Sources and profiles of toxins in shellfish from the south-central coast of Chile (36°â€’ 43° S).

The study of marine toxins in shellfish is of the utmost importance to ensure people's food safety. Marine toxins in shellfish and microalgae in the water column off the south-central coast of Chile (36°â€’43° S) were studied in a network of 64 stations over a 14-month period. The relative abundance of harmful species Alexandrium catenella, Alexandrium ostenfeldii, Protoceratium reticulatum, Dinophysis acuminata, Dinophysis acuta, Pseudo-nitzschia seriata group and P. delicatissima group was analyzed. The detection and quantification of lipophilic toxins and domoic acid (DA) in shellfish was determined by UHPLC-MS/MS, and for Paralytic Shellfish Toxins (PSTs) by HPLC-FD with post-column oxidation, while for a culture of A. ostenfeldii a Hylic-UHPLC-MS/MS was used. Results showed that DA, gonyautoxin (GTX)-2, GTX-3 and pectenotoxin (PTX)-2 were detected below the permitted limits, while Gymnodimine (GYM)-A and 13-desmethylespirolide C (SPX-1) were below the limit of quantitation. According to the distribution and abundance record of microalgae, DA would be associated to P. seriata and P. delicatissima-groups, PTX-2 to D. acuminata, and GTX-2, GTX-3, GYM-A, and SPX-1 to A. ostenfeldii. However, the toxin analysis of an A. ostenfeldii culture from the Biobío region only showed the presence of the paralytic toxins C2, GTX-2, GTX-3, GTX-5 and saxitoxin, therefore, the source of production of GYM and SPX is still undetermined.

Using solid phase adsorption toxin tracking and extended local similarity analysis to monitor lipophilic shellfish toxins in a mussel culture ranch in the Yangtze River Estuary.

Harmful algal blooms (HABs) and their associated phycotoxins are increasing globally, posing great threats to local coastal ecosystems and human health. Nutrients have been carried by the freshwater Yangtze River and have entered the estuary, which was reported to be a biodiversity-rich but HAB-frequent region. Here, in situ solid phase adsorption toxin tracking (SPATT) was used to monitor lipophilic shellfish toxins (LSTs) in seawaters, and extended local similarity analysis (eLSA) was conducted to trace the temporal and special regions of those LSTs in a one-year trail in a mussel culture ranch in the Yangtze River Estuary. Nine analogs of LSTs, including okadaic acid (OA), dinophysistoxin-1 (DTX1), yessotoxin (YTX), homoyessotoxin (homoYTX), 45-OH-homoYTX, pectenotoxin-2 (PTX2), 7-epi-PTX2 seco acid (7-epi-PTX2sa), gymnodimine (GYM) and azaspiracids-3 (AZA3), were detected in seawater (SPATT) or rope farmed mussels. The concentrations of OA + DTX1 and homoYTX in mussels were positively correlated with those in SPATT samplers (Pearson test, p < 0.05), indicating that SPATT (with resin HP20) would be a good monitoring tool and potential indicator for OA + DTX1 and homoYTX in mussel Mytilus coruscus. The eLSA results indicated that late summer and early autumn were the most phycotoxin-contaminated seasons in the Yangtze River Estuary. OA + DTX1, homoYTX, PTX2 and GYM were most likely driven by the local growing HAB species in spring and summer, while Yangtze River diluted water may impact the accumulation of HAB species, causing potential phycotoxin contamination in the Yangtze River Estuary in autumn and winter. Together, the results showed that the mussel harvesting season, late summer and early autumn, would be the season with the greatest phycotoxin risk and would be the most contaminated by local growing toxic algae. Routine monitoring sites should be set up close to the local seawaters.

Risk characteristics of shellfish toxins in Mytilus unguiculatus around the Zhoushan Islands, East China Sea.

