Two commonly used methods for cyanotoxin analysis are enzyme-linked immunosorbent assay (ELISA) and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Each method has its advantages and ...disadvantages, and discrepancies are commonly observed between the two methods due to various factors including the ELISA antibody cross-reacting to different cyanotoxin congeners. However, reliable cyanotoxin monitoring methods and accurate interpretation of results are needed for water utilities to guide recreational water planning and drinking water treatment operations. In this study, we explored an innovative “effective concentration-equivalent concentration” (EC-EQ) approach to improve the interpretation of ELISA results and the comparison to LC-MS/MS results. The precision of ELISA results was first improved by reporting the sample ECs and EQs derived from their ELISA dose curves. Concentrations of each cyanotoxin as measured by LC-MS/MS were then combined with their respective ELISA cross-reactivities to calculate their theoretical ELISA responses. Finally, instead of comparing the results from the two methods directly, the equivalent concentration based on one single reference cyanotoxin was used for reporting and comparison. This integrated mass balance-based approach provides a more reliable interpretation of results by considering the reactivity differences between toxins as well as their mixture effects. This approach has been successfully applied to microcystin (one main group of cyanotoxins) standard mixtures and cyanobacterial bloom samples to interpret and compare their ELISA and LC-MS/MS detection results. The study provides guidance to utilities on how to obtain more accurate cyanotoxin monitoring results and better understand the discrepancy between the two methods.
Enzyme‐linked immunosorbent assay (ELISA) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were compared for analyzing microcystins in water. ELISA results of microcystin‐LR spiked into ...raw water samples were close to the spike concentrations, but method variability was ±25%. However, ELISA‐derived microcystin‐LA concentrations were two to three times higher than the spike concentrations obtained using the kit‐provided microcystin‐LR standards, indicating the need for variant‐appropriate ELISA standards. LC/MS/MS results agreed with spike concentrations for all variants in reagent water, but matrix suppression was observed in some raw waters. In bench‐scale studies, ozonated microcystins generated low‐level positive responses by ELISA and a protein phosphatase inhibition assay, even though microcystins were not detected by LC/MS/MS. These findings indicate that ELISA results—particularly in treated water—should be interpreted with caution because of the possibility of false‐positives, relatively high variability, and differential detection of some variants.
Sucralose is an artificial sweetener and an indicator of wastewater impacts in drinking water. N-nitrosodimethyamine (NDMA) is a disinfection by-product with wastewater-derived precursors. In two ...studies conducted in the United States and Canada, data showed watershed and region-specific relationships between sucralose occurrence, stream flow, and NDMA formation potential (FP). In addition, other water supplies have been identified with high NDMA FP that were low in sucralose, which appeared to be impacted by other sources of precursors in the watershed during high-flow events (e.g., runoff). In these studies, seasonal and climatic effects were explored where changes in stream flow (e.g., storm events, droughts) and sucralose and NDMA FP have been well correlated in many watersheds. These studies demonstrate the usefulness of measuring sucralose, including the determination of site-specific correlations with NDMA FP and temporal variability, as well as determining the likely percentage of treated wastewater in the influent of drinking water treatment plants.
An evaluation of existing analytical methods used to measure contaminants of emerging concern (CECs) was performed through an interlaboratory comparison involving 25 research and commercial ...laboratories. In total, 52 methods were used in the single-blind study to determine method accuracy and comparability for 22 target compounds, including pharmaceuticals, personal care products, and steroid hormones, all at ng/L levels in surface and drinking water. Method biases ranged from <10% to well over 100% in both matrixes, suggesting that while some methods are accurate, others can be considerably inaccurate. In addition, the number and degree of outliers identified suggest a high degree of variability may be present between methods currently in use. Three compounds, ciprofloxacin, 4-nonylphenol (NP), and 4-tert-octylphenol (OP), were especially difficult to measure accurately. While most compounds had overall false positive rates of ≤5%, bisphenol A, caffeine, NP, OP, and triclosan had false positive rates >15%. In addition, some methods reported false positives for 17β-estradiol and 17α-ethynylestradiol in unspiked drinking water and deionized water, respectively, at levels higher than published predicted no-effect concentrations for these compounds in the environment. False negative rates were also generally <5%; however, rates were higher for the steroid hormones and some of the more challenging compounds, such as ciprofloxacin. The elevated false positive/negative rates of some analytes emphasize the susceptibility of many current methods to blank contamination, misinterpretation of background interferences, and/or inappropriate setting of detection/quantification levels for analysis at low ng/L levels. The results of both comparisons were collectively assessed to identify parameters that resulted in the best overall method performance. Liquid chromatography–tandem mass spectrometry coupled with the calibration technique of isotope dilution were able to accurately quantify most compounds with an average bias of <10% for both matrixes. These findings suggest that this method of analysis is suitable at environmentally relevant levels for most of the compounds studied. This work underscores the need for robust, standardized analytical methods for CECs to improve data quality, increase comparability between studies, and help reduce false positive and false negative rates.
Variabilities in commercial cyanotoxin standards Jia, Ai; Prescott, Matthew D.; Guo, Yingbo C. ...
AWWA water science,
March/April 2023, 2023-03-00, 20230301, Volume:
5, Issue:
2
Journal Article
Peer reviewed
Cyanotoxin standards are commercially available from various suppliers. To investigate the potential impact of different sources on the comparability of cyanotoxin monitoring results among different ...methods and studies, this study evaluated the quality of 86 cyanotoxin standards from nine vendors via enzyme‐linked immunosorbent assay and liquid chromatography–tandem mass spectrometry. Substantial variabilities between vendors (up to 60%) and between lots (up to 98%) were observed for most of the standards. In addition, some of the microcystin standards had up to 11% of other microcystins as impurities. Compared to non‐certified analytical standards, certified standards showed much better agreement, with standards variations below 15% for all tested toxins. This study highlights the importance of developing and applying unified and certified standards for cyanotoxin analysis to improve the consistency and comparability of results. If different sources/lots of standards are used, they need to be cross‐checked to evaluate the potential impacts on results.
Two commonly used methods for cyanotoxin analysis are enzyme‐linked immunosorbent assay (ELISA) and liquid chromatography/tandem mass spectrometry (LC–MS/MS). Two rounds of interlaboratory ...comparisons of ELISA and LC–MS/MS analyses were conducted with 12 participating laboratories to evaluate method performances in various matrices, including cyanobacterial bloom and drinking water samples. Fifteen cyanotoxins, including 12 microcystin variants, nodularin, anatoxin‐a, and cylindrospermopsin were evaluated. The impact of sample matrices, preservatives, and quenching reagents was assessed, and no substantial effects were observed. Overall, comparable results were obtained among laboratories performing ELISA and LC–MS/MS analyses, respectively. ELISA results for fortified samples matched more closely with those from LC–MS/MS when microcystin cross‐reactivities were considered, providing data 26% closer to theoretical values on average. This study demonstrates that understanding the effect of cross‐reactivities when comparing ELISA and LC–MS/MS results and considering potential variabilities in commercial standards is important when interpreting data from these two methods.
Winter wheat is one of China's most important staple food crops, and its growth and productivity are influenced by climate. Given its importance, we investigated the influence of excess precipitation ...under recent climate change on winter wheat in east-central China during 1961–2017. Although annual precipitation in the studied region decreased slightly, it increased during the winter wheat flowering and maturity period (May to June). Concurrently, the number of late growing season sunshine hours decreased. Our results showed that about 44% of the years with excess precipitation and less than normal radiation (16 years) were associated with decreasing winter wheat yields. Furthermore, during most years, precipitation of 50% above normal resulted in large decreases in winter wheat production in Jiangsu and Anhui provinces, some of the wetter parts of the studied region. These results indicated that the grain yield variability of winter wheat was mainly influenced by excess precipitation in May, where precipitation could explain 70%–78% of yield variability in the wet parts. Moreover, excess precipitation can induce Fusarium head blight as well as wheat sprouting of pre-harvest, both affecting the grain quality of winter wheat. Projected increases in precipitation throughout the 21st century in the studied region, warrants further studies of how to maintain the winter wheat production in a changing climate.
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•In wetter regions of China, wheat yield could be induced to decrease when precipitation of May was more 50% than normal.•Excess rain can induce Fusarium head blight as well as wheat sprouting of pre-harvest, both affecting the grain quality.•The contribution rate of precipitation on yield variability was 70%-78% in wet region of China.
N‐nitrosodimethylamine (NDMA) and other nitrosamines have been reported in drinking and recycled water at low parts‐per‐trillion levels, both as contaminants and disinfection byproducts. Many of ...these nitrosamines cause cancer in animals and are probable human carcinogens. In this study, three existing analytical methods were expanded, refined, and validated for the analysis of NDMA and seven other nitrosamines in potable, waste, and recycled water. Method detection limits for a solid‐phase extraction (SPE) method and a cartridge SPE (CSPE) coupled with gas chromatography–tandem mass spectrometry were 0.7–0.8 ng/L for NDMA and 0.3–1.4 ng/L for other nitrosamines. The micro‐liquid–liquid extraction (MLLE) method that was developed used less volume and was effective at analyzing the different sample matrixes but yielded higher detection limits (2–4 ng/L). NDMA was the only nitrosamine detected in potable water; however N‐nitrosodiethylamine, N‐nitrosomorpholine, N‐nitrosopiperidine, N‐nitrosodi‐N‐butylamine, and N‐nitrosopyrrolidine were present in other matrixes tested. The SPE methods and the MLLE method have been submitted for inclusion in the next revision of Standard Methods for the Examination of Water and Wastewater, and the CSPE method will be submitted for approval in the future.
•Cepharanthine (CEP) was identified as an inhibitor of PRRSV infection in vitro.•CEP suppressed PRRSV infection and reduced PRRSV titer in Marc-145 cells.•CEP protected Marc-145 cells from severe ...cytopathic effects.•CEP inhibited Integrins/ILK/RACK1/PKCα/NF-κB signalling axis•CEP was suggested as a potential promising drug candidate for PRRS control.
Porcine Reproductive and Respiratory Syndrome (PRRS) is a devastating disease among the most notorious threats to the swine industry worldwide and is characterized by respiratory distress and reproductive failure. Highly evolving porcine reproductive and respiratory syndrome virus (PRRSV) strains with complicated genetic diversity make the current vaccination strategy far from cost-effective and thus urge identification of potent lead candidates to provide prevention and treatment approaches. From an in vitro small molecule screening with the TargetMol Natural Compound Library comprising 623 small molecules, cytopathic effect (CPE) observations and RT-qPCR analysis of viral ORF7 gene expression identified cepharanthine (CEP) to be one of the most protent inhibitors of PRRSV infection in Marc-145 cells. When compared with tilmicosin, which is one of the most commonly used antibiotics in swine industry to inhibit infections, CEP more prominently inhibited PRRSV infection represented by both RNA and protein levels, further reduced the TCID50 by 5.6 times, and thus more remarkably protected Marc-145 cells against PRRSV infection. Mechanistically, western blot analyses of the Marc-145 cells and the porcine alveolar macrophages (PAMs) with or without CEP treatment and PRRSV infection at various time points revealed that CEP can inhibit the expression of integrins β1 and β3, integrin-linked kinase (ILK), RACK1 and PKCα, leading to NF-κB suppression and consequent alleviation of PRRSV infection. Collectively, our small molecule screening identified cepharanthine as an inhibitor of PRRSV infection in vitro by suppressing Integrins/ILK/RACK1/PKCα/NF-κB signalling axis, which may enlighten the deeper understanding of the molecular pathogenesis of PRRSV infection and more importantly, suggested CEP as a potential promising drug for PRRS control in veterinary clinics.
Epithelial-mesenchymal transition (EMT) is a biological process that is associated with cancer metastasis and invasion. In cancer, EMT promotes cell motility, invasion and distant metastasis. ...Interleukin (IL)-8 is highly expressed in tumors and may induce EMT. The IL-8/IL-8R axis has a vital role in EMT in carcinoma, which is regulated by several signaling pathways, including the transforming growth factor β-spleen associated tyrosine kinase/Src-AKT/extracellular signal-regulated kinase, p38/Jun N-terminal kinase-activating transcription factor-2, phosphoinositide 3-kinase/AKT, nuclear factor-κB and Wnt signaling pathways. Blocking the IL-8/IL-8R signaling pathway may be a novel strategy to reduce metastasis and improve patient survival rates. This review will cover IL-8-IL-8R signaling pathway in tumor epithelial-mesenchymal transition.
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