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Microcystins (MCs) are hepatotoxic heptapeptides produced by cyanobacteria and are potent inhibitors of protein phosphatases in eukaryotic cells. Algae for dietary supplements are harvested from outdoor environments and can be contaminated with MCs. Monitoring of MCs in these products is necessary but is complicated by their structural diversity (>250 congeners). We used a combination of protein phosphatase inhibition assay (PPIA), ELISA, LC-MS/MS, and nontargeted LC-high-resolution MS (LC-HRMS) with thiol derivatization to characterize the total MCs in 18 algal dietary supplements. LC-MS/MS revealed that some products contained >40 times the maximum acceptable concentration (MAC) of 1 μg/g MCs, but ELISA and PPIA showed up to 50-60 times the MAC. LC-HRMS identified all congeners targeted by LC-MS/MS plus MC-(H4)YR contributing up to 18% of total MCs, along with numerous minor MCs. Recommended dosages of the products greater than the MAC would result in 2.6-75 times the tolerable daily intake, presenting a risk to consumers. This study confirms the need for monitoring these products and presents strategies to fully describe the total MC pool in environmental samples and algal products.

Azaspiracids (AZAs) are a group of biotoxins that appear periodically in shellfish and can cause food poisoning in humans. Current methods for quantifying the regulated AZAs are restricted to LC-MS but are not well suited to detecting novel and unregulated AZAs. An ELISA method for total AZAs in shellfish was reported recently, but unfortunately, it used relatively large amounts of the AZA-1-containing plate-coating conjugate, consuming significant amounts of pure AZA-1 per assay. Therefore, a new plate-coater, OVA-cdiAZA1 was produced, resulting in an ELISA with a working range of 0.30-4.1 ng/mL and a limit of quantification of 37 μg/kg for AZA-1 in shellfish. This ELISA was nearly twice as sensitive as the previous ELISA while using 5-fold less plate-coater. The new ELISA displayed broad cross-reactivity toward AZAs, detecting all available quantitative AZA reference materials as well as the precursors to AZA-3 and AZA-6, and results from shellfish analyzed with the new ELISA showed excellent correlation ( R 2 = 0.99) with total AZA-1-10 by LC-MS. The results suggest that the new ELISA is suitable for screening samples for total AZAs, even in cases where novel AZAs are present and regulated AZAs are absent, such as was reported recently from Puget Sound and the Bay of Naples.

In the summer of 2012, over 750 dead and dying birds were observed at the Paul S. Sarbanes Ecosystem Restoration Project at Poplar Island, Maryland, USA (Chesapeake Bay). Clinical signs suggested avian botulism, but an ongoing dense Microcystis bloom was present in an impoundment on the island. Enzyme-linked immunosorbent assay (ELISA) analysis of a water sample indicated 6000 ng mL -1 of microcystins (MCs). LC-UV/MS analysis confirmed the presence of MC-LR and a high concentration of an unknown MC congener (m/z 1037.5). The unknown MC was purified and confirmed to be [D-Leu 1 ]MC-LR using NMR spectroscopy, LC-HRMS and LC-MS 2 , which slowly converted to [D-Leu 1 ,Glu(OMe) 6 ]MC-LR during storage in MeOH. Lyophilized algal material from the bloom was further characterized using LC-HRMS and LC-MS 2 in combination with chemical derivatizations, and an additional 24 variants were detected, including MCs conjugated to Cys, GSH and γ-GluCys and their corresponding sulfoxides. Mallard (Anas platyrhynchos) livers were tested to confirm MC exposure. Two broad-specificity MC ELISAs and LC-MS 2 were used to measure free MCs, while 'total' MCs were estimated by both MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) and thiol de-conjugation techniques. Free microcystins in the livers (63-112 ng g -1 ) accounted for 33-41% of total microcystins detected by de-conjugation and MMPB techniques. Free [D-Leu 1 ]MC-LR was quantitated in tissues at 25-67 ng g -1 (LC-MS 2 ). The levels of microcystin varied based on analytical method used, highlighting the need to develop a comprehensive analysis strategy to elucidate the etiology of bird mortality events when microcystin-producing HABs are present., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Azaspiracids belong to a family of more than 50 polyether toxins originating from marine dinoflagellates such as Azadinium spinosum. All of the azaspiracids reported thus far contain a 21,22-dihydroxy group. Boric acid gel can bind selectively to compounds containing vic-diols or α-hydroxycarboxylic acids via formation of reversible boronate complexes. Here we report use of the gel to selectively capture and release azaspiracids from extracts of blue mussels. Analysis of the extracts and fractions by liquid chromatography-tandem mass spectrometry (LC-MS) showed that this procedure resulted in an excellent cleanup of the azaspiracids in the extract. Analysis by enzyme-linked immunoasorbent assay (ELISA) and LC-MS indicated that most azaspiracid analogues were recovered in good yield by this procedure. The capacity of boric acid gel for azaspiracids was at least 50 μg/g, making this procedure suitable for use in the early stages of preparative purification of azaspiracids. In addition to its potential for concentration of dilute samples, the extensive cleanup provided by boric acid gel fractionation of azaspiracids in mussel samples almost eliminated matrix effects during subsequent LC-MS and could be expected to reduce matrix effects during ELISA analysis. The method may therefore prove useful for quantitative analysis of azaspiracids as part of monitoring programs. Although LC-MS data showed that okadaic acid analogues also bound to the gel, this was much less efficient than for azaspiracids under the conditions used. The boric acid gel methodology is potentially applicable to other important groups of natural toxins containing diols including ciguatoxins, palytoxins, pectenotoxins, tetrodotoxin, trichothecenes, and toxin glycosides.

Azaspiracids (AZAs) are a group of biotoxins that cause food poisoning in humans. These toxins are produced by small marine dinoflagellates such as Azadinium spinosum and accumulate in shellfish. Ovine polyclonal antibodies were produced and used to develop an ELISA for quantitating AZAs in shellfish, algal cells, and culture supernatants. Immunizing antigens were prepared from synthetic fragments of the constant region of AZAs, while plate coating antigen was prepared from AZA-1. The ELISA provides a sensitive and rapid analytical method for screening large numbers of samples. It has a working range of 0.45-8.6 ng/mL and a limit of quantitation for total AZAs in whole shellfish at 57 μg/kg, well below the maximum permitted level set by the European Commission. The ELISA has good cross-reactivity to AZA-1-10, -33, and -34 and 37-epi-AZA-1. Naturally contaminated Irish mussels gave similar results whether they were cooked or uncooked, indicating that the ELISA also detects 22-carboxy-AZA metabolites (e.g., AZA-17 and AZA-19). ELISA results showed excellent correlation with LC-MS/MS analysis, both for mussel extract spiked with AZA-1 and for naturally contaminated Irish mussels. The assay is therefore well suited to screening for AZAs in shellfish samples intended for human consumption, as well as for studies on AZA metabolism.

The peroxidase-like activity of Ti3C2 nanosheets (Ti3C2 NSs) was evaluated by catalytic oxidation of colorless o-phenylenediamine (OPD) into orange-yellow 2,3-diaminophenazine (DAP) with the aid of H2O2. The catalytic behavior followed the typical Michaelis–Menten kinetics. Systematic studies about the catalytic activity of Ti3C2 NSs including cytochrome C (Cyt C) electron transfer experiments, radical capture experiments, and fluorescence analysis were conducted, revealing that the catalytic mechanism of Ti3C2 NSs was attributed to nanozyme-accelerated electron transfer between substrates and nanozyme-promoted generation of active species (superoxide anion free radical (·O2−) and holes (h+)). Single-stranded DNA (ssDNA) inhibited the peroxidase-like activity of Ti3C2 NSs, and the reduced catalytic activity was ascribed to DNA-hindered substrate accessibility to nanozyme surface. Based on the DNA controllable peroxidase-mimicking activity of Ti3C2 NSs, taking microcystin-LR (MC-LR) aptamer as an example, a label-free colorimetric aptasensor was proposed for the sensitive detection of MC-LR. The colorimetric aptasensor showed a wide linear range (0.01–60 ng mL−1), low limit of detection (6.5 pg mL−1), and high selectivity. The practicality of the colorimetric aptasensor was demonstrated by detecting different levels of MC-LR in spiked real water samples; satisfactory recoveries (97.2–102.1%) and low relative standard deviations (1.16–3.72%) were obtained. [ABSTRACT FROM AUTHOR]

Molecular-based approaches for harmful algal bloom species, which can be difficult to identify by light microscopy, are important tools for detecting and predicting the blooms. In this study, 84 samples from Aotearoa/New Zealand coastal waters that contained Azadinium-like cells were analysed using an Amphidomataceae real-time PCR assay. Nineteen samples were positive, and 24 Azadinium-like clonal isolates were established from these samples. Subsequently, only five isolates from one sampling point were positive using the real-time PCR assay. Four of the five isolates were genetically identified as Amphidoma languida and one as Azadinium dalianense. The field samples were analysed retrospectively using the species-specific real-time PCR assays; Am. languida was detected from the temperate areas, while Az. poporum and Az. spinosum were detected at one site. Two representative strains of each species were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), including Am. languida, which is known as toxic in the previous studies, but neither produced the currently monitored azaspiracids (AZAs). These two newly recorded species for New Zealand are important findings. The results highlight the need for more information on the spatio-temporal variations of potentially toxic Amphidomataceae species and the risk of AZAs in New Zealand coastal waters. [ABSTRACT FROM AUTHOR]

In this study, the contents of microcystin-LR (MC-LR) of Microcystis aeruginosa cultures in the laboratory and natural water samples from the Huangpu River in different seasons were detected through enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC), respectively. Excellent correlation between the two methods was obtained (R2 > 0.99). ELISA was a reliable and simple method with high reproducibility (coefficient of variation < 25%) and satisfactory recovery for the monitoring of low levels of MC-LR. MC-LR concentrations in Huangpu River varied with the seasonal variation, which peaked in August with the temperature over 30 °C and then gradually declined with the decreasing temperature after August. The highest MC-LR concentration in the Huangpu River was below the WHO drinking water quality standard (1 µg/L). These results indicated that warm temperature accelerated the MC-LR synthesis and release, and it is necessary to regularly monitor the MC-LR levels, especially during the high algae period in summer. ELISA can be applied to detect the low levels of MC-LR in the field without complex treatment, avoiding the samples from denaturation and degradation during the transportation. Hence, ELISA is a better alternative of HPLC when HPLC is unavailable, especially when rapid testing is required in routine MC-LR analysis. [ABSTRACT FROM AUTHOR]

The peroxidase-like activity of Ti 3 C 2 nanosheets (Ti 3 C 2 NSs) was evaluated by catalytic oxidation of colorless o-phenylenediamine (OPD) into orange-yellow 2,3-diaminophenazine (DAP) with the aid of H 2 O 2 . The catalytic behavior followed the typical Michaelis-Menten kinetics. Systematic studies about the catalytic activity of Ti 3 C 2 NSs including cytochrome C (Cyt C) electron transfer experiments, radical capture experiments, and fluorescence analysis were conducted, revealing that the catalytic mechanism of Ti 3 C 2 NSs was attributed to nanozyme-accelerated electron transfer between substrates and nanozyme-promoted generation of active species (superoxide anion free radical (·O 2 - ) and holes (h + )). Single-stranded DNA (ssDNA) inhibited the peroxidase-like activity of Ti 3 C 2 NSs, and the reduced catalytic activity was ascribed to DNA-hindered substrate accessibility to nanozyme surface. Based on the DNA controllable peroxidase-mimicking activity of Ti 3 C 2 NSs, taking microcystin-LR (MC-LR) aptamer as an example, a label-free colorimetric aptasensor was proposed for the sensitive detection of MC-LR. The colorimetric aptasensor showed a wide linear range (0.01-60 ng mL -1 ), low limit of detection (6.5 pg mL -1 ), and high selectivity. The practicality of the colorimetric aptasensor was demonstrated by detecting different levels of MC-LR in spiked real water samples; satisfactory recoveries (97.2-102.1%) and low relative standard deviations (1.16-3.72%) were obtained., (© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.)

Food allergies are life-threatening conditions that allergic individuals can avoid by consulting the food labels before consuming. Precautionary allergen labelling (or PAL), to warn against possible allergen cross-contamination, is widely used by food industries, reducing the food choices for allergic individuals. In Canada, there is limited information on the actual occurrence of allergens in products with a PAL related to the given allergen. This study attempted to fill the data gap by evaluating the occurrence of milk and egg allergens in Canadian products with PAL. A total of 1125 samples were analysed for milk and 840 for eggs, with 23% and 7% of these samples showing positive detection of ≥2.5 mg kg−1 for milk and ≥0.245 mg kg−1 for eggs. Chocolate products gave the largest number of positive results. Although the proportion of positive results was low, the levels detected reached 6471 mg kg−1 in a chocolate sample and were indicative of possible health consequences, if PAL was ignored by allergic consumers. The occurrence data generated is destinated to be used in exposure and risk assessments, to support allergen management linked to cross-contamination, with the possible development of allergen action levels that would be used by food industries, thus improving a risk-based approach for the application of PAL. [ABSTRACT FROM AUTHOR]

Microcystis is a frequent cyanobacterium bloom-forming with cosmopolitan distribution which can produce a hepatotoxin group called microcystins (MCs). These MCs are resistant to the traditional processes employed in the water treatment plants and they are often detected after conventional treatments. Because of this, the bio-removal studies have obtained a great interest in the last decades. In this work, a bacterial strain namely LG1 with the ability to remove microcystin-LR (MC-LR) under laboratory conditions was isolated from Rio de la Plata River and it was identified as Achromobacter spp. This ubiquitous bacterium was able to remove 79.5% MC-LR in 7 days with average removal time of 3.33 ± 0.08, 3.06 ± 0.05, and 2.77 ± 0.05 days at 28, 32, and 36 ± 1 °C, being higher at high temperature (36 °C) with an activation energy = 16.79 ± 1.99 kJ mol−1. LG1 grew better at higher temperature (from 28 to 36 ± 1 °C) increasing the specific growth rate (μ) and reducing 2-fold the lag phase duration (LPD) without significant differences (p > 0.05) between maximum population density (MPD). In addition, LG1 showed a lysis activity on two M. aeruginosa native strains in 7 days measured as chlorophyll a (Chl-a) concentration. The lysis activity increased around 2-fold when increasing the temperature from 28 to 36 ± 1 °C. This is the first report of an indigenous bacterium belonging to the genus Achromobacter spp. isolated from the Rio de la Plata River with the capacity to remove MC-LR and lysis activity on M. aeruginosa. [ABSTRACT FROM AUTHOR]

Anisakiasis is an underrecognized condition globally, and accurate diagnosis remains problematic even in countries where the condition is well known. Our "systematic review" was conducted according to Prisma guidelines. The stated basis of our study was "syndromic surveillance." Both methods are recognized in published literature as valid to identify or predict disease and to make accessible large amounts of evidence from published literature. Our study identified Anisakis allergy "hot spots" and other geographical areas where fish are highly infected with Anisakis without commensurate studies of human allergy. Results of our study will open up new lines of enquiry. Norway, used as an example to discredit the scientific integrity of our article, has a cuisine thriving with raw fish dishes and many sushi restaurants. The peer reviewed data sets, confirmed A. simplex sensitization among the Norwegian population, although this has been overlooked by the authors of the "Letter to the Editor." The identification of hot spots in our study may be influential in many ways not the least in raising diagnostic suspicion to expedite accurate diagnosis. [ABSTRACT FROM AUTHOR]

This study characterized the mineral composition of 15 common Portuguese seaweed (green, brown, and red) species. Total measured mineral content ranged from 10.9 g kg dry matter (DM) in Gracilaria vermiculophylla to 71.0 g kg DM in Codium adhaerens, calcium being the mineral generally found in higher amounts. Overall, the results suggest that seaweeds have great potential as mineral sources for animal feeding, but a great variability between species was observed regarding their mineral profile. Compared to common animal feed ingredients, the studied seaweeds can be considered as good sources of calcium, magnesium, iron, iodine, copper, manganese, and selenium but are poor sources of phosphorous and zinc. The maximum level of dietary inclusion will be strongly dependent on the mineral profile of the seaweeds. Depending on the seaweed, the upper level of inclusion in poultry and swine diets may reach more than 40 %. The high iodine content of studied seaweeds limits their use in diets for horses, and, to a lesser extent, for ruminants. This work constitutes a paramount contribution regarding the use of seaweeds as mineral sources in animal diets, allowing a more precise choice of the algae species and level of inclusion to be used, thus assuring animal health and strengthening the seaweed industry through this underexploited application field. [ABSTRACT FROM AUTHOR]

Microcystins (MCs) and nodularin (NOD) are cyanobacterial hepatotoxins that can greatly harm human health. Multi-analyte immunoassays provide efficient and cheap methods of screening these toxins. To develop a multi-analyte immunoassay, an antibody with both broad specificity and high affinity for structurally similar algal toxins is urgently needed. In this study, microcystin-leucine-arginine (MC-LR) and NOD were conjugated to carrier proteins using a one-step active ester (AE) method and multistep thiol-ene click chemistry and glutaraldehyde method, respectively. The immunogens obtained from these two conjugation methods were evaluated for their effectiveness in producing antibodies. The results demonstrated that the antisera derived from AE immunogens showed better performance in terms of affinity and titer. Using this simple AE method, we prepared a new immunogen for NOD and successfully produced a monoclonal antibody (mAb), 2G5, which could recognize not only NOD but also all eight of the tested MCs (MC-LR, MC-RR, MC-YR, MC-WR, MC-LA, MC-LF, MC-LY, and MC-LW) with high sensitivity and improved uniform affinities (0.23 ≤ IC ≤ 0.68 ng mL) compared with previously described mAbs. Under optimal conditions, one indirect competitive enzyme-linked immunosorbent assay was developed based on mAb2G5 for the detection of MC-LR and NOD, with limits of detection of 0.16 and 0.10 μg L, respectively, and a recovery of 62-86 % with a coefficient of variation below 12.6 % in water samples. [ABSTRACT FROM AUTHOR]

Since the number of recalls involving undeclared allergens is commonly associated with bakery and snack foods, we aimed to determine the frequency of egg allergens in a large number of these products using two commercial enzyme-linked immunosorbent assay (ELISA) methods. Samples were chosen that either had no egg identified on the product label or which had an egg precautionary statement. Among all samples, egg protein was detected in 5% of products using a Morinaga (MO) kit and 1% of products using a R-Biopharm (RB) kit. For bakery samples, egg protein was detected in 6% of 363 samples with no precautionary labelling (6% by MO and 1% by RB kit) and 12% of 80 samples which had precautionary labelling. For snack samples, egg protein was detected in 2% of 371 samples with no precautionary labelling (2% by MO and < 1% by RB kit) and 5% of 21 samples which had precautionary labelling. The disagreement rates between two methods were 5.2% for bakery products and 2.6% for snack products. The sample repeatability was at an acceptable level for bakery (< 12.5%) and snack foods (< 7.5%) for each method. The relative standard deviation between test kits was high (103.1%) for bakery foods. Four bakery products without precautionary labelling had a higher level of egg protein per serving compared with the eliciting dose (ED10 of 3.7 mg protein) for egg allergic patients. These results highlight the fact that detection methodology plays a vital role for accurate labelling control and mitigation of risk for egg allergic consumers. [ABSTRACT FROM AUTHOR]

Background: Sensitization to Anisakis spp. can produce allergic reactions after eating raw or undercooked parasitized fish. Specific IgE is detected long after the onset of symptoms, but the changes in specific IgE levels over a long follow-up period are unknown; furthermore, the influence of Anisakis spp. allergen exposure through consumption of fishery products is also unknown. Objective: To analyse the changes in IgE sensitization to Anisakis spp. allergens over several years of follow-up and the influence of the consumption of fishery products in IgE sensitization. Methods: Total IgE, Anisakis spp.-specific IgE, anti-Ani s 1 and anti-Ani s 4 IgE were repeatedly measured over a median follow-up duration of 49 months in 17 sensitized patients. Results: Anisakis spp.-specific IgE was detected in 16/17 patients throughout the follow-up period. The comparison between baseline and last visit measurements showed significant decreases in both total IgE and specific IgE. The specific IgE values had an exponential or polynomial decay trend in 13/17 patients. In 4/17 patients, an increase in specific IgE level with the introduction of fish to the diet was observed. Three patients reported symptoms after eating aquaculture or previously frozen fish, and in two of those patients, symptom presentation was coincident with an increase in specific IgE level. Conclusions: IgE sensitization to Anisakis spp. allergens lasts for many years since specific IgE was detectable in some patients after more than 8 years from the allergic episode. Specific IgE monitoring showed that specific IgE titres increase in some allergic patients and that allergen contamination of fishery products can account for the observed increase in Anisakis spp.-specific IgE level. Clinical Relevance: Following sensitization to Anisakis spp. allergens, the absence of additional exposure to those allergens does not result in the loss of IgE sensitization. Exposure to Anisakis spp. allergens in fishery products can increase the specific IgE level in some sensitized patients. [ABSTRACT FROM AUTHOR]

A study of the seasonal dynamics of copper (Cu) deficiency and its effects on the antioxidant system in Wumeng semi-fine wool sheep in Weining county was conducted. In addition, the concentrations of mineral elements in soil, grass, and blood of affected and healthy Wumeng semi-fine wool sheep were determined, and blood indices were measured. The results showed that Cu contents in soil, grass, and sheep blood from the experimental pasture were significantly lower than those from the control pasture (P < 0.05), while the Mo content in the samples from the experimental pasture were higher than those from the control pasture (P < 0.05). Hb, RBC, PCV, and MDA values in the blood of affected sheep were significantly higher than those of healthy sheep, the content of TP was significantly lower (P < 0.05), and the activities of CAT, GSH-Px, CP, SOD, POD, and TP in the blood of affected sheep were lower than those in healthy sheep (P < 0.05). A comparison of different months showed that the Cu content in soil was higher in November than in June and September. Hb, RBC, PCV, WBC, and the content of MDA in the blood of affected sheep was higher in November than in June and September; however, the change in CP, CAT, GSH-Px, POD, and TP showed the opposite trend. The content of Cu and Mo in soil, grass, and blood showed seasonal changes. Therefore, it is concluded that the seasonal dynamics of Cu deficiency in Wumeng semi-fine wool sheep in Weining was due to the content of Mo and Cu in soil and grass in different seasons, and high Mo concentration in grass affected Cu deficiency in sheep. The function of the antioxidant system of Wumeng semi-fine wool sheep was reduced as a result.