The Zhoushan Islands, are an important area for Mytilus unguiculatus aquaculture, and are threatened by potentially harmful algal blooms. However, a full understanding of the risks posed by their toxin residues is still lacking. M. unguiculatus samples were collected from the area between 2020 and 2021 and analyzed for their toxin profiles to assess the contamination status of shellfish toxins. The main toxins detected were the paralytic shellfish toxins (PSTs), gymnodimine (GYM), and domoic acid (DA). Nine PSTs components were detected, the dominant ones being C1, C2, and GTX5, with an overall detection rate of 85.7 %. The detection rate of DA was 55.05 %, and GYM was detected in all samples. The toxin levels in the samples were significantly lower than the European Union regulatory limits, but toxin contamination was generally universal.

Stability and Chemical Conversion of the Purified Reference Material of Gymnodimine-A under Different Temperature and pH Conditions.

Gymnodimines (GYMs) are a group of fast-acting phycotoxins and their toxicological effects on human beings are still unclear due to the lack of sufficiently well-characterized large quantities of purified toxins for toxicology studies. In this study, a certified reference material (CRM) of GYM-A was prepared from the dinoflagellate Karenia selliformis, followed by multi-step chromatography separation and purification. Subsequently, the stability of GYM-A in methanolic media was evaluated at different temperature (-20, 4, and 20 °C) and pH (3, 5, and 7) conditions for 8 months, and the conversion products of GYM-A were explored by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The results show that the stability of GYM-A decreased with increasing temperature and pH values. The GYM-A was stable during storage at -20 °C regardless of pH, but it decreased rapidly (81.8% ± 9.3%) at 20 °C in pH 7 solution after 8 months. Moreover, the concentrations of GYM-A did not significantly change at all temperatures in solutions with pH 3 (p > 0.05). It is recommended that GYM-A should be stored at low temperature (≤-20 °C) and pH (≤3) conditions for long-term storage in aqueous methanolic media. In addition, two conversion products of GYM-A, tentatively named as GYM-K (m/z 540) and GYM-L (m/z 524), were identified in the samples stored at high levels of pH and temperature. Based on the LC-HRMS data, the hypothetical chemical structures of both converting derivatives were proposed. A useful strategy for long-term storage of GYM-A CRM in aqueous methanolic media was suggested and two hypothesized conversion products of GYM-A were discovered in this study.

Understanding the Liquid States of Cyclic Hydrocarbons Containing N, O, and S Atoms via the 3D-RISM-KH Molecular Solvation Theory.

The 3D-reference interaction site model (3D-RISM) molecular solvation theory in combination with the Kovalenko-Hirata (KH) closure is extended to seven heterocyclic liquids to understand their liquid states and to test the performance of the theory in solvation free energy (SFE) calculations of solutes in select solvents. The computed solvent site distribution profiles were compared with the all-atom molecular dynamics (MD) simulations, showing comparable performances. The computational results were compared against the structural parameters for liquids, whenever available, as well as against the experimental SFEs. The liquids are found to have local ordered structures held together via weak interactions in both the RISM and MD simulations. The 3D-RISM-KH computed SFEs are in good agreement with the benchmark values for the tetrahydrothiophene-S,S-dioxide, and showed comparatively larger deviations in the case of the SFEs in the tetrahydrofuran continuum.

Comparative study on the esterification of gymnodimine in different shellfish exposed to the dissolved toxin in seawater.

Some lipophilic phycotoxins dissolved in seawater can be accumulated by bivalves via the filtering process. To explore the relationship between the bioaccumulation of gymnodimine-A (GYM-A) and free fatty acids (FFAs) of shellfish, three species of bivalves (venus clam Meretrix meretrix, mussel Mytilus galloprovincialis, and ark shell Anadara kagoshimensis) were exposed to dissolved GYM-A for 7 days in the same seawater system. Results indicated that GYM-A can be accumulated by these bivalves from the dissolved phase and esterified with FFAs reaching over 90% in most tissues of bivalves. Gymnodimine-A and its esters mainly distributed in the gills of shellfish, and the highest concentration of toxins occurred in mussel, followed by ark shell and venus clam. Similar percent of different fatty acid esters occurred in the experimental shellfish, in which the C16:0, C17:0, C18:0, C18:1, C20:1, C20:2, C22:2, and C22:6-GYM-A esters were the main metabolites of GYM-A. The binding capacity of fatty acids and GYM-A varied in different FFAs, which can explain why the C20:1-GYM-A ester dominated the ester profile while C16:0 was the most abundant fatty acid in all samples. Comparing with the FFA profile of shellfish in the control groups, overexpression of some FFAs occurred in the tissues of shellfish exposed to GYM-A in the experimental groups, which suggested that biosynthesis of FFAs was affected by the accumulation and metabolism of GYM-A in bivalves. Multiple fatty acids including some valuably nutritional FFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were consumed in the esterification metabolism of GYM-A, which hinted that the lipid metabolism and nutritional quality of shellfish affected by the contamination of GYMs should be explored and assessed in future works.

Selection, Characterization, and Optimization of DNA Aptamers against Challenging Marine Biotoxin Gymnodimine-A for Biosensing Application.

Gymnodimines (GYMs), belonging to cyclic imines (CIs), are characterized as fast-acting toxins, and may pose potential risks to human health and the aquaculture industry through the contamination of sea food. The existing detection methods of GYMs have certain defects in practice, such as ethical problems or the requirement of complicated equipment. As novel molecular recognition elements, aptamers have been applied in many areas, including the detection of marine biotoxins. However, GYMs are liposoluble molecules with low molecular weight and limited numbers of chemical groups, which are considered as "challenging" targets for aptamers selection. In this study, Capture-SELEX was used as the main strategy in screening aptamers targeting gymnodimine-A (GYM-A), and an aptamer named G48nop, with the highest KD value of 95.30 nM, was successfully obtained by screening and optimization. G48nop showed high specificity towards GYM-A. Based on this, a novel aptasensor based on biolayer interferometry (BLI) technology was established in detecting GYM-A. This aptasensor showed a detection range from 55 to 1400 nM (linear range from 55 to 875 nM) and a limit of detection (LOD) of 6.21 nM. Spiking experiments in real samples indicated the recovery rate of this aptasensor, ranging from 96.65% to 109.67%. This is the first study to report an aptamer with high affinity and specificity for the challenging marine biotoxin GYM-A, and the new established aptasensor may be used as a reliable and efficient tool for the detection and monitoring of GYMs in the future.

Violations. How Nature Circumvents the Woodward-Hoffmann Rules and Promotes the Forbidden Conrotatory 4n + 2 Electron Electrocyclization of Prinzbach's Vinylogous Sesquifulvalene.

Woodward and Hoffmann, in their treatise on orbital symmetry in 1969, stated "Violations. There are none!" Prinzbach reported in 1978 that the electrocyclization of vinylogous sesquifulvalene occurs exclusively through the Woodward-Hoffmann orbital-symmetry-forbidden 14π-electron conrotatory pathway, despite the availability of a variety of orbital-symmetry-allowed processes. Prinzbach later demonstrated that an 18π-electron homologue exhibits the same forbidden behavior. And yet, the analogous vinylogous pentafulvalene and heptafulvalene both follow the orbital symmetry rules, each proceeding through its allowed conrotatory 12π and 16π process, respectively. We report the investigation of these reactions with ωB97X-D DFT. The physical origins of the flagrant Prinzbach violations of the Woodward-Hoffmann orbital symmetry selection rules have now been elucidated by these calculations in conjunction with extensive analyses and comparisons to electrocyclizations that obey the Woodward-Hoffmann rules. This remarkable reversal of the Rules (the 14π-electron-forbidden process is found to be 11 kcal/mol more energetically facile than the allowed process) occurs due to the high degree of polarization of this hydrocarbon, such that conrotatory electrocyclization of vinylogous sesquifulvalene behaves like a cyclopentadienide combining with a tropylium. These results are compared to other forbidden pericyclic processes driven by steric constraints and strain release or by diradical character of the reactants that facilitates the formation of diradical transition states for symmetry-forbidden reactions. We predict how strong donor-acceptor substitution can modify nodal properties to level the difference between allowed and forbidden electrocyclic reaction barriers, and we provide computational predictions of two such cases.

A General, Multimetallic Cross-Ullmann Biheteroaryl Synthesis from Heteroaryl Halides and Heteroaryl Triflates.

