium acetate buffer, spiked PBS buffer and spiked mussel extracts was determined. The effects of a range of toxin concentrations (20-300 ng/mL), incubation times and temperature on the capability of the immuno-capture of the STX from the spiked mussel extracts were investigated. Finally, the coated microspheres were tested to determine their efficiency at extracting PSP toxins from naturally contaminated mussel samples. Toxin recovery after each experiment was determined by HPLC analysis. This study on using a highly novel immunoaffinity based extraction procedure, using STX as a model, has indicated that it could be a convenient alternative to conventional extraction procedures used in toxin purification prior to sample analysis.     

Extraction of microalgal toxins by large-scale pumping of seawater in Spain and Norway, and isolation of okadaic acid and dinophysistoxin-2.

Marine biotoxins from microalgae can accumulate in shellfish and lead to poisoning of human consumers as well as fish, marine mammals and sea birds. Toxicological assessment of the toxins and development of analytical methods require large amounts of high-purity toxins and their metabolites. Although these toxins can be obtained in limited amounts from contaminated shellfish or from microalgal cultures, difficulties arise when the toxin-producing microalga is difficult to culture or its identity is not known. To circumvent this problem, we have developed a large-scale method for solid-phase extraction (SPE) of lipophilic biotoxins from natural microalgal blooms in seawater. To enhance subsequent purification of toxins adsorbed on the column, we included a filtration step to release the toxins from the cells while removing insoluble compounds and cellular debris. The efficacy of the method was illustrated by extraction and purification of okadaic acid and dinophysistoxin-2 from a high-density Dinophysis acuta bloom in Spain and from a mixed bloom containing low densities of D. acuta in Norway. Isolation of the toxins adsorbed on the SPE column was simple and efficient, and results obtained so far indicate that the method is potentially applicable to a wide range of microalgal toxins such as azaspiracids, pectenotoxins, spirolides and microcystins. The method should also be useful for harvesting toxins from large-scale microalgal cultures, and for bioprospecting for and isolation of bioactive natural products from marine and freshwater environments.     

Azaspiracid: first evidence of protein binding in shellfish.

The occurrence of azaspiracid (AZA) toxins in contaminated shellfish has been the focus of much research. The present study investigated the binding properties of these toxins in mussels of the species Mytilus edulis. The work involved extraction of proteins and AZAs from contaminated mussel hepatopancreas and examination of the extracts by isoelectric focusing (IEF), size exclusion chromatography (SEC) and sodium docecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Liquid chromatography coupled with tandem mass spectrometry analysis (LC-MS/MS) was also performed in this study to identify AZAs. Blank mussels were subjected to the same purification and analytical procedures. AZAs were found to be weakly bound to a protein with a molecular weight of 45 kDa, in samples of contaminated mussels. This protein, which was abundant in contaminated mussels, was also present in blank mussels, albeit at much lower concentrations. It was further noted that a 22 kDa protein was also present only in contaminated mussel samples.     

Uremic Toxins and Blood Purification: A Review of Current Evidence and Future Perspectives

Accumulation of uremic toxins represents one of the major contributors to the rapid progression of chronic kidney disease (CKD), especially in patients with end-stage renal disease that are undergoing dialysis treatment. In particular, protein-bound uremic toxins (PBUTs) seem to have an important key pathophysiologic role in CKD, inducing various cardiovascular complications. The removal of uremic toxins from the blood with dialytic techniques represents a proved approach to limit the CKD-related complications. However, conventional dialysis mainly focuses on the removal of water-soluble compounds of low and middle molecular weight, whereas PBTUs are strongly protein-bound, thus not efficiently eliminated. Therefore, over the years, dialysis techniques have been adapted by improving membranes structures or using combined strategies to maximize PBTUs removal and eventually prevent CKD-related complications. Recent findings showed that adsorption-based extracorporeal techniques, in addition to conventional dialysis treatment, may effectively adsorb a significant amount of PBTUs during the course of the sessions. This review is focused on the analysis of the current state of the art for blood purification strategies in order to highlight their potentialities and limits and identify the most feasible solution to improve toxins removal effectiveness, exploring possible future strategies and applications, such as the study of a synergic approach by reducing PBTUs production and increasing their blood clearance.     

Diversity of shellfish toxins of "diarrhetic" type revealed by biological and chemical assays.

Extracts of "diarrhetic" shellfish toxins from the edible mussel (Mytilus edulis) were tested with biological and chemical assays to determine toxin content. When tested with the standard mouse bioassay, a higher toxicity was detected in most samples compared to that revealed from detection of the diarrhea-causing substances okadaic acid and dinophysistoxin-1 by high-performance liquid chromatography. Routine extraction of toxins for the two assays was carried out with two different solvents, acetone versus aqueous methanol. Accordingly, we questioned whether the variation in results between the methods could be due to differences in chemical properties of these two solvents. When tested, the two solvent systems showed practically the same efficiency concerning the extraction of okadaic acid and dinophysistoxin-1. This demonstrated that toxins other than those causing diarrhea were present in the samples, and that the mouse bioassay was sensitive to these additional toxins. Subsequent testing of the samples with the mouse bioassay, employing both acetone and methanol extracts, revealed that at least two classes of toxins were present in the mussel samples in addition to okadaic acid and dinophysistoxin-1. It is unclear whether the shellfish toxins revealed in this study are partially from known, nondiarrhetic types, such as pectenotoxins or yessotoxins, or from unknown toxin groups exhibiting ichthyotoxic and hemolytic properties.     

Toxicity of French strains of the dinoflagellate Prorocentrum minimum experimental and natural contaminations of mussels.

Mediterranean strains of Prorocentrum minimum do not appear to have the same toxic component as Japanese strains since they showed no cytotoxicity for hepatocytes in culture. However, their toxic components, which appear to block calcium channels, were detectable by the immobilisation test on Diptera larvae. A bio-accumulation experiment in the laboratory showed that the toxins could accumulate in nearly equivalent amounts in the hepatopancreas and meat of cultured mussels. The same toxicity was found in natural samples collected in a period of bloom of P. minimum. These results suggest that P. minimum could be responsible for shellfish toxicity in the natural environment and thus present a risk for human health.     

Development and optimization of modified nucleosides and deoxynucleosides simultaneous extraction with the use of Design of Experiments approach

Modified nucleosides and deoxynucleosides are investigated as potential markers of pathological disorders. Due to different molecular properties, their concurrent extraction from biological matrices is challenging. The aim of the present work was the development of sample preparation procedure allowing for simultaneous extraction of 12 selected modified nucleosides and deoxynucleosides with the use of solid phase extraction (SPE) technique. Commercially available mixed-mode SPE cartridges based on phenylboronic acid and cation-exchange sorbents were utilized. In order to decrease the number of experiments and, consequently the volume of applied solvents, the developed method was optimized with the use of Design of Experiments approach. Optimization of SPE method was carried out by employing Fractional Factorial Design with selected input variables such as composition of conditioning, washing and elution solvents. Next, Full Factorial Design with conditioning, extraction and elution duration time as factors was employed. Evaluated responses in these two experimental sets were peak areas of selected analytes. Levels of assessed factors were determined. Developed and optimized SPE procedure allowed for simultaneous extraction of modified nucleosides and deoxynucleosides with mean recovery of 76.0%. Procedure was applied on the exemplary urine samples with the use of high performance liquid chromatography hyphenated with triple quadrupole mass spectrometry (HPLC-QqQ/MS).     

植物内源激素检测方法新进展

植物内源激素是植物体内产生的一类小分子化合物,在极低的浓度下就能发挥多种生理作用,调控植物生长发育的每一过程。植物内源激素在植物体内含量极低、性质不稳定,其定量检测一直是当前研究的热点和难点。随着分析技术的进步及新仪器的出现,植物内源激素的提取和纯化技术及检测方法也在不断更新。目前,有机溶剂提取、固相萃取柱纯化是常用的提取和纯化步骤,检测方法主要包括生物鉴定法、理化检测法和免疫检测法3大类,其中理化检测是最常用方法。本文对近几年植物内源激素提取、纯化和检测方法 3个方面的新进展进行综述,以期为植物内源激素的深入研究提供参考。     

Immunoenzymatic assay of dyes used in affinity chromatography of proteins]

Immobilized reactive dyes are frequently used in pseudoaffinity chromatography since it has been evidenced that they could specifically interact with some biologicals of interest, and, as a consequence, be separated and purified by this way. This specificity was so high in some cases, that a single step purification process was possible. In addition to these features, the wide variety of dye molecules available associated to their chemical stability and low cost make possible their use as ligands for industrial applications. Nevertheless, one drawback of the use of dye sorbents for protein purification is the possible leakage of dye molecules, with a risk of biological properties alteration of the purified product. We describe a new quantitative assay method for dyes that could contaminate biologicals separated through special affinity columns. This method based on the specific recognition of dyes by antibodies showed a high specificity and a much better sensitivity than the classical spectrophotometric approaches. It has been applied to the quantification of dyes used in affinity chromatography (Cibacron Blue F3-GA, Basilen Bleue E3-G and Procion Red HE-3B) in the presence of proteins having a special affinity for these dyes. This immunoenzymatic assay is also easily applicable to the detection of leachables from dye affinity columns submitted to drastic regeneration treatments.     

Harnessing the Membrane Translocation Properties of AB Toxins for Therapeutic Applications

Over the last few decades, proteins and peptides have become increasingly more common as FDA-approved drugs, despite their inefficient delivery due to their inability to cross the plasma membrane. In this context, bacterial two-component systems, termed AB toxins, use various protein-based membrane translocation mechanisms to deliver toxins into cells, and these mechanisms could provide new insights into the development of bio-based drug delivery systems. These toxins have great potential as therapies both because of their intrinsic properties as well as the modular characteristics of both subunits, which make them highly amenable to conjugation with various drug classes. This review focuses on the therapeutical approaches involving the internalization mechanisms of three representative AB toxins: botulinum toxin type A, anthrax toxin, and cholera toxin. We showcase several specific examples of the use of these toxins to develop new therapeutic strategies for numerous diseases and explain what makes these toxins promising tools in the development of drugs and drug delivery systems.     

Toxicity of French strains of the dinoflagellate Prorocentrum minimum experimental and natural contaminations of mussels

Mediterranean strains of Prorocentrum minimum do not appear to have the same toxic component as Japanese strains since they showed no cytotoxicity for hepatocytes in culture. However, their toxic components, which appear to block calcium channels, were detectable by the immobilisation test on Diptera larvae. A bio-accumulation experiment in the laboratory showed that the toxins could accumulate in nearly equivalent amounts in the hepatopancreas and meat of cultured mussels. The same toxicity was found in natural samples collected in a period of bloom of P. minimum. These results suggest that P. minimum could be responsible for shellfish toxicity in the natural environment and thus present a risk for human health.     

Bacterial protein toxins: current and potential clinical use

Natural toxins are the product of a long-term evolution, and act on essential mechanisms in the most crucial and vital processes of living organisms. They can attack components of the protein synthesis machinery, actin polymerization, signal transduction pathways, intracellular trafficking of vesicles as well as immune and inflammatory responses. For this reason, toxins have increasingly being used as valuable tools for analysis of cellular physiology, and in the recent years, some of them are used medicinally for the treatment of human diseases. This review is devoted to protein toxins of bacterial origin, specifically those toxins that are currently used in therapy or those under study for their potential clinical applications. Bacterial protein toxins are all characterized by a specific mechanism of action that involves the central molecular pathways in the eukaryotic cell. Knowledge of their properties could be used for medical purposes.     

Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced.     

Interrelationships between toxins: studies on the cross-reactivity between bacterial or animal toxins and monoclonal antibodies to two jellyfish venoms

Affinity chromatography on columns of immobilized anti-Chrysaora and anti-Physalia monoclonal antibodies can be an effective purification tool for animal and bacterial toxins. Furthermore, the fact that specific fractions of a given species obtained from immunochromatography columns prepared with either monoclonal antibody possessed identical protein bands, were quantitatively similar in in vitro cardiotoxicity and bound like amounts of antibody, as indicated by the enzyme-linked immunosorbent assay, suggested that antigenic targets of the two monoclonal antibodies are cross-reactive and/or are located on the same molecule. Additional enzyme-linked immunosorbent assays were conducted using non-coelenterate toxins. The significant binding of brown recluse spider venom and purified cholera toxin to both our monoclonal antibodies indicated that these toxic substances shared a common or cross-reacting antigenic site(s) with some coelenterate venoms.     

Tarantulas: eight-legged pharmacists and combinatorial chemists.

Tarantula venoms represent a cornucopia of novel ligands for a variety of cell receptors and ion channels. The diversity of peptide toxin pharmacology has been barely explored as indicated by pharmacological, toxicological and mass spectrometry investigations on more than 55 tarantula venoms. MALDI-TOF MS analysis reveals that the pharmacological diversity is based on relatively small size peptides, which seem to fall into a limited number of structural patterns. Properties and biological activities of the 33 known peptide toxins from tarantula venoms are described. Most known toxins conform to the Inhibitory Cystine Knot (ICK) motif, with differences in the length of intercysteine loops. Recently described peptides show that tarantula toxins can fold according to an elaboration of the Disulfide-Directed beta-Hairpin (DDH) motif which is also the canonical motif for the ICK fold. The ICK fold itself offers many variations leading to differing toxin properties. Examination of pharmacological data gives insights on the possible conserved site of action of toxins acting on voltage-gated ion channels and other toxins acting by a pore-blocking mechanism. Structure-activity data shows the versatility of the toxin scaffolds and the importance of surface features in the selectivity and specificity of these toxins. Tarantulas appear to be a good model for the discovery of novel compounds with important therapeutic potential, and for the study of the molecular evolution of peptide toxins.     

Nanoparticle-based microextraction techniques in bioanalysis

Nanoparticles (NPs) have attracted a great deal of attention in the last decade due to their exceptional mechanical, optical and electronic properties. This article deals with the use of NPs as probes for the extraction of biomolecules from biological samples. In this context, NPs present some advantages compared with conventional sorbents. Their high surface-to-volume ratio, easy synthetic (especially for inorganic NPs) and derivatization procedures, and their biocompatibility make them a powerful alternative. In order to provide a systematic approach to the topic, NPs have been divided into two general groups attending to their chemical nature. Carbon-based (e.g., fullerene and nanotubes) and inorganic NPs (e.g., gold and magnetic NPs) are considered in depth, explaining their main properties and applications. After these critical considerations, the most important conclusions and essential trends in this field are also outlined.     

Effective on-line purification for cationic compounds in rat bile using a column-switching LC technique

An on-line purification method for cationic compounds and their metabolites in rat bile was investigated using a column-switching technique. 8-Hydroxyquinoline and its glucuronide were used as test compounds. Bile samples were injected directly into the system and successful on-line extraction with high purification efficiency for analytes was achieved using two-dimensional extraction LC; that is, reversed-phase chromatography followed by cation-exchange chromatography. After removal of the endogenous component by extraction LC, chromatographic separation of the target analyte was performed on an analytical ODS column, followed by identification using UV detection. The quantitative ability of the method was evaluated on the basis of injection repeatability, linearity and accuracy. This novel method was also applied to LC/MS analysis in order to characterise the pharmacokinetics of propranolol in rats, and the metabolites were successfully identified.     

Evaluation of solid-phase extraction of basic drugs from human milk

This article evaluates the use of commercially available cyanopropyl and octadecyl sorbents for the extraction of basic drugs from breast milk. Twenty drugs were selected from different pharmacological groups (beta-blocking agents, antidepressants, anxiolytic sedatives and neuroleptics, antihistamines, alkaloids and an anthelmintic) and subjected to a general solid-phase extraction (SPE) procedure described earlier for plasma samples. This SPE method was developed on a cyanopropyl cartridge and consisted of a conditioning step with methanol and water, the adsorption of the deproteinized matrix, washing with water and/or methanol, and finally the elution of the basic compounds with 0.1% propylamine in methanol. The extracts were further analysed by reversed-phase liquid chromatography (RP-LC). The application of SPE to human milk samples utilized cyanopropyl and octadecyl cartridges. The latter can be applied more generally because it better retains the basic compounds. For 14 out of 17 drugs extracted from breast milk, recoveries of greater than 70% were obtained. Standard deviations were, with the exception of three drugs, in the same range as those observed for plasma samples, i.e. 2-8%. The development of a strategy for SPE of drugs from human milk was difficult. For a number of drugs, in particular those present in human milk at low concentrations and/or detected in a non-selective way, matrix compounds interfered with the subsequent LC analysis. Therefore, SPE on CN or C18-sorbent for the analysis of basic compounds in breast milk was found to be useful as one of the steps in an extraction procedure, but not as a single technique. A major drawback of SPE is the batch-to-batch variation of the sorbents.     

A simple capture-release strategy based on an instantly formed mixed metal hydroxide sorbent for determination of salicylic acid in cosmetics

We present a simple eco-friendly extraction-release strategy for enhancing the assessment of salicylic acid (SA) in cosmetic samples. Given that the extraction and the formation of mixed metal hydroxides (MMHs) acting as sorbent occurred at the same time, the in situ solid phase extraction (is-SPE) of SA was achieved by mixing magnesium and aluminium ions in an alkaline sample solution. After the is-SPE, the MMHs precipitates were isolated by centrifugation and dissolved completely in sulfuric acid. Subsequently, the released SA was quantified by spectrophotometry at 237 nm. The is-SPE parameters including the amount of hydroxide ions, amount of metal ions, extraction time, and centrifugation time were studied thoroughly. Under the optimized conditions, this method provided the linear range of 0.15–2 mg L^?1 with the relative standard deviations of less than 7.20% and the limit of detection of 45 μg L^?1. In addition, the proposed method was successfully applied for SA analysis in toner, foam, and serum samples with reasonable relative recoveries in the range of 76.3–100.3%. This method eliminated the use of organic solvents and avoided the synthesis step of sorbents as well as their characterization, thus saving time, chemicals, and energy.     

Antibodies against anthrax: mechanisms of action and clinical applications

B. anthracis is a bioweapon of primary importance and its pathogenicity depends on its lethal and edema toxins, which belong to the A-B model of bacterial toxins, and on its capsule. These toxins are secreted early in the course of the anthrax disease and for this reason antibiotics must be administered early, in addition to other limitations. Antibodies (Abs) may however neutralize those toxins and target this capsule to improve anthrax treatment, and many Abs have been developed in that perspective. These Abs act at various steps of the cell intoxication and their mechanisms of action are detailed in the present review, presented in correlation with structural and functional data. The potential for clinical application is discussed for Abs targeting each step of entry, with four of these molecules already advancing to clinical trials. Paradoxically, certain Abs may also enhance the lethal toxin activity and this aspect will also be presented. The unique paradigm of Abs neutralizing anthrax toxins thus exemplifies how they may act to neutralize A-B toxins and, more generally, be active against infectious diseases.