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The receptor-binding assay (RBA) method for the detection of paralytic shellfish poisoning (PSP) toxins was evaluated for its overall performance in comparison with the mouse bioassay (MBA). An initial study to evaluate the effects of filtering shellfish extracts prior to running the RBA indicated no significant difference between filtered and unfiltered extracts on the determined saxitoxin (STX) concentrations. Next, we tested the RBA assay on 295 naturally contaminated mussel tissue samples, ranging in concentrations from 320 µg STX equiv. kg-1 to 13,000 µg STX equiv. kg-1 by MBA. An overall trend was observed with the RBA giving higher results (256 µg STX equiv. kg-1 on average) than the MBA; however, at low concentrations (< 500 µg STX equiv. kg-1) the RBA results were marginally lower. A third study was conducted using spiked mussel tissue analysed by three independent laboratories, two of which performed the RBA and one the MBA. This multi-laboratory study again showed the RBA to give higher results than the MBA; however, it also revealed that STX determination was accurate by the RBA, unlike the MBA. To optimise the assay for efficient usage under regulatory practice, three suggestions have been made: the use of an initial screening plate to separate those samples that exceed the alert level; use of rapid PSP test kits in the field and in the laboratory for screening negative samples and for early detection of toxicity; and use of an alternate commercially available porcine membrane in place of the laboratory-prepared rat membrane homogenate. The large number of samples analysed and the diversity of the tests conducted in this study further support the RBA as an affordable rapid method for STX detection that is also free of the routine sacrifice of live animals.

To study the potential relationship between melatonin and beta‐amyloid (Abeta), we established a liquid chromatography–mass spectrometry (LC‐MS) method to quantitatively analyze melatonin, deuterated isotopes (melatonin‐D4), and internal standard 6‐iodo‐2‐(4'‐dimethylamino‐) phenyl‐imidazo(1,2) pyridine (IMPY) under positive (+) mode. The gradient elution was set to 6 min, and the corresponding peak time of melatonin and its isotope melatonin‐D4 was 3.14 min, while the peak time for the internal standard IMPY was 3.24 min. Next, we established and optimized the molecule receptor saturation binding assay based on LC‐MS to determine the melatonin affinity for beta‐amyloid (Abeta). Melatonin showed a high and specific binding for Abeta. The corresponding equilibrium dissociation constant (Kd) of melatonin with Abeta 1‐40 and Abeta 1‐42 was 814.37 ± 36.62 and 628.33 ± 13.57 nmol·L−1; besides, the Kd of melatonin with mixed plaques (1‐40 and 1‐42) was 461.13 ± 45.37 nmol·L−1. The results may suggest the potential mechanism of action of MT on Abeta and provide a theoretical basis for the improvement of MT treatment of Alzheimer's disease.

A liquid chromatography-tandem mass spectrometry method is presented for the quantitative determination of the in vivo deamidation of the biopharmaceutical proteins trastuzumab and pertuzumab at an asparagine in their complementarity determining regions (CDRs). For each analyte, two surrogate peptides are quantified after tryptic digestion of the entire plasma protein content: one from a stable part of the molecule, representing the total concentration, and one containing the deamidation-sensitive asparagine, corresponding to the remaining non-deamidated concentration. Using a plasma volume of 10 µL and a 2-h digestion at pH 7, concentrations between 2 and 1000 µg/mL can be determined for the various protein forms with values for bias and CV below 15% and without unacceptable in vitro deamidation taking place. A considerable difference between the total and non-deamidated concentrations, and thus a substantial degree of deamidation, was observed in plasma for both trastuzumab and pertuzumab. After a 56-day forced deamidation test 40% of trastuzumab and 68% of pertuzumab was deamidated, while trastuzumab and pertuzumab showed up to 47% and 35% of deamidation, respectively, in samples collected from breast cancer patients during treatment with a combination of both drugs. A good correlation between the non-deamidated concentration results and those of a receptor binding assay indicate a loss of receptor binding for both trastuzumab and pertuzumab along with the deamidation in their CDRs. Deamidated trastuzumab also lost its capability to inhibit the growth of breast cancer cells in a cell-based viability assay, suggesting a relation between the degree of deamidation and pharmacological activity. [ABSTRACT FROM AUTHOR]

Native interactions between lysophospholipids (LPs) and their cognate LP receptors are difficult to measure because of lipophilicity and/or the adhesive properties of lipids, which contribute to high levels of nonspecific binding in cell membrane preparations. Here, we report development of a free-solution assay (FSA) where label-free LPs bind to their cognate G protein-coupled receptors (GPCRs), combined with a recently reported compensated interferometric reader (CIR) to quantify native binding interactions between receptors and ligands. As a test case, the binding parameters between lysophosphatidic acid (LPA) receptor 1 (LPA1; one of six cognate LPA GPCRs) and LPA were determined. FSA-CIR detected specific binding through the simultaneous real-time comparison of bound versus unbound species by measuring the change in the solution dipole moment produced by binding-induced conformational and/or hydration changes. FSA-CIR identified KD values for chemically distinct LPA species binding to human LPA1 and required only a few nanograms of protein: 1-oleoyl (18:1; KD = 2.08 ± 1.32 nM), 1-linoleoyl (18:2; KD = 2.83 ± 1.64 nM), 1-arachidonoyl (20:4; KD = 2.59 ± 0.481 nM), and 1-palmitoyl (16:0; KD = 1.69 ± 0.1 nM) LPA. These KD values compared favorably to those obtained using the previous generation back-scattering interferometry system, a chip-based technique with low-throughput and temperature sensitivity. In conclusion, FSA-CIR offers a new increased-throughput approach to assess quantitatively label-free lipid ligand-receptor binding, including nonactivating antagonist binding, under near-native conditions.

To study the potential relationship between melatonin and beta‐amyloid (Abeta), we established a liquid chromatography–mass spectrometry (LC‐MS) method to quantitatively analyze melatonin, deuterated isotopes (melatonin‐D4), and internal standard 6‐iodo‐2‐(4'‐dimethylamino‐) phenyl‐imidazo(1,2) pyridine (IMPY) under positive (+) mode. The gradient elution was set to 6 min, and the corresponding peak time of melatonin and its isotope melatonin‐D4 was 3.14 min, while the peak time for the internal standard IMPY was 3.24 min. Next, we established and optimized the molecule receptor saturation binding assay based on LC‐MS to determine the melatonin affinity for beta‐amyloid (Abeta). Melatonin showed a high and specific binding for Abeta. The corresponding equilibrium dissociation constant (Kd) of melatonin with Abeta 1‐40 and Abeta 1‐42 was 814.37 ± 36.62 and 628.33 ± 13.57 nmol·L−1; besides, the Kd of melatonin with mixed plaques (1‐40 and 1‐42) was 461.13 ± 45.37 nmol·L−1. The results may suggest the potential mechanism of action of MT on Abeta and provide a theoretical basis for the improvement of MT treatment of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

In order to investigate the effects of metallic nanoparticles (NPs) on the performance of in vitro bioassay, zinc oxide NP (ZnO NP), aluminum oxide NP (Al2O3 NP), bare silver NP (Ag NP), and Ag NP capped with citrate (Agcit NP) were evaluated with yeast (Saccharomyces cerevisiae Y190) two-hybrid system (YES assay), carrying Japanese medaka estrogen receptors (mERs) in the presence of 17β-estradiol (E2, 10−6 M), a reference chemical for estrogenic activity. The distribution of NPs in the yeast was also examined by field-emission transmission electron microscopy (FE-TEM). The results show that TEM analysis revealed that NPs were present inside the yeast and accumulated deep inside the cell organelles, suggesting that cell death was caused by NPs. However, despite no significant change of mortality, the E2 estrogenic activities in yeast exposed to ZnO NP and Al2O3 NP were dose-dependently reduced. For Ag NP and Agcit NP, such phenomenon observed in the exposure of ZnO NP and Al2O3 NP did not occur. From the observations, we found that ZnO NP and Al2O3 NP in the environmental media could result in underestimated estrogenicity of endocrine-disrupting compounds when evaluated by YES assay. [ABSTRACT FROM AUTHOR]