Despite their importance to medicine and materials science, the synthesis of biheteroaryls by cross-coupling remains challenging. We describe here a new, general approach to biheteroaryls: the Ni- and Pd-catalyzed multimetallic cross-Ullmann coupling of heteroaryl halides with triflates. An array of 5-membered, 6-membered, and fused heteroaryl bromides and chlorides, as well as aryl triflates derived from heterocyclic phenols, proved to be viable substrates in this reaction (62 examples, 63 ± 17% average yield). The generality of this approach to biheteroaryls was further demonstrated in 96-well plate format at 10 µmol scale. An array of 96 possible products provided >90% hit rate under a single set of conditions. Further, low-yielding combinations could be rapidly optimized with a single "Toolbox Plate" of ligands, additives, and reductants.

Development of an Efficient Extraction Method for Harvesting Gymnodimine-A from Large-Scale Cultures of Karenia selliformis.

Gymnodimine-A (GYM-A) is a fast-acting microalgal toxin and its production of certified materials requires an efficient harvesting technology from the large-scale cultures of toxigenic microalgae. In this study the recoveries of GYM-A were compared between several liquid-liquid extraction (LLE) treatments including solvents, ratios and stirring times to optimize the LLE technique for harvesting GYM-A from Karenia selliformis cultures, of which the dichloromethane was selected as the extractant and added to microalgal cultures at the ratio 55 mL L-1 (5.5%, v/v). The recovery of GYM-A obtained by the LLE technique was also compared with filtration and centrifugation methods. The stability of GYM-A in culture media were also tested under different pH conditions. Results showed that both the conventional filter filtration and centrifugation methods led to fragmentation of microalgal cells and loss of GYM-A in the harvesting processes. A total of 5.1 µg of GYM-A were obtained from 2 L of K. selliformis cultures with a satisfactory recovery of 88%. Interestingly, GYM-A obviously degraded in the culture media with the initial pH 8.2 and the adjusted pH of 7.0 after 7 days, but there was no obvious degradation in the acidic medium at pH 5.0. Therefore, the LLE method developed here permits the collection of large-volume cultures of K. selliformis and the high-efficiency extraction of GYM-A. This work provides a simple and valuable technique for harvesting toxins from large-scale cultures of GYM-producing microalgae.

MLatom 2: An Integrative Platform for Atomistic Machine Learning.

Atomistic machine learning (AML) simulations are used in chemistry at an ever-increasing pace. A large number of AML models has been developed, but their implementations are scattered among different packages, each with its own conventions for input and output. Thus, here we give an overview of our MLatom 2 software package, which provides an integrative platform for a wide variety of AML simulations by implementing from scratch and interfacing existing software for a range of state-of-the-art models. These include kernel method-based model types such as KREG (native implementation), sGDML, and GAP-SOAP as well as neural-network-based model types such as ANI, DeepPot-SE, and PhysNet. The theoretical foundations behind these methods are overviewed too. The modular structure of MLatom allows for easy extension to more AML model types. MLatom 2 also has many other capabilities useful for AML simulations, such as the support of custom descriptors, farthest-point and structure-based sampling, hyperparameter optimization, model evaluation, and automatic learning curve generation. It can also be used for such multi-step tasks as Δ-learning, self-correction approaches, and absorption spectrum simulation within the machine-learning nuclear-ensemble approach. Several of these MLatom 2 capabilities are showcased in application examples.

Smokeless tobacco analysis: Simultaneous extraction and purification of alkaloids, volatile N-nitrosamines, and polycyclic hydrocarbons for GC-MS/MS.

Several smokeless tobacco products are available in the market and comprise complex chemical matrices. Sample preparation for analysis of the multiple classes of harmful compounds in smokeless tobacco products is highly cumbersome. In this study, a simultaneous extraction scheme was developed for three toxic analyte classes in smokeless tobacco products using a two-phase solution consisting of 5% aqueous NaOH and dichloromethane in a 1:4 ratio. The dichloromethane extract was used to analyze four alkaloids directly at levels greater than parts per million; however, passing the layer through a silica cartridge for further purification and concentration was necessary for determining 18 polycyclic aromatic hydrocarbons and four volatile N-nitrosoamines at the ppt level. The multitargets were determined by using gas chromatography with tandem mass spectrometry. The limits of detection for the 18 polycyclic aromatic hydrocarbons, four volatile N-nitrosoamines, three minor alkaloids, and nicotine were 0.2-1.2, 0.2-0.4, 0.6-1.0, and 10.2 µg/g, respectively. Four different smokeless tobacco substrates were fortified with three levels of mixed standards, and the recoveries ranged between 83 and 110%. The method was highly efficient, reduced the sample amounts, solvents, and the time required by approximately 60%. The method was used to assay 18 smokeless tobacco products, and showed potentials in assaying drugs and other plant-based substrates.

A rapid method and mechanism to identify the active compounds in Malus micromalus Makino fruit with spectrum-effect relationship, components knock-out and molecular docking technology.

Fingerprints of 20 batches of Malus micromalus Makino fruit were established by HPLC coupled with hierarchical cluster analysis (HCA) and principal component analysis (PCA) to estimate the common peaks on the basis of traditional similarity evaluation methods. Chromatographic peaks were identified as p-coumaric acid (P2), ferulic acid glycoside (P6), 4-O-ß-Glucopyranosyl-cis-coumaric acid (P8), phloretin-2'-xyloglucoside (P10), phloridzin (P11) and quercetin-3-O-α-rhamnoside (P12) by UPLC-MS/MS method. The results of tyrosinase kinetics experiments showed that: P2 and the concentration of P11 was greater than 0.50 mmol/L mainly had a competitive inhibitory effect on tyrosinase, and the concentration of phlorizin was less than at 0.25 mmol/L, it has a mixed inhibitory effect. P8 was mainly a non-competitive activation type in the concentration range, while P12 was a mixed activation type. The results of tyrosinase molecular docking showed that: P2, P8, P11, P12 was located in the active center of the hydrophobic pocket of the enzyme. They bound to tyrosinase residues by hydrogen bonds and interacted with many hydrophobic residues around them to maintain the structure of the complex. This research provides a rapid method to determine the active compounds in edible plants with the technology of spectrum-effect relationship, component knock-out and molecular docking.

Lipophilic Toxins in Wild Bivalves from the Southern Gulf of California, Mexico.

Most of the shellfish fisheries of Mexico occur in the Gulf of California. In this region, known for its high primary productivity, blooms of diatoms and dinoflagellates are common, occurring mainly during upwelling events. Dinoflagellates that produce lipophilic toxins are present, where some outbreaks related to okadaic acid and dinophisystoxins have been recorded. From January 2015 to November 2017 samples of three species of wild bivalve mollusks were collected monthly in five sites in the southern region of Bahía de La Paz. Pooled tissue extracts were analyzed using LC-MS/MS to detect lipophilic toxins. Eighteen analogs of seven toxin groups, including cyclic imines were identified, fortunately individual toxins did not exceed regulatory levels and also the total toxin concentration for each bivalve species was lower than the maximum permitted level for human consumption. Interspecific differences in toxin number and concentration were observed in three species of bivalves even when the samples were collected at the same site. Okadaic acid was detected in low concentrations, while yessotoxins and gymnodimines had the highest concentrations in bivalve tissues. Although in low quantities, the presence of cyclic imines and other lipophilic toxins in bivalves from the southern Gulf of California was constant.

Tricyclic-Carbocyclic RORγt Inverse Agonists-Discovery of BMS-986313.

SAR efforts directed at identifying RORγt inverse agonists structurally different from our clinical compound 1 (BMS-986251) led to tricyclic-carbocyclic analogues represented by 3-7 and culminated in the identification of 3d (BMS-986313), with structural differences distinct from 1. The X-ray co-crystal structure of 3d with the ligand binding domain of RORγt revealed several key interactions, which are different from 1. The in vitro and in vivo PK profiles of 3d are described. In addition, we demonstrate robust efficacy of 3d in two preclinical models of psoriasis-the IMQ-induced skin lesion model and the IL-23-induced acanthosis model. The efficacy seen with 3d in these models is comparable to the results observed with 1.

Desorption Temperature, Solid-Phase Microextraction (SPME), and Natural Product Analyses, how Low Can we Go?

Solid phase microextraction (SPME) has become a common technique for volatile sampling due to its ease of use and limited technical requirements. The solvent-free nature of SPME is also exceptionally attractive for gas chromatography mass spectrometry (GC/MS) analysis. To ensure efficient transfer of the sample to the GC, the manufacturer recommend injector desorption temperatures in the range of 200 to 320 °C. A high desorption temperature can, however, have unwanted effects on analyses of plant and insect produced semiochemicals. By investigating the quantitative and qualitative chromatographic responses at varying inlet temperatures for a component blend of seven plant produced volatile compounds, we found the thermally labile plant-nematode signaling compound, pregeijerene to degrade to geijerene at all tested temperatures within the recommended range (200, 240, and 280 °C), but that it did not break down with an inlet temperature below 200 °C (100 °C and 150 °C). Degradation was also detected for the sesquiterpene germacrene D, but only at the highest inlet temperature tested (280 °C). Surprisingly, an inlet temperature of 200 °C gave the highest sample recovery, measured as total peak area while an inlet temperature of 100 °C as well as 280 °C gave the lowest total area values. An increase in desorption time from 3 to 5 min. Resulted in a recovery at 100 °C close to that obtained at 200 °C. Peak broadening was minimal, and only observed at the 100 °C inlet temperature. Based on these results, we highly recommend that SPME users include desorption temperature as one variable when developing sampling procedures for novel biological systems to ensure that potentially present thermally labile compounds are not degraded.

Levels of PAHs, PCBs, and toxic metals in Ruditapes philippinarum and Donax trunculus in Marmara Sea, Turkey.

BACKGROUND: The manila clam Ruditapes philippinarum and the wedge clam Donax trunculus are economic bivalve species which constitute an important part of the natural bivalve beds in the Marmara Sea, Turkey. Toxic chemicals such as, dioxins, dioxin-like polychlorinated biphenyls (PCBs), polycyclic hydrocarbons (PAHs), and toxic metals are recognized as important health risk factors that threaten public health via food or directly in the environment. In the present study, we aimed to determine and monitor levels of these toxic chemicals in both clam species, R. philippinarum and D. trunculus, between 2013 and 2017. RESULTS: According to the results, maximum levels of dioxins, PAHs, and toxic metals (lead, cadmium, mercury) during the period were determined as 0.18 pg g-1 , 2.43 mg kg-1 , and 0.44-0.53-0.1 mg kg-1 , respectively. The level of contaminants in both clam species were determined to be below the threshold or tolerable daily intake values established by the European Commission, European Food Safety Authority (EFSA), and Food and Agriculture Organization/World Health Organization (FAO/WHO). Seasonal fluctuations were observed to be similar in both species and also in seasons, but elevated levels of PCBs and PAHs were detected in warmer months during the period. CONCLUSION: As a consequence, toxic chemicals in both clam species were found at low concentrations in this study from the point of view of public health concerns; however, the contaminants should be closely monitored in the future due to their elevated levels in samples. © 2020 Society of Chemical Industry.

Click and Bio-Orthogonal Reactions with Mesoionic Compounds.

Click and bio-orthogonal reactions are dominated by cycloaddition reactions in general and 1,3-dipolar cycloadditions in particular. Among the dipoles routinely used for click chemistry, azides, nitrones, isonitriles, and nitrile oxides are the most popular. This review is focused on the emerging click chemistry that uses mesoionic compounds as dipole partners. Mesoionics are a very old family of molecules, but their use as reactants for click and bio-orthogonal chemistry is quite recent. The facility to derivatize these dipoles and to tune their reactivity toward cycloaddition reactions makes mesoionics an attractive opportunity for future click chemistry development. In addition, some compounds from this family are able to undergo click-and-release reactions, finding interesting applications in cells, as well as in animals. This review covers the synthetic access to main mesoionics, their reaction with dipolarophiles, and recent applications in chemical biology and heterocycle synthesis.