Residue behavior and efficacy of benzothiazole in grains under different fumigation conditions.

BACKGROUND: Benzothiazole is a potential grain fumigant for Tribolium castaneum. However, its safety profile and suitable fumigation conditions remain unknown. We therefore investigated the insecticidal efficacy, accumulation and dissipation of benzothiazole in grains (wheat, corn and rice) under different temperatures. RESULTS: We established a universal detection method (modified QuEChERS coupled with GC-MS/MS) of benzothiazole residues in three grains, which provided high linearity (R2 > 0.999), sensitivity (limits of detection = 0.001 mg/kg, limits of quantification = 0.002-0.005 mg/kg), accuracy (recoveries = 88.18-118.75%) and precision (relative standard deviations < 4.78%). The insecticidal efficacy order of benzothiazole was 30 ≥ 10 > 20 °C and corn > wheat > rice. Temperature positively affected the accumulation/dissipation rate of benzothiazole. Rice was the most easily accumulated and dissipated grain for benzothiazole residues, while corn accumulated benzothiazole more than wheat but less than rice, with dissipation slower than wheat and rice. CONCLUSION: Our results provide important references for the application of benzothiazole and other fumigants. © 2023 Society of Chemical Industry.

Nonenantioselective environmental behavior of a chiral antiviral pesticide dufulin in aerobic soils.

Dufulin is a promising chiral antiviral agent, but little is known about its fate in soils. In this study, the fate of dufulin enantiomers in aerobic soils was investigated using radioisotope tracing techniques. The result of the four-compartment model showed no significant differences in dissipation, generation of bound residues (BR) and mineralization between S-dufulin and R-dufulin during incubation. Dufulin dissipated most quickly in cinnamon soils, followed by fluvo-aquic and black soils and the half-lives of dufulin in these soils obtained by the modified model were 4.92-5.23, 32.39-33.32 and 60.80-61.34 d, respectively. After 120 d incubation, the percentage of radioactivity of BR increased to 18.2-38.4 % in the three soils. Dufulin formed most bound residues in the black soil, least in the cinnamon soil, and BRs rapidly formed in the cinnamon soil during the early culture period. In these three soils, the cumulative mineralization of 14CO2 ranged from 25.0 to 26.7 %, 42.1 to 43.4 % and 33.8 to 34.4 %, respectively, which indicated that the environmental fate of dufulin was primarily influenced by soil characteristics. The study of microbial community structure revealed that the phyla Ascomycota, Proteobacteria and genus Mortierella might be related to the degradation of dufulin. These findings provide a reference for assessing the environmental impact and ecological safety of dufulin application.

Determination of benzothiazoles, benzotriazoles and benzenesulfonamides in seafood using quick, easy, cheap, effective, rugged and safe extraction followed by gas chromatography - tandem mass spectrometry: Method development and risk assessment.

The common use of benzothiazoles, benzotriazoles and benzenesulfonamides has led to widespread ubiquity in several environmental matrices. Their occurrence in edible fish could represent an additional exposure route for the population. The present study aims to develop a method for the simultaneous determination of these three compound families in seafood samples. Based on QuEChERS extraction, different salt combinations and clean-up strategies have been evaluated to achieve the highest recoveries while reducing the matrix effect in low and high lipidic content species. The best results were obtained with the original method salts and the lipid-selective push-through clean-up, which combined with gas chromatography-tandem mass spectrometry led to recoveries between 50 and 112% with negligible matrix effects and method detection limits between 0.15-9.50 ng g-1 dw. The application of the method to commercially available samples confirmed the presence of BTs as well as BSAs, with the latter being determined in seafood for the first time. Exposure and risk assessment calculations indicated a minor risk for the population when consuming fish.

Prenatal exposure to benzotriazoles and benzothiazoles and child neurodevelopment: A longitudinal study.

Benzotriazoles (BTRs) and benzothiazoles (BTHs) are emerging benzo-heterocyclic compounds that may induce neurotoxicity. However, the effect of prenatal exposure to BTs (BTRs and BTHs) on child neurodevelopment has not been elucidated. We aimed to explore the associations between maternal urinary concentrations of BTs in single or in mixture with child neurodevelopment at the age of two. This study recruited 513 mother-child pairs based on a birth cohort from 2014 to 2015 in Wuhan. Maternal urinary concentrations of eight BTs (four BTRs and four BTHs) in the first, second, and third trimesters were measured. The mental development index (MDI) and psychomotor development index (PDI) of children, as two indexes of neurodevelopment, were assessed at two years old by the Bayley Scales. In the analyses of single BTs, prenatal average tolyltriazole (TTR) exposure level was associated with decreased boys' MDI scores (ß = -2.84, 95 % CI: -5.11, -0.57) and prenatal average 1-H-benzotriazole (1-H-BTR) exposure level was associated with decreased boys' PDI scores (ß = -1.44, 95 % CI: -2.70, -0.17), respectively. Maternal urinary concentrations of benzothiazole (BTH) in the 1st trimester (ß = -1.79, 95 % CI: -2.78, -0.80), 2nd trimester (ß = -1.14, 95 % CI: -2.19, -0.09), and the prenatal average exposure (ß = -2.15, 95 % CI: -3.69, -0.61) were also negatively associated with boys' PDI scores. However, no significantly negative association was observed among girls. In the further mixture analysis, the quantile g-computation model found a significant negative association between prenatal average concentrations of BTs in mixture and boys' PDI scores [ß = -4.80 (95 % CI: -9.08, -0.52)], and BTH weighted the highest in the negative association. As far as we know, this is the first research to estimate the effect of prenatal exposure to BTs on child neurodevelopment. The findings showed that prenatal exposure to BTs was negatively associated with neurodevelopment among boys, suggesting that the associations may be modified by infant sex.

Understanding the metabolism of the novel plant antiviral agent dufulin by different positional 14C labeling in cherry radishes.

Dufulin is a new type of plant antiviral agent. However, its metabolism in plants, which is very important for environmental risk assessment, is still unclear. In this study, we used 14C markers at different positions and high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (HPLC-QTOF-MS) to qualitatively and quantitatively analyze dufulin metabolites in cherry radish. By combining ion pairs with unique abundance ratios, we clarified the metabolite structures, inferred the metabolic pathway of dufulin, and clarified the criteria for residues. The extractable residue of dufulin from cherry radish stem and leaf tissues was above 98 % and that in the succulent root was above 87 %. In the stem and leaf tissues and succulent root, dufulin underwent both phase I and phase II metabolism, and four metabolites were produced, including a conjugate of glucose malonate and hydroxylated dufulin, which was confirmed by comparison with a standard. However, the proportions and concentrations of the four metabolites did not meet the residue criteria, so only the dufulin precursor compound was included as a residue. This study provides reliable data for evaluating the impacts of dufulin on the environment and human health and for objectively examining the safety of dufulin.

Aquatic environmental fates and risks of benzotriazoles, benzothiazoles, and p-phenylenediamines in a catchment providing water to a megacity of China.

Wearing of vehicle parts could release many chemical additives into the environment, such as benzotriazoles (BTRs), benzothiazoles (BTHs), and p-phenylenediamines (PPDs), which are potentially toxic to wildlife and humans. This study investigated the occurrence, source, and risks of BTRs, BTHs, and PPDs in a source catchment providing water to Guangzhou, a megacity in South China, covering groundwater, surface water, and stormwater. The results showed that BTRs and BTHs were predominant in surface water and groundwater. Unexpectedly, the BTR and BTH concentrations were lower in surface water than groundwater in a third of the paired samples. For the first time, 6PPD-quinone, a toxic ozonation product of N-(1,3-dimethylbutyl)-N'-phenyl-1,4-phenylenediamine (6PPD), was extensively detected in source waters. Stormwater decreased the BTR concentrations but increased the 6PPD-quinone concentrations in surface water owing to their affiliation to suspended particles. From natural to urban segments of Liuxi river, a downstream increasing trend in BTR and BTH concentrations was observed, confirming that they are indicative of urban anthropogenic activities. Strong correlations between industrial activities and BTR or BTH concentrations in surface water indicated that industrial activities were their main sources. Six compounds were prioritized as potentially persistent, mobile, and toxic (PMT) chemicals, combing our monitoring results and REACH criterion. This study improves our understanding of the environmental fates and risks of water-soluble tire-wear chemicals, which provides important information for chemical management, and indicates attention should be paid to the risk posed by 6PPD-quinone in the source water.

Occurrence and removal of benzotriazole and benzothiazole in drinking water treatment plants.

The occurrence and removal of four benzotriazoles (BTRs) and five benzothiazoles (BTHs) in drinking water treatment plants (DWTPs) and bottled water were investigated. The mean total BTR and BTH concentrations were 390 and 117 ng/L in raw water, 51.2 and 66.5 ng/L in treated water, and 0.758 and 48.4 ng/L in bottled water, respectively. Different distribution patterns were observed according to the water type, with the dominant BTR being 1H-BTR (mean: 57.8%) in raw water and a predominance of BTH in bottled water (mean: 84.6%). In the DWTPs, the mean removal of BTRs (90.9%) was better than that of BTHs (29.3%). The BTRs were efficiently removed in DWTPs, and in particular during adsorption processes. 5Cl-BTR had a high removal efficiency (75.7%) in the adsorption processes, followed by 5M-BTR (70.0%), 5,6-di-MeBTR (58.4%), and 1H-BTR (50.1%). By contrast, BTHs were not efficiently removed in DWTPs, although relatively high removal efficiencies were achieved with an ozonation process (43.1%) compared to other treatment processes. In treated drinking and bottled water, the hazard quotients (HQs) of the representative BTRs and BTHs were acceptable (defined as HQ < 1), with a safety margin of 2-5 orders of magnitude.

Air monitoring of tire-derived chemicals in global megacities using passive samplers.

Pollution from vehicle tires has received world-wide research attention due to its ubiquity and toxicity. In this study, we measured various tire-derived contaminants semi-quantitatively in archived extracts of passive air samplers deployed in 18 major cities that comprise the Global Atmospheric Passive Sampling (GAPS) Network (GAPS-Megacities). Analysis was done on archived samples, which represent one-time weighted passive air samples from each of the 18 monitoring sites. The target analytes included cyclic amines, benzotriazoles, benzothiazoles, and p-phenylenediamine (PPD) derivatives. Of the analyzed tire-derived contaminants, diphenylguanidine was the most frequently detected analyte across the globe, with estimated concentrations ranging from 45.0 pg/m3 in Beijing, China to 199 pg/m3 in Kolkata, India. The estimated concentrations of 6PPD-quinone and total benzothiazoles (including benzothiazole, 2-methylthio-benzothiazole, 2-methyl-benzothiazole, 2-hydroxy-benzothiazole) peaked in the Latin American and the Caribbean region at 1 pg/m3 and 100 pg/m3, respectively. In addition, other known tire-derived compounds, such as hexa(methoxymethyl)melamine, phenylguanidine, and various transformation products of 6PPD, were also monitored and characterized semi-quantitatively or qualitatively. This study presents some of the earliest data on airborne concentrations of chemicals associated with tire-wear and shows that passive sampling is a viable techniquefor monitoring airborne tire-wear contamination. Due to the presence of many tire-derived contaminants in urban air across the globe as highlighted by this study, there is a need to determine the associated exposure and toxicity of these chemicals to humans.

Development and validation of a simultaneous method for the analysis of benzothiazoles and organic ultraviolet filters in various environmental matrices by GC-MS/MS.

The presence of benzothiazoles (BTHs) and organic ultraviolet filters (UV filters) in aquatic ecosystems has emerged as a significant environmental issue, requiring urgent and efficient determination methods. A new, rapid, and sensitive determination method using gas chromatography triple quadrupole mass spectrometer (GC-MS/MS) was developed for the simultaneous extraction and analysis of 10 commonly used BTHs and 10 organic UV filters in surface water, wastewater, sediment, and sludge. For aqueous samples, solid-phase extraction (SPE) method was employed with optimizing of SPE cartridge type, pH, and elution solvent. For solid samples, ultrasonic extraction-solid-phase extraction purification (UE-SPE) and pressurized liquid extraction (PLE) methods were compared. And extraction conditions for ultrasonic extraction method (extraction solvents and extraction times) and PLE method (extraction temperatures and extraction cycles) were optimized. The limits of quantification for the 20 target compounds in surface water and wastewater were 0.01-2.12 ng/L and 0.05-6.14 ng/L, while those for sediment and sludge with UE-SPE method were 0.04-5.88 ng/g and 0.22-6.61 ng/g, respectively. Among the 20 target compounds, the recoveries ranged from 70 to 130% were obtained for 16, 15, 15, and 15 analytes in the matrix-spiked samples of surface water, wastewater, sediment, and sludge with three levels, respectively. And the precision was also acceptable with relative standard deviation (RSD) below 20% for all analytes. The developed methods were applied for the determination and quantification of target compounds in surface water, sediment, wastewater, and sludge samples collected from two wastewater treatment plants (WWTPs) and the Pearl River in Guangzhou, China. BTHs were frequently detected in surface water and wastewater, while UV filters were mainly found in sediment and sludge. Benzotriazole (BT) and 2-hydroxybenzothiazole (2-OH-BTH) were the two major BTHs in influent wastewater and surface water, respectively, with concentrations up to 966 and 189 ng/L. As for sediment and sludge, 2-(2'-hydroxy-5'-octylphenyl)-benzotriazole (UV-329) was a predominant chemical, detected at concentrations of 111 and 151 ng/g, respectively.

Associations of benzotriazoles and benzothiazoles with estrogens and androgens among pregnant women: A cohort study with repeated measurements.

People are extensively exposed to benzotriazoles (BTRs) and benzothiazoles (BTHs) derivatives, which are environmental pollutants that may possess endocrine-disrupting potential; however, no epidemiological evidence is available on the associations of BTRs and BTHs with estrogens and androgens. This study aimed at investigating the associations of BTRs and BTHs with estrogens and androgens among pregnant women. Based on a prospective cohort study, we included 459 pregnant women who donated a complete serial of urine samples at each trimester and had repeated measurements of four BTRs, four BTHs, three estrogens (estrone, 17ß-estradiol, and estrio), and two androgens (dehydroepiandrosterone and testosterone) in the urine samples. Associations of repeatedly measured BTRs and BTHs with maternal urinary estrogens and androgens were analyzed, and the cross-sectional associations were also analyzed. Tolyltriazole (TTR) (≥59.3%) and benzothiazole (BTH) (≥93.5%) had the highest detection rate among the BTRs and BTHs, respectively. Repeated measurement analysis and cross-sectional analysis consistently found the target BTRs and BTHs were positively associated with 17ß-estradiol, estriol, and testosterone, while the trend of the associations with estrone and dehydroepiandrosterone was inconsistent. Among the positive associations with 17ß-estradiol, estriol, and testosterone, the percent of change in estriol associated with TTR was the most prominent [28.5% (95% confidential interval: 24.2%, 32.9%) for each doubling in TTR]. The significant associations with estrone, estriol, testosterone, and dehydroepiandrosterone were stronger among pregnant women who gave birth to a boy than those who gave birth to a girl. These findings add epidemiological evidence on the endocrine-disrupting potential of BTRs and BTHs and highlight the importance of focusing on the health outcomes of BTRs and BTHs related to disturbed estrogens and androgens. Future studies are needed to validate these findings and explore the underlying mechanisms.

Suspect and non-target screening of chemicals in clothing textiles by reversed-phase liquid chromatography/hybrid quadrupole-Orbitrap mass spectrometry.

The global manufacturing of clothing is usually composed of multistep processes, which include a large number of chemicals. However, there is generally no information regarding the chemical content remaining in the finished clothes. Clothes in close and prolonged skin contact may thus be a significant source of daily human exposure to hazardous compounds depending on their ability to migrate from the textiles and be absorbed by the skin. In the present study, twenty-four imported garments on the Swedish market were investigated with respect to their content of organic compounds, using a screening workflow. Reversed-phase liquid chromatography coupled to electrospray ionization/high-resolution mass spectrometry was used for both suspect and non-target screening. The most frequently detected compound was benzothiazole followed by quinoline. Nitroanilines with suspected mutagenic and possible skin sensitization properties, and quinoline, a carcinogenic compound, were among the compounds occurring at the highest concentrations. In some garments, the level of quinoline was estimated to be close to or higher than 50,000 ng/g, the limit set by the REACH regulation. Other detected compounds were acridine, benzotriazoles, benzothiazoles, phthalates, nitrophenols, and organophosphates. Several of the identified compounds have logP and molecular weight values enabling skin uptake. This pilot study indicates which chemicals and compound classes should be prioritized for future quantitative surveys and control of the chemical content in clothing as well as research on skin transfer, skin absorption, and systemic exposure. The results also show that the current control and prevention from chemicals in imported garments on the Swedish market is insufficient.

An innovative green protocol for the quantification of benzothiazoles, benzotriazoles and benzosulfonamides in PM10 using microwave-assisted extraction coupled with solid-phase microextraction gas chromatography tandem-mass spectrometry.

Benzothiazoles (BTHs), benzotriazoles (BTRs), and benzenesulfonamides (BSAs) are chemicals used in several industrial and household applications. Despite these compounds are emerging pollutants, there is still a lack of information about their presence in outdoor air samples. In this paper, we developed a new method for the quantification of BTHs, BTRs, and BSAs in airborne particulate matter (PM10). The extraction of fourteen analytes from PM10 was accomplished by microwave-assisted extraction (MAE) using an environmentally friendly mixture of water and ethanol. SPME was used to analyze the target compounds from the MAE extract by gas chromatography-tandem mass spectrometry (SPME-GC-MS/MS), eliminating additional sample clean-up steps. The best working conditions for MAE and SPME were examined multivariately by experimental design techniques. The target compounds were quantified in selected reaction monitoring acquisition mode. The proposed method was carefully validated, and the achieved results were satisfactory in terms of linearity, lower limit of quantification (picograms per cubic meter), intra- and inter-day accuracy (81-118% and 82-114%, respectively), and precision (repeatability and reproducibility in the range 2.3-17% and 7.4-19%, respectively). The application in a real monitoring campaign showed that the developed protocol is a valuable and eco-friendly alternative to the methods proposed so far.

Competitive and noncompetitive fluorescence polarization immunoassays for the detection of benzothiostrobin using FITC-labeled dendrimer-like peptides.

Peptides obtained from phage display libraries are valuable reagents for small-molecule immunoassays. However, their application in fluorescence polarization immunoassays (FPIAs) is limited by phage particles. Here, monomer, dendrimer-like dimer, tetramer peptidomimetic and anti-immunocomplex tracers were designed and synthesized using lysine as special scaffolds and spacers to develop competitive and noncompetitive FPIAs for benzothiostrobin. The affinity between tracers and monoclonal antibodies or immunocomplexes increased with the tracer valence. A higher signal-to-noise ratio and sensitivity could be generated in the FPIAs based on tetramer tracers. The sensitivities of competitive (50% inhibitory concentration) and noncompetitive (50% saturation concentration) FPIAs were 19.71 ± 4.65 and 40.43 ± 2.73 ng mL-1, respectively. The spiked recoveries were 78.3%-105.2% with relative standard deviations (RSDs) of 0.7%-15.4% for the competitive FPIA, while 78.7%-115.3% with RSDs of 0.7%-12.5% for the noncompetitive FPIA. The amounts of benzothiostrobin in rice detected by the FPIAs were consistent with those detected by high performance liquid chromatography.

Solvent-free mechanochemical preparation of graphene oxide-Fe3 O4 and its application in magnetic dispersive solid-phase extraction of illegal dyes in food samples.

A simple, green, and efficient mechanochemical approach was developed herein to prepare tunable magnetic graphene oxide nanoparticles. The obtained nanoparticles were successfully used as adsorbents in a magnetic dispersive solid-phase extraction method to extract three cationic dyes (i.e., thioflavine T, auramine-O, and basic orange 2) found in food samples. Our proposed approach also utilized high-performance liquid chromatography with ultraviolet detection. Several key variables affecting the extraction recovery were investigated. These included the sample pH, amount of extractant, extraction time, sample volume, elution solvent type and volume, and the stability and reusability of the magnetic graphene oxide nanoparticles. Under optimized conditions, the calibration curve was linear at a concentration range of 0.005-1.0 µg/mL with a correlation coefficient of 0.9992-0.9996. Moreover, the limits of detection were determined at 0.97-1.35 µg/mL. The extraction mechanism was investigated via ultraviolet-visible spectrophotometry and zeta-potential analyses. The developed method was used to analyze the above-mentioned cationic dyes in bean products and yellow fish samples. Notably, satisfactory spiked recoveries ranging from 90.7 to 104.9% were achieved.

Epigallocatechin-3-Gallate Promotes the in vitro Maturation and Embryo Development Following IVF of Porcine Oocytes.

PURPOSE: Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols and exerts protective effects because of its strong antioxidant properties. As far as we know, there is still a lack of systematic research on the effects of EGCG on the in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes. The present study aimed to determine the effects of EGCG on the IVM and IVF of porcine oocytes. METHODS: Porcine oocytes were treated with different concentrations of EGCG (5, 10 and 20 µM), and the cumulus cell expansion, oocyte maturation rate, reactive oxygen species (ROS), glutathione (GSH) and malondialdehyde (MDA) levels, total antioxidant capacity were determined. The mRNA expression levels of oxidative stress- and apoptosis-associated genes were determined by quantitative real-time PCR. The cleavage rate and blastocyst rate of oocytes after 10 µM EGCG treatment during IVM and IVF were also evaluated. RESULTS: EