Influence of biochar on the removal of Microcystin-LR and Saxitoxin from aqueous solutions.

The present study assessed the effective use of biochar for the adsorption of two potent HAB toxins namely, Microcystin-LR (MCLR) and Saxitoxin (STX) through a combination of dosage, kinetic, equilibrium, initial pH, and competitive adsorption experiments. The adsorption results suggest that biochar has excellent capabilities for removing MCLR and STX, with STX reporting higher adsorption capacities (622.53-3507.46 µg/g). STX removal required a minimal dosage of 0.02 g/L, while MCLR removal needed 0.4 g/L for > 90%. Similarly, a shorter contact time was required for STX removal compared to MCLR for > 90% of toxin removed from water. Initial pH study revealed that for MCLR acidic conditions favored higher uptake while STX favored basic conditions. Kinetic studies revealed that the Elovich model to be most suitable for both toxins, while STX also showed suitable fittings for Pseudo-First Order and Pseudo-Second Order in individual toxin systems. Similarly, for the Elovich model the most suited kinetic model for both toxins in presence of each other. Isotherm studies confirmed the Langmuir-Freundlich model as the best fit for both toxins. These results suggest adsorption mechanisms including pore filling, hydrogen bonding, π-π interactions, hydrophobic interactions, electrostatic attraction, and dispersive interactions.

Synthesis and Identification of decarbamoyloxySaxitoxins in Toxic Microalgae and their Reactions with the Oxygenase, SxtT, Reveal Saxitoxin Biosynthesis.

Saxitoxin (STX, 1) is a representative compound of paralytic shellfish toxins (PSTs) that are produced by marine dinoflagellates and freshwater cyanobacteria. Although several pathways have been proposed for the biosynthesis of STX, the order of ring and side chain hydroxylation, and formation of the tricyclic skeleton have not been well established. In this study, 12,12-dideoxy-decarbamoyloxySTX (dd-doSTX, 2), the most reduced STX analogue having the tricyclic skeleton, and its analogues, 12ß-deoxy-doSTX (12ß-d-doSTX, 3), 12α-deoxy-doSTX (12α-d-doSTX, 4), and doSTX (5), were synthesized, and these compounds were screened in the toxic microalgae using high-resolution LCMSMS. dd-doSTX (2) and 12ß-d-doSTX (3) were identified in the PSTs-producing dinoflagellates (Alexandrium catenella, A. pacificum, and/or Gymnodinium catenatum) and in the cyanobacterium Dolichospermum circinale (TA04). doSTX (5), previously isolated from the dinoflagellate G. catenatum, was also identified in D. circinale (TA04). Furthermore, the conversion of 2 to 3, and 4 to 5, by SxtT with VanB, a reported Rieske oxygenase and its redox partner in STX biosynthesis, was confirmed. These results support that 2 is a possible biosynthetic precursor of STX, and that ring and side-chain hydroxylations proceed after cyclization.

Aptamers-functionalized nanoscale MOFs for saxitoxin and tetrodotoxin sensing in sea foods through FRET.

Saxitoxin (STX) and tetrodotoxin (TTX) are widely distributed and extremely harmful marine toxins, it is certainly worth to spend effort to develop facile methods to detect them in sea food for human safety. In this work, two nano-sensors were developed by combining with two zirconium fluorescence Nanoscale metal-organic frameworks (NMOFs) with two emissions and TAMRA-labelled aptamers for STX and TTX sensing, respectively. The recognition of STX and TTX by these nano-sensors could change the structure of aptamer, which caused the blue or green emissions from NMOFs (energy donor) decreased while red emission from TAMRA-labelled aptamers (energy acceptor) increased owing to fluorescence resonance energy transfer (FRET) effect. Based on this strategy, NMOFs-Aptasensor 1 and NMOFs-Aptasensor 2 were developed for the ratiometric detection, with detection limits of 1.17 nM and 3.07 nM for STX and TTX, respectively. Moreover, NMOFs-Aptasensors displayed significant stability, pH-independence, selectivity and NMOFs-Aptasensors were successfully applied in shellfish sample for toxin sensing.

First Identification of 12ß-Deoxygonyautoxin 5 (12α-Gonyautoxinol 5) in the Cyanobacterium Dolichospermum circinale (TA04) and 12ß-Deoxysaxitoxin (12α-Saxitoxinol) in D. circinale (TA04) and the Dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC).

Saxitoxin and its analogues, paralytic shellfish toxins (PSTs), are potent and specific voltage-gated sodium channel blockers. These toxins are produced by some species of freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates of PSTs, as well as new analogues, from such organisms and proposed the biosynthetic and metabolic pathways of PSTs. In this study, 12ß-deoxygonyautoxin 5 (12α-gonyautoxinol 5 = gonyautoxin 5-12(R)-ol) was identified in the freshwater cyanobacterium, Dolichospermum circinale (TA04), and 12ß-deoxysaxitoxin (12α-saxitoxinol = saxitoxin-12(R)-ol) was identified in the same cyanobacterium and in the marine dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC) for the first time from natural sources. The authentic standards of these compounds and 12α-deoxygonyautoxin 5 (12ß-gonyautoxinol 5 = gonyautoxin 5-12(S)-ol) were prepared by chemical derivatization from the major PSTs, C1/C2, produced in D. circinale (TA04). These standards were used to identify the deoxy analogues by comparing the retention times and MS/MS spectra using high-resolution LC-MS/MS. Biosynthetic or metabolic pathways for these analogues have also been proposed based on their structures. The identification of these compounds supports the α-oriented stereoselective oxidation at C12 in the biosynthetic pathway towards PSTs.

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.

Nuclease-assisted target recycling signal amplification strategy for graphene quantum dot-based fluorescent detection of marine biotoxins.

Saxitoxin (STX) is a major marine toxin from shellfish, and it is responsible for paralytic shellfish poisoning (PSP). In this study, a highly sensitive and rapid aptamer assay was developed for STX detection by combining fluorescence resonance energy transfer (FRET) and nuclease-assisted target recycling signal amplification. The aptamer STX-41 conjugated with graphene quantum dots (GQDs) was adsorbed on magnetic reduced graphene oxide (MRGO) to establish a fluorescence quenching system. Then, the binding between STX and aptamer induced the desorption of GQD-aptamer from MRGO and the restoring of fluorescence for the fluorescent determination of STX. The digestion of the target bound aptamer by DNase I could release the target for recycling thus achieving signal amplification. Under the optimized conditions, the aptamer assay showed a wide detection range (0.1-100 ng·mL-1), low detection limit (LOD of 0.035 ng·mL-1), high specificity, good recovery (86.75-94.08% in STX-spiked clam samples) and repeatability (RSD of 4.27-7.34%). Combined with fluorescent detection technology, signal amplification technology, and magnetic separation technology, the proposed method can be used to detect STX in seafood products successfully.

Interplay of Nutrients, Temperature, and Competition of Native and Alien Cyanobacteria Species Growth and Cyanotoxin Production in Temperate Lakes.

Global warming and eutrophication contribute to formation of HABs and distribution of alien cyanobacteria northward. The current study assessed how alien to Europe Sphaerospermopsis aphanizomenoides and Chrysosporum bergii will co-occur with dominant native Planktothrix agardhii and Aphanizomenon gracile species under changing conditions in temperate freshwaters. The experiments were carried out to examine the effect of nutrients and temperature on the growth rate of cyanobacteria, production of cyanotoxins, and interspecies competition. The highest growth rate was determined for A. gracile (0.43 day-1) and S. aphanizomenoides (0.40 day-1) strains at all the tested nutrient concentrations (IP and IN were significant factors). S. aphanizomenoides adapted to the wide range of nutrient concentrations and temperature due to high species ecological plasticity; however, A. gracile was able to suppress its dominance under changing conditions. Regularity between tested variables and STX concentration in A. gracile was not found, but IP concentration negatively correlated with the amount of dmMC-RR and other non-ribosomal peptides (NRPs) in P. agardhii strains. The relative concentration of NRPs in nontoxic P. agardhii strain was up to 3-fold higher than in MC-producing strain. Our study indicated that nutrients, temperature, and species had significant effects on interspecies competition. A. gracile had a negative effect on biomass of both alien species and P. agardhii.

The chemistry and biology of guanidine secondary metabolites.

Covering: 2017-2019Guanidine natural products isolated from microorganisms, marine invertebrates and terrestrial plants, amphibians and spiders, represented by non-ribosomal peptides, guanidine-bearing polyketides, alkaloids, terpenoids and shikimic acid derived, are the subject of this review. The topics include the discovery of new metabolites, total synthesis of natural guanidine compounds, biological activity and mechanism-of-action, biosynthesis and ecological functions.

Biosynthesis of marine toxins.

Throughout history, humans have encountered natural toxic chemicals from the ocean environment, often through contaminated seafood. Although marine toxins can be harmful to human health and devastate local environments when they are produced during algal bloom events, they are also important biochemical research reagents and drug leads in medicine. In spite of their long history, the biosynthetic origin of many well-known marine toxins has remained elusive. New biosynthetic insights have shed light on the chemical transformations that create the complex structures of several iconic oceanic toxins. To that end, this review highlights advances made in the biosynthetic understanding of five important environmental toxins of marine origin: domoic acid, kainic acid, saxitoxin, tetrodotoxin, and polyether polyketides such as brevetoxin.

Biosynthesis of Saxitoxin in Marine Dinoflagellates: An Omics Perspective.

Saxitoxin is an alkaloid neurotoxin originally isolated from the clam Saxidomus giganteus in 1957. This group of neurotoxins is produced by several species of freshwater cyanobacteria and marine dinoflagellates. The saxitoxin biosynthesis pathway was described for the first time in the 1980s and, since then, it was studied in more than seven cyanobacterial genera, comprising 26 genes that form a cluster ranging from 25.7 kb to 35 kb in sequence length. Due to the complexity of the genomic landscape, saxitoxin biosynthesis in dinoflagellates remains unknown. In order to reveal and understand the dynamics of the activity in such impressive unicellular organisms with a complex genome, a strategy that can carefully engage them in a systems view is necessary. Advances in omics technology (the collective tools of biological sciences) facilitated high-throughput studies of the genome, transcriptome, proteome, and metabolome of dinoflagellates. The omics approach was utilized to address saxitoxin-producing dinoflagellates in response to environmental stresses to improve understanding of dinoflagellates gene-environment interactions. Therefore, in this review, the progress in understanding dinoflagellate saxitoxin biosynthesis using an omics approach is emphasized. Further potential applications of metabolomics and genomics to unravel novel insights into saxitoxin biosynthesis in dinoflagellates are also reviewed.

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.

Synthesis of C12-Keto Saxitoxin Derivatives with Unusual Inhibitory Activity Against Voltage-Gated Sodium Channels.

A novel series of C12-keto-type saxitoxin (STX) derivatives bearing an unusual nonhydrated form of the ketone at C12 has been synthesized, and their NaV -inhibitory activity has been evaluated in a cell-based assay as well as whole-cell patch-clamp recording. Among these compounds, 11-benzylidene STX (3 a) showed potent inhibitory activity against neuroblastoma Neuro 2A in both cell-based and electrophysiological analyses, with EC50 and IC50 values of 8.5 and 30.7 nm, respectively. Interestingly, the compound showed potent inhibitory activity against tetrodotoxin-resistant subtype of NaV 1.5, with an IC50 value of 94.1 nm. Derivatives 3 a-d and 3 f showed low recovery rates from NaV 1.2 subtype (ca 45-79 %) compared to natural dcSTX (2), strongly suggesting an irreversible mode of interaction. We propose an interaction model for the C12-keto derivatives with NaV in which the enone moiety in the STX derivatives 3 works as Michael acceptor for the carboxylate of Asp1717 .

Structural characterization of the saxitoxin-targeting APTSTX1 aptamer using optical tweezers and molecular dynamics simulations.

Optical tweezers have enabled the exploration of picoNewton forces and dynamics in single-molecule systems such as DNA and molecular motors. In this work, we used optical tweezers to study the folding/unfolding dynamics of the APTSTX1-aptamer, a single-stranded DNA molecule with high affinity for saxitoxin (STX), a lethal neurotoxin. By measuring the transition force during (un)folding processes, we were able to characterize and distinguish the conformational changes of this aptamer in the presence of magnesium ions and toxin. This work was supported by molecular dynamics (MD) simulations to propose an unfolding mechanism of the aptamer-Mg+2 complex. Our results are a step towards the development of new aptamer-based STX sensors that are potentially cheaper and more sensitive than current alternatives.

Identification of a Novel Saxitoxin Analogue, 12ß-Deoxygonyautoxin 3, in the Cyanobacterium, Anabaena circinalis (TA04).

Saxitoxin (STX) and its analogues, the potent voltage-gated sodium channel blockers, are biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates in the extract of the cyanobacterium, Anabaena circinalis (TA04), that are primarily produced during the early and middle stages in the biosynthetic pathway to produce STX. These findings allowed us to propose a putative biosynthetic pathway responsible for STX production based on the structures of these intermediates. In the present study, we identified 12ß-deoxygonyautoxin 3 (12ß-deoxyGTX3), a novel STX analogue produced by A. circinalis (TA04), by comparing the retention time and MS/MS fragmentation pattern with those of synthetic standards using LC-MS. The presence of this compound in A. circinalis (TA04) is consistent with stereoselective enzymatic oxidations at C11 and C12, and 11-O-sulfation, during the late stage of STX biosynthesis, as proposed in previous studies.

A Direct C11 Alkylation Strategy on the Saxitoxin Core: A Synthesis of (+)-11-Saxitoxinethanoic Acid.

The bis-guanidinium ion family of natural products are revered for their utility in the study of ion channel physiology. While many congeners have been isolated with various oxidation and sulfation patterns, only two members of this family have been isolated bearing a carbon-carbon bond at C11, namely 11-saxitoxinethanoic acid and zetekitoxin AB. Herein we described a synthetic platform capable of efficiently targeting (+)-saxitoxin and 11-saxitoxinethanoic acid with an embedded C11 carbon-carbon bond. We demonstrate that this strategy enables direct enolate coupling in both an inter- and intramolecular fashion to create the C11-C15 carbon-carbon bond.

Prevalence of phycotoxin contamination in shellfish from the Northern Bering Sea and the Chukchi Sea.

To understand phycotoxin contamination in shellfish in the sub-Arctic and Arctic areas, scanning for the presence of 13 hydrophilic and lipophilic toxin components each was by liquid chromatography tandem quadrupole mass spectrometry analysis in shellfish samples collected from the Northern Bering Sea and the Chukchi Sea in 2014. The results showed that shellfish collected in both areas werecontaminated to different extents. Saxitoxin (STX), decarbamoylsaxitoxin (dcSTX) and decarbamoylneosaxitoxin (dcNEO) were the most frequently detected hydrophilic components, with maximum concentrations of 90.1 µg/kg, 112.25 µg/kg and 23.09 µg/kg, respectively. Although gonyautoxins (GTXs) were only detected in 3 samples, they were the main contributors to overall toxicity of high-latitude samples, especially GTX1. For lipophilic toxins, spirolide-1 (SPX1) and yessotoxin (YTX) were present in all samples at low levels (< 7 µg/kg and < 50 µg/kg, respectively). Only 5 samples showed evidence of okadaic acid (OA) and dinophysistoxin-2 (DTX-2) at low concentrations, ranging from 0.42 µg/kg to 7.23 µg/kg and 3.03 µg/kg to 30.59 µg/kg, respectively. Notably, a high level of pectenotoxin-1 (PTX-1) at 467.40 µg/kg was found in the shellfish collected at the northernmost station, exceeding the safety regulation standard by nearly 3 times. For both lipophilic and hydrophilic toxins, contamination in shellfish in the sub-Arctic and the Arctic area may be much more widespread and severe than was previously thought. This study highlighted the need to monitor toxins in a wider variety of shellfish, especially economic or commercial species, and across a wider range of sub-Arctic and Arctic waters, as well as the potential sources of these toxins.

Structures of human Nav1.7 channel in complex with auxiliary subunits and animal toxins.

Voltage-gated sodium channel Nav1.7 represents a promising target for pain relief. Here we report the cryo-electron microscopy structures of the human Nav1.7-ß1-ß2 complex bound to two combinations of pore blockers and gating modifier toxins (GMTs), tetrodotoxin with protoxin-II and saxitoxin with huwentoxin-IV, both determined at overall resolutions of 3.2 angstroms. The two structures are nearly identical except for minor shifts of voltage-sensing domain II (VSDII), whose S3-S4 linker accommodates the two GMTs in a similar manner. One additional protoxin-II sits on top of the S3-S4 linker in VSDIV The structures may represent an inactivated state with all four VSDs "up" and the intracellular gate closed. The structures illuminate the path toward mechanistic understanding of the function and disease of Nav1.7 and establish the foundation for structure-aided development of analgesics.

A signal-on magnetic electrochemical immunosensor for ultra-sensitive detection of saxitoxin using palladium-doped graphitic carbon nitride-based non-competitive strategy.

Saxitoxin (STX) has high toxicity, and is water soluble, acid stable and thermostable. Therefore, STX in seawater can be accumulated by marine organism to form bioaccumulation. To ensure the safety of seafood for consumption, it is crucial to accurately determine trace STX in seawater and seafood. We herein developed a novel magnetic electrochemical immunosensor for ultra-sensitive detection of STX in seawater and seafood by using non-competitive strategy. The immunosensor employs STX-specific antibody-functionalized magnetic beads (MBs) for STX recognition, palladium-doped graphitic carbon nitride (g-C3N4-PdNPs) peroxidase mimetic for catalyzing H2O2-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate signal. The immunosensor combines the merits of g-C3N4-PdNPs peroxidase mimetic, non-competitive strategy, MBs-based antibody recognition and magnetic gold electrode, and thus has excellent stability, lower cost, no risk of false positive result, high sensitivity and strong ability resist to matrix interference. The proposed immunosensor has been successfully used to detect trace STX in seawater and shellfish samples with a detection limit of 1.2 pg/mL (4.0 × 10-12 M), a recovery of 93-107% and a relative standard deviation (RSD, n = 5) < 5%. The success of this study provided a promising approach for the rapid and on-site detection of trace STX in seawater and seafood.

Divergent Synthesis of Natural Derivatives of (+)-Saxitoxin Including 11-Saxitoxinethanoic Acid.

The bis-guanidinium toxins are a collection of natural products that display nanomolar potency against select isoforms of eukaryotic voltage-gated Na+ ion channels. We describe a synthetic strategy that enables access to four of these poisons, namely 11-saxitoxinethanoic acid, C13-acetoxy saxitoxin, decarbamoyl saxitoxin, and saxitoxin. Highlights of this work include an unusual Mislow-Evans rearrangement and a late-stage Stille ketene acetal coupling. The IC50 value of 11-saxitoxinethanoic acid was measured against rat NaV 1.4, and found to be 17.0 nm, similar to those of the sulfated toxins gonyautoxin II and III.

Synthetic Approaches to Zetekitoxin AB, a Potent Voltage-Gated Sodium Channel Inhibitor.

Voltage-gated sodium channels (NaVs) are membrane proteins that are involved in the generation and propagation of action potentials in neurons. Recently, the structure of a complex made of a tetrodotoxin-sensitive (TTX-s) NaV subtype with saxitoxin (STX), a shellfish toxin, was determined. STX potently inhibits TTX-s NaV, and is used as a biological tool to investigate the function of NaVs. More than 50 analogs of STX have been isolated from nature. Among them, zetekitoxin AB (ZTX) has a distinctive chemical structure, and is the most potent inhibitor of NaVs, including tetrodotoxin-resistant (TTX-r) NaV. Despite intensive synthetic studies, total synthesis of ZTX has not yet been achieved. Here, we review recent efforts directed toward the total synthesis of ZTX, including syntheses of 11-saxitoxinethanoic acid (SEA), which is considered a useful synthetic model for ZTX, since it contains a key carbon-carbon bond at the C11 position.