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Biosensors May 2024In this study, we report a multiplexed platform for the simultaneous determination of five marine toxins. The proposed biosensor is based on a disposable electrical...
In this study, we report a multiplexed platform for the simultaneous determination of five marine toxins. The proposed biosensor is based on a disposable electrical printed (DEP) microarray composed of eight individually addressable carbon electrodes. The electrodeposition of gold nanoparticles on the carbon surface offers high conductivity and enlarges the electroactive area. The immobilization of thiolated aptamers on the AuNP-decorated carbon electrodes provides a stable, well-orientated and organized binary self-assembled monolayer for sensitive and accurate detection. A simple electrochemical multiplexed aptasensor based on AuNPs was designed to synchronously detect multiple cyanotoxins, namely, microcystin-LR (MC-LR), Cylindrospermopsin (CYL), anatoxin-α, saxitoxin and okadaic acid (OA). The choice of the five toxins was based on their widespread presence and toxicity to aquatic ecosystems and humans. Taking advantage of the conformational change of the aptamers upon target binding, cyanotoxin detection was achieved by monitoring the resulting electron transfer increase by square-wave voltammetry. Under the optimal conditions, the linear range of the proposed aptasensor was estimated to be from 0.018 nM to 200 nM for all the toxins, except for MC-LR where detection was possible within the range of 0.073 to 150 nM. Excellent sensitivity was achieved with the limits of detection of 0.0033, 0.0045, 0.0034, 0.0053 and 0.0048 nM for MC-LR, CYL, anatoxin-α, saxitoxin and OA, respectively. Selectivity studies were performed to show the absence of cross-reactivity between the five analytes. Finally, the application of the multiplexed aptasensor to tap water samples revealed very good agreement with the calibration curves obtained in buffer. This simple and accurate multiplexed platform could open the window for the simultaneous detection of multiple pollutants in different matrices.
Topics: Marine Toxins; Biosensing Techniques; Microcystins; Gold; Aptamers, Nucleotide; Electrochemical Techniques; Saxitoxin; Metal Nanoparticles; Cyanobacteria Toxins; Bacterial Toxins; Uracil; Tropanes; Alkaloids; Okadaic Acid; Electrodes; Limit of Detection
PubMed: 38920572
DOI: 10.3390/bios14060268 -
Ecotoxicology and Environmental Safety Jun 2024Microplastics (MPs) and okadaic acid (OA) are known to coexist in marine organisms, potentially impacting humans through food chain. However, the combined toxicity of OA...
Microplastics (MPs) and okadaic acid (OA) are known to coexist in marine organisms, potentially impacting humans through food chain. However, the combined toxicity of OA and MPs remains unknown. In this study, mice were orally administered OA at 200 μg/kg bw and MPs at 2 mg/kg bw. The co-exposure group showed a significant increase in malondialdehyde (MDA) content and significant decreases in superoxide dismutase (SOD) activity and glutathione (GSH) level compared to the control, MPs and OA groups (p < 0.05). Additionally, the co-exposure group exhibited significantly higher levels of IL-1β and IL-18 compared to other groups (p < 0.05). These results demonstrated that co-exposure to MPs and OA induces oxidative stress and exacerbates inflammation. Histological and cellular ultrastructure analyses suggested that this combined exposure may enhance gut damage and compromise barrier integrity. Consequently, the concentration of OA in the small intestine of the co-exposure group was significantly higher than that in the OA group. Furthermore, MPs were observed in the lamina propria of the gut in the co-exposure group. Transcriptomic analysis revealed that the co-exposure led to increased expression of certain genes related to the NF-κB/NLRP3 pathway compared to the OA and MPs groups. Overall, this combined exposure may disrupt the intestinal barrier, and promote inflammation through the NF-κB/NLRP3 pathway. These findings provide precious information for the understanding of health risks associated with MPs and phycotoxins.
PubMed: 38905936
DOI: 10.1016/j.ecoenv.2024.116628 -
Molecules (Basel, Switzerland) Jun 2024GSK-3β, IKK-β, and ROCK-1 kinases are implicated in the pathomechanism of Alzheimer's disease due to their involvement in the misfolding and accumulation of amyloid β...
Connecting GSK-3β Inhibitory Activity with IKK-β or ROCK-1 Inhibition to Target Tau Aggregation and Neuroinflammation in Alzheimer's Disease-Discovery, In Vitro and In Cellulo Activity of Thiazole-Based Inhibitors.