• First report of six Gambierdiscus and one Fukuyoa species in Cuba at low to moderate abundances. • First isolation and culture of low toxicity strains of G. caribaeus and G. carolinianus from Cuba. • Dictyota /Canistrocarpus macroalgal complex identified as target substrate or location to deploy artificial substrate. Ciguatera poisoning is caused by the consumption of reef fish or shellfish that have accumulated ciguatoxins, neurotoxins produced by benthic dinoflagellates of the genera Gambierdiscus or Fukuyoa. Although ciguatera constitutes the primary cause of seafood intoxication in Cuba, very little information is available on the occurrence of ciguatoxins in the marine food web and the causative benthic dinoflagellate species. This study conducted on the south-central coast of Cuba reports the occurrence of Gambierdiscus and Fukuyoa genera and the associated benthic genera Ostreopsis and Prorocentrum. Gambierdiscus / Fukuyoa cells were present at low to moderate abundances depending on the site and month of sampling. This genus was notably higher on Dictyota ceae than on other macrophytes. PCR analysis of field-collected samples revealed the presence of six different Gambierdiscus and one Fukuyoa species, including G. caribaeus , G. carolinianus , G. carpenteri , G. belizeanus , F. ruetzleri , G. silvae , and Gambierdiscus sp. ribotype 2. Only Gambierdiscus excentricus was absent from the eight Gambierdiscus / Fukuyoa species known in the wider Caribbean region. Eleven clonal cultures were established and confirmed by PCR and SEM as being either G. carolinianus or G. caribaeus. Toxin production in each isolate was assessed by a radioligand receptor binding assay and found to be below the assay quantification limit. These novel findings augment the knowledge of the ciguatoxin-source dinoflagellates that are present in Cuba, however further studies are needed to better understand the correlation between their abundance, species-specific toxin production in the environment, and the risk for fish contamination, in order to develop better informed ciguatera risk management strategies. [ABSTRACT FROM AUTHOR]

Environmental occurrence and human exposure of emerging organophosphate esters (eOPEs) have increased significantly in recent years. Resorcinol bis(diphenyl) phosphate (RDP) is one of the major eOPEs detected in indoor dust, but the knowledge on its toxicities and health risks is rather limited. In this study, we investigated the in vitro estrogenic effects and underlying mechanism of RDP in comparison with a legacy OPE triphenyl phosphate (TPHP). Our results showed that RDP promoted MCF-7 cell proliferation with the lowest effect concentration of 2.5 μM, and the maximum enhancement of 1.6 folds is greater than that of TPHP (1.3 folds). The effect was inhibited completely by an estrogen receptor (ER) antagonist, suggesting that ER activation was responsible for the enhancement. In luciferase reporter gene assays both RDP and TPHP activated ER transcriptional activity at 2.5 μM, but RDP activity was higher than TPHP. Competitive fluorescence binding assays showed that RDP bound to ER with an IC 10 of 0.26 μM, which is 20 folds lower than TPHP (5.6 μM). Molecular docking simulation revealed that both RDP and TPHP interacted with ER at the binding pocket of estradiol, although the hydrogen bonds were different. Taken together, RDP exerted stronger estrogenic effects than TPHP through ER-mediated pathways and may pose more health risks. [Display omitted] [ABSTRACT FROM AUTHOR]

In order to investigate the effects of metallic nanoparticles (NPs) on the performance of in vitro bioassay, zinc oxide NP (ZnO NP), aluminum oxide NP (Al2O3 NP), bare silver NP (Ag NP), and Ag NP capped with citrate (Agcit NP) were evaluated with yeast (Saccharomyces cerevisiae Y190) two-hybrid system (YES assay), carrying Japanese medaka estrogen receptors (mERs) in the presence of 17β-estradiol (E2, 10−6 M), a reference chemical for estrogenic activity. The distribution of NPs in the yeast was also examined by field-emission transmission electron microscopy (FE-TEM). The results show that TEM analysis revealed that NPs were present inside the yeast and accumulated deep inside the cell organelles, suggesting that cell death was caused by NPs. However, despite no significant change of mortality, the E2 estrogenic activities in yeast exposed to ZnO NP and Al2O3 NP were dose-dependently reduced. For Ag NP and Agcit NP, such phenomenon observed in the exposure of ZnO NP and Al2O3 NP did not occur. From the observations, we found that ZnO NP and Al2O3 NP in the environmental media could result in underestimated estrogenicity of endocrine-disrupting compounds when evaluated by YES assay.

In late October and early November 2013 and 2017, hundreds of sea turtles were found dead along the Pacific coastline of El Salvador. The dead turtles were in good body condition and did not have any injuries or other major anomalies. In order to determine the role of paralytic shellfish toxins (PST) in this mass mortality, tissue samples, including blood, flipper, liver, kidney, stomach and intestinal contents, of dead green turtles (Chelonia mydas) and olive ridley turtles (Lepidochelys olivacea) were analyzed for PST using a radioactive receptor binding assay, enzyme-linked immunosorbent assay, and high performance liquid chromatography. Highest values of PST were detected in enteric contents in the 2013 event (7,304.1 μg STX eq kg−1) and in gastric contents during the 2017 event (16,165.0 μg STX eq kg−1). During these events, remotely-sensed chlorophyll-a and fluorescence line height imagery revealed anomalies suggestive of algal blooms off the coast of El Salvador. In the 2017 event, Pyrodinium bahamense was observed in samples of gastrointestinal contents from affected sea turtles. Seawater from the region where dead sea turtles were found was also analyzed, but saxitoxin-producing species were found in low abundance (5400 cell/L in 2013 and 672 cell/L in 2017), which may reflect limited sampling. Although threshold levels of toxicity in sea turtle species are not well-characterized, our evidence suggests that these large events were the result of PST-producing algal blooms and that these blooms are a major cause of sea turtle mortality in this region.

Ciguatera fish poisoning (CFP) is a foodborne illness affecting > 50,000 people worldwide annually. It is caused by eating marine invertebrates and fish that have accumulated ciguatoxins (CTXs). Recently, the risk of CFP to human health, the local economy, and fishery resources have increased; therefore, detection methods are urgently needed. Functional assays for detecting ciguatoxins in fish include receptor binding (RBA) and neuroblastoma cell-based assay (N2a assay), which can detect all CTX congeners. In this study, we made these assays easier to use. For RBA, an assay was developed using a novel near-infrared fluorescent ligand, PREX710-BTX, to save valuable CTXs. In the N2a assay, a 1-day assay was developed with the same detection performance as the conventional 2-day assay. Additionally, in these assays, we used calibrated CTX standards from the Pacific determined by quantitative NMR for the first time to compare the relative potency of congeners, which differed significantly among previous studies. In the RBA, there was almost no difference in the binding affinity among congeners, showing that the differences in side chains, stereochemistry, and backbone structure of CTXs did not affect the binding affinity. However, this result did not correlate with the toxic equivalency factors (TEFs) based on acute toxicity in mice. In contrast, the N2a assay showed a good correlation with TEFs based on acute toxicity in mice, except for CTX3C. These findings, obtained with calibrated toxin standards, provide important insights into evaluating the total toxicity of CTXs using functional assays. [Display omitted] • Receptor binding assay using near-infrared fluorescent-labeled brevetoxin was effective in saving valuable ciguatoxins. • Assay time of neuroblastoma cell-based assay was shortened by omitting the 24 h pre-culture without lowering detectability. • Differences in binding affinities of calibrated ciguatoxin congeners to voltage-gated sodium channels were slight. • The relative potency of neuroblastoma cell-based assay for ciguatoxin congeners correlated well with acute toxicity in mice. [ABSTRACT FROM AUTHOR]

Human norovirus is a leading cause of gastroenteritis worldwide. Although two in vitro cultivation methods have been reported, they cannot provide mechanistic insights into viral inactivation. Receptor-binding assays supplement these assays and give insight into capsid integrity. We present a streamlined version of a receptor-binding assay with minimal time-to-result while maintaining accuracy and high throughput. We validate assay performance for physical and chemical inactivation treatments of a norovirus GII.4 capsid. The assay produces a high positive/negative ratio (25.3 ± 4.9) in <2.5 h and has a limit of detection of 0.1 μg/ml capsid. This method is a valuable additional tool for understanding human norovirus inactivation. [ABSTRACT FROM AUTHOR]

The aim of the present work is to study the in vitro screening of psychoactive potential of F. macrophylla using neurotransmitter receptor binding assay and in vivo screening of anti-dementia activity in mouse model scopolamine induced memory deficits .Different extracts were prepared on the basis of polarity and these extracts were assessed for psychoactive potential. It was found thatin vitro receptor binding assay exhibited more than 60% inhibition on anti-dementia and anti-anxiety activity while in vivo it was not significantly active on anti-dementia activity in the model of scopolamine(i.p) induced memory deficits in passive avoidance test in mice. [ABSTRACT FROM AUTHOR]

Development of novel analytical tools to detect marine biotoxins has been warranted in view of the apparent global pervasiveness of algal-derived shellfish poisoning, and the limitations of existing methods. Here, we describe the initial phase in the development and evaluation of a tyrosine-containing analog of μ-conotoxin (μ-CTX) GIIIA as an alternative to saxitoxin (STX) in a receptor binding assay (RBA) for paralytic shellfish poisons. The peptide analog was synthesized and characterized for structure and bioactivity. The major product of oxidation elicited paralytic symptoms in mice at a minimum dose of 1.31 mg kg −1 (i.p.). Mass spectrometry analysis of the bioactive peptide gave a molecular mass of 2637.52 Da that was close to the predicted value. Iodination via chloramine-T produced non-, mono- and di-iodinated peptides (respectively, NIP, MIP and DIP). Competition assays against 3 H-STX revealed higher Ki and EC50 (P < 0.0001, ANOVA) indicating reduced affinity for the receptor, and limited displacement of receptor-bound STX. However, subsequent use of MIP may extend the application of RBA to detect small changes in toxin levels owing to its likely enhanced displacement by STX. This may be useful in analyzing samples with toxicities near the regulatory limit, or in establishing baseline values in high risk environments. [ABSTRACT FROM AUTHOR]

In the present study, we discovered a novel IL-1 family member (nIL-1F) from grass carp that possessed the ability to bind with grass carp IL-1β receptor type 1 (gcIL-1R1) and attenuate grass carp IL-1β activity in head kidney leukocytes (HKLs), suggesting that it may function as an IL-1β receptor antagonist. Grass carp nIL-1F transcript was constitutively expressed with the highest levels in some lymphoid organs, including head kidney, spleen and intestine, implying its potential in grass carp immunity. In agreement with this notion, in vitro and in vivo studies showed that nIL-1F mRNA was inductively expressed in grass carp with a rapid kinetics, indicating that it may be an early response gene during immune challenges. In addition, recombinant grass carp IL-1β (rgcIL-1β) induced nIL-1F mRNA expression via NF-κB and MAPK (JNK, p38 and p42/44) signaling pathways in HKLs. Particularly, the orthologs of nIL-1F found in other fish species, including zebrafish, pufferfish and rainbow trout are not homologous to mammalian IL-1β receptor antagonist (IL-1Ra), indicating that fish nIL-1F and mammalian IL-1Ra may not share a common evolutionary ancestor. Taken together, our data suggest the existence of a naturally occurring fish nIL-1F, which may function like mammalian IL-1Ra, being beneficial to understand the auto-regulatory mechanism of IL-1β activity in fish immunity. [ABSTRACT FROM AUTHOR]

Cytokinin receptors play a key role in cytokinin-dependent processes regulating plant growth, development, and adaptation; therefore, the functional properties of these receptors are of great importance. Previously the properties of cytokinin receptors were investigated in heterologous assay systems using unicellular microorganisms, mainly bacteria, expressing receptor proteins. However, within microorganisms receptors reside in an alien environment that might distort the receptor properties. Therefore, a new assay system has been developed allowing studies of individual receptors within plant membranes (i.e. closer to their natural environment). The main ligand-binding characteristics of receptors from Arabidopsis [AHK2, AHK3, and AHK4] and maize (ZmHK1) were refined in this new system, and the properties of full-length Arabidopsis receptor AHK2 were characterized for the first time. Ligand specificity profiles of receptors towards cytokinin bases were comparable with the profiles retrieved in bacterial assay systems. In contrast, cytokinin- 9-ribosides displayed a strongly reduced affinity for receptors in the plant assay system, indicating that ribosides as the common transport form of cytokinins have no or very weak cytokinin activity. This underpins the central role of free bases as the sole biologically active cytokinin compounds. According to molecular modelling and docking studies, N9-ribosylation alters the bonding pattern in cytokinin-receptor interaction and prevents β6-β7 loop movement important for tight hormone binding. A common feature of all receptors was a greatly reduced ligand binding at low (5.0-5.5) pH. The particularly high sensitivity of ZmHK1 to pH changes leads to the suggestion that some cytokinin receptors may play an additional role as pH sensors in the lumen of the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts. [ABSTRACT FROM AUTHOR]

Accumulation of ciguatoxins (CTXs) in tropical reef fish tissues during their life is responsible of the most prevalent human seafood intoxication in the South Pacific called Ciguatera Fish Poisoning (CFP). It has been assumed for a long time that CTXs are transferred and accumulated along the trophic food chain, and consequently that smaller individuals within a given fish species are safer to eat than larger ones. However, the relationship between toxicity and fish size has been studied for a limited number of species only and the conclusions are often contradictory. The toxicity of 856 fishes from 59 different species sampled in six islands in French Polynesia between 2003 and 2011 was assessed by Receptor Binding Assay. Among them, 45 species × island and 32 families × island for which the number of individuals was ≥6 allowed testing the relationship between toxicity and size. Except for six specimens of Lutjanus bohar caught in Fakarava (P < 0.01; R 2 = 0.854), the 44 remaining species × island showed no significant increase of CTXs concentration with fish total length (TL). Moreover, the proportion of toxic individuals decreased significantly for Epinephelus polyphekadion from Fakarava (n = 24; P < 0.05) and Kyphosus cinerascens from Raivavae (n = 29; P < 0.05), while no significant variation was detected for the other 43 species × island. At the family level, only three positive and three negative relationships between size and CTXs concentration were observed among the 32 family × island analyzed. No relationship between the proportion of toxic fish within a family and the relative total length of individuals were observed. The lack of relationship between toxicity and size observed for most of the species and families from the six islands suggests that fish size cannot be used as an efficient predictor of fish toxicity in French Polynesia. These results highlight the need for improving our knowledge about metabolic processes which may play a role in CTXs bio-accumulation and depuration among the different trophic levels of fishes. [ABSTRACT FROM AUTHOR]