GSK-3β, IKK-β, and ROCK-1 kinases are implicated in the pathomechanism of Alzheimer's disease due to their involvement in the misfolding and accumulation of amyloid β (Aβ) and tau proteins, as well as inflammatory processes. Among these kinases, GSK-3β plays the most crucial role. In this study, we present compound , a novel, remarkably potent, competitive GSK-3β inhibitor (IC = 8 nM, K = 2 nM) that also exhibits additional ROCK-1 inhibitory activity (IC = 2.3 µM) and demonstrates anti-inflammatory and neuroprotective properties. Compound effectively suppresses the production of nitric oxide (NO) and pro-inflammatory cytokines in the lipopolysaccharide-induced model of inflammation in the microglial BV-2 cell line. Furthermore, it shows neuroprotective effects in an okadaic-acid-induced tau hyperphosphorylation cell model of neurodegeneration. The compound also demonstrates the potential for further development, characterized by its chemical and metabolic stability in mouse microsomes and fair solubility.
Topics: tau Proteins; Glycogen Synthase Kinase 3 beta; Alzheimer Disease; Animals; Thiazoles; Humans; rho-Associated Kinases; Mice; I-kappa B Kinase; Neuroprotective Agents; Protein Kinase Inhibitors; Cell Line; Anti-Inflammatory Agents; Microglia; Nitric Oxide; Lipopolysaccharides; Protein Aggregates; Neuroinflammatory Diseases
PubMed: 38893493
DOI: 10.3390/molecules29112616 -
Bioscience, Biotechnology, and... Jun 2024Dinophysistoxin 1 (DTX1, 1) and okadaic acid (OA, 2), produced by the dinoflagellates Dinophysis spp. and Prorocentrum spp., are primary diarrhetic shellfish toxins...
Dinophysistoxin 1 (DTX1, 1) and okadaic acid (OA, 2), produced by the dinoflagellates Dinophysis spp. and Prorocentrum spp., are primary diarrhetic shellfish toxins (DSTs), which may cause gastric illness in people consuming such as bivalves. Both compounds convert to dinophysistoxin 3 (DTX3, 3; generic name for 1 and 2 with fatty acids conjugated at 7-OH) in bivalves. The enzyme okadaic acid O-acyl transferase (OOAT) is a membrane protein found in the microsomes of the digestive glands of bivalves. In this study, we established an in vitro enzymatic conversion reaction using 4-nitro-2,1,3-benzoxadiazole (NBD)-OA (4), an OA derivative conjugated with (R)-(-)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (NBD-APy) on 1-CO2H, as a substrate. We detected the enzymatically produced 3, NBD-7-O-palmitoyl-OA (NBD-Pal-OA), using high-performance liquid chromatography-fluorescence detection. We believe that an OOAT assay using 4 will facilitate the fractionation and isolation of OOAT in the future.
PubMed: 38886126
DOI: 10.1093/bbb/zbae085 -
Analytica Chimica Acta Jul 2024Okadaic acid (OA), as a diarrhetic shellfish poisoning, can increase the risk of acute carcinogenic or teratogenic effects for the ingestion of OA contaminated...
BACKGROUND
Okadaic acid (OA), as a diarrhetic shellfish poisoning, can increase the risk of acute carcinogenic or teratogenic effects for the ingestion of OA contaminated shellfish. At present, much effort has been made to graft immunoassay onto a paper substrate to make paper-based sensors for rapid and simple detection of shellfish toxin. However, the complicated washing steps and low protein fixation efficiency on the paper substrate need to be further addressed.
RESULTS
A novel paper-tip immunosensor for detecting OA was developed combined with smartphone and naked eye readout. The trapezoid paper tip was consisted of quantitative and qualitative detection zones. To improve the OA antigen immobilization efficiency on the paper substrate, graphene oxide (GO)-assisted protein immobilization method was introduced. Meanwhile, Au nanoparticles composite probe combined with the lateral flow washing was developed to simplify the washing step. The OA antigen-immobilized zone, as the detection zone Ⅰ, was used for quantitative assay by smartphone imaging. The paper-tip front, as the detection zone Ⅱ, which could qualitatively differentiate OA pollution level within 45 min using the naked eye. The competitive immunoassay on the paper tip exhibited a wide linear range for detecting OA (0.02-50 ng∙mL) with low detection limit of 0.02 ng∙mL. The recovery of OA in spiked shellfish samples was in the range of 90.3 %-113.%.
SIGNIFICANCE
These results demonstrated that the proposed paper-tip immunosensor could provide a simple, low-cost and high-sensitivity test for OA detection without the need for additional large-scale equipment or expertise. We anticipate that this paper-tip immunosensor will be a flexible and versatile tool for on-site detecting the pollution of marine products.
Topics: Graphite; Smartphone; Okadaic Acid; Immunoassay; Paper; Biosensing Techniques; Gold; Metal Nanoparticles; Immobilized Proteins; Limit of Detection; Animals; Antibodies, Immobilized
PubMed: 38876519
DOI: 10.1016/j.aca.2024.342781 -
Molecular Diversity Jun 2024Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is implicated in accumulation of amyloid β-protein (Aβ) and phosphorylation of Tau proteins, and...
Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is implicated in accumulation of amyloid β-protein (Aβ) and phosphorylation of Tau proteins, and thus represents an important therapeutic target for neurodegenerative diseases. Though many DYRK1A inhibitors have been discovered, there is still no marketed drug targeting DYRK1A. This is partly due to the lack of effective and safe chemotypes. Therefore, it is still necessary to identify new classes of DYRK1A inhibitors. By performing virtual screening with the workflow mainly composed of pharmacophore modeling and molecular docking as well as the following DYRK1A inhibition assay, we identified compound L9, ((Z)-1-(((5-phenyl-1H-pyrazol-4-yl)methylene)-amino)-1H-tetrazol-5-amine), as a moderately active DYRK1A inhibitor (IC: 1.67 μM). This compound was structurally different from the known DYRK1A inhibitors, showed a unique binding mode to DYRK1A. Furthermore, compound L9 showed neuroprotective activity against okadaic acid (OA)-induced injury in the human neuroblastoma cell line SH-SY5Y by regulating the expression of Aβ and phosphorylation of Tau protein. This compound was neither toxic to the SH-SY5Y cells nor to the human normal liver cell line HL-7702 (IC: >100 μM). In conclusion, we have identified a novel DYRK1A inhibitor with neuroprotective activity through virtual screening and in vitro biological evaluation, which holds the promise for further study.
PubMed: 38833123
DOI: 10.1007/s11030-024-10856-2 -
Archives of Toxicology Jun 2024Okadaic acid (OA), a prevalent marine biotoxin found in shellfish, is known for causing acute gastrointestinal symptoms. Despite its potential to reach the bloodstream...
Okadaic acid (OA), a prevalent marine biotoxin found in shellfish, is known for causing acute gastrointestinal symptoms. Despite its potential to reach the bloodstream and the liver, the hepatic effects of OA are not well understood, highlighting a significant research gap. This study aims to comprehensively elucidate the impact of OA on the liver by examining the transcriptome, proteome, and phosphoproteome alterations in human HepaRG liver cells exposed to non-cytotoxic OA concentrations. We employed an integrative multi-omics approach, encompassing RNA sequencing, shotgun proteomics, phosphoproteomics, and targeted DigiWest analysis. This enabled a detailed exploration of gene and protein expression changes, alongside phosphorylation patterns under OA treatment. The study reveals concentration- and time-dependent deregulation in gene and protein expression, with a significant down-regulation of xenobiotic and lipid metabolism pathways. Up-regulated pathways include actin crosslink formation and a deregulation of apoptotic pathways. Notably, our results revealed that OA, as a potent phosphatase inhibitor, induces alterations in actin filament organization. Phosphoproteomics data highlighted the importance of phosphorylation in enzyme activity regulation, particularly affecting proteins involved in the regulation of the cytoskeleton. OA's inhibition of PP2A further leads to various downstream effects, including alterations in protein translation and energy metabolism. This research expands the understanding of OA's systemic impact, emphasizing its role in modulating the phosphorylation landscape, which influences crucial cellular processes. The results underscore OA's multifaceted effects on the liver, particularly through PP2A inhibition, impacting xenobiotic metabolism, cytoskeletal dynamics, and energy homeostasis. These insights enhance our comprehension of OA's biological significance and potential health risks.
PubMed: 38832940
DOI: 10.1007/s00204-024-03796-1 -
Harmful Algae May 2024This article presents the first results on shellfish toxicity in the Slovenian sea (Gulf of Trieste, Adriatic Sea) since the analytical methods for the detection of...
Okadaic acid as a major problem for the seafood safety (Mytilus galloprovincialis) and the dynamics of toxic phytoplankton in the Slovenian coastal sea (Gulf of Trieste, Adriatic Sea).
This article presents the first results on shellfish toxicity in the Slovenian sea (Gulf of Trieste, Adriatic Sea) since the analytical methods for the detection of biotoxins (PSP, ASP, DSP and other lipophilic toxins) in bivalve molluscs were included in the national monitoring program in 2013. In addition to toxins, the composition and abundance of toxic phytoplankton and general environmental characteristics of the seawater (surface temperature and salinity) were also monitored. During the 2014-2019 study period, only lipophilic toxins were detected (78 positive tests out of 446 runs), of which okadaic acid (OA) predominated in 97 % of cases, while dinophysistoxin-2 and yessotoxins only gave a positive result in one sampling event each. The number of samples that did not comply with the EC Regulation for the OA group was 17 or 3.8 % of all tests performed, all of which took place from September to November, while a few positive OA tests were also recorded in December, April, and May. This toxicity pattern was consistent with the occurrence pattern of the five most common DSP-producing dinoflagellates, which was supported by the development of warm and thermohaline stratified waters: Dinophysis caudata, D. fortii, D. sacculus, D. tripos and Phalacroma rotundatum. The strong correlation (r = 0.611, p < 0.001) between D. fortii, reaching abundances of up to 950 cells L, and OA suggests that D. fortii is the main cause of OA production in Slovenian waters. Strong interannual variations in OA and phytoplankton dynamics, exacerbated by the effects of anthropogenic impacts in this coastal ecosystem, reduce the predictability of toxicity events and require continuous and efficient monitoring. Our results also show that the introduction of the LC-MS/MS method for lipophilic toxins has improved the management of aquaculture activities, which was not as accurate based on mouse bioassays.
Topics: Okadaic Acid; Animals; Phytoplankton; Marine Toxins; Slovenia; Mytilus; Seafood; Seawater; Dinoflagellida
PubMed: 38830710
DOI: 10.1016/j.hal.2024.102632 -
Food Chemistry May 2024In this study, a sensitive dual-signal electrochemiluminescence (ECL) immunosensor was developed for okadaic acid (OA) detection utilizing copper nanoclusters (CuNCs)...
In this study, a sensitive dual-signal electrochemiluminescence (ECL) immunosensor was developed for okadaic acid (OA) detection utilizing copper nanoclusters (CuNCs) and Ru(bpy)-doped silica nanoparticles (RuSiNPs). Interestingly, the CuNCs could simultaneously enhance both cathodic (-0.95 V) and anodic (+1.15 V) ECL signals of RuSiNPs, forming a dual-signal ECL sensing platform. Further, RuSiNPs@CuNCs were used as immunomarkers by covalently conjugating them with an anti-OA monoclonal antibody (mAb) to form probes. Finally, dual ECL signals of the immunosensor were fabricated and showed good linear relationships with OA concentrations in the range of 0.05-70 ng mL, having a median inhibitory concentration (IC) of 1.972 ng mL and a limit of detection of 0.039 ng mL. Moreover, the constant ratio of the cathodic and anodic ECL peaks achieved self-calibration of the detection signal and improved the reliability of the results. Finally, we successfully applied the ECL sensor to detect OA in spiked oyster samples.
PubMed: 38823134
DOI: 10.1016/j.foodchem.2024.139844 -
Analytica Chimica Acta Jun 2024Reliability and robustness have been recognized as key challenges for Surface-enhanced Raman scattering (SERS) analytical techniques. Quantifying the concentration of an...
BACKGROUND
Reliability and robustness have been recognized as key challenges for Surface-enhanced Raman scattering (SERS) analytical techniques. Quantifying the concentration of an analyte using a single characteristic peak from SERS has been a controversial topic because the Raman signal is susceptible to highly concentrated electromagnetic hotspots, inhomogeneity of SERS substrate, or non-standardization of measurement conditions. Ratiometric SERS strategies have been demonstrated as a promising solution to effectively balance and compensate for signal fluctuations caused by matrix heterogeneity. However, it is not easy to construct ratiometric SERS sensors with monitoring the ratio of two different signal intensities for target analysis.
RESULTS
An attempt has been made to develop a novel ratiometric biosensor that can be applied to detect okadaic acid (OA). Aptamer-anchored magnetic particles were first combined with gold-tagged short complementary DNA (Au-cDNA) to create heterogeneous nanostructures. When the target was present, the Au-cDNA was dissociated from nanostructures, and 4-nitrothiophenol (4-NTP) was initiated to reduce to 4-aminothiophenol (4-ATP) in the presence of hydrogen sources. The SERS ratio change of 4-NTP and 4-ATP was finally detected by AuNPs-coated film. OA was successfully quantified, and the detection limit was as low as 2.4524 ng/mL. The constructed biosensor had good stability and reproducibility with a relative standard deviation of less than 4.47%. The proposed method used gold nanoparticles as an intermediate to achieve catalytic signal amplification and subsequently increased the sensitivity of the biosensor.
SIGNIFICANCE AND NOVELTY
Catalytic reaction-based ratiometric SERS biosensors combine the multiple advantages of catalytic signal amplification and signal self-calibration and provide new insights into the development of stable, reproducible, and reliable SERS detection techniques. This ratiometric SERS technique offered a universal method that is anticipated to be applicable for the detection of other targets by substituting the aptamer.
Topics: Spectrum Analysis, Raman; Gold; Biosensing Techniques; Okadaic Acid; Metal Nanoparticles; Aptamers, Nucleotide; Food Contamination; Limit of Detection; Food Analysis; Surface Properties
PubMed: 38811142
DOI: 10.1016/j.aca.2024.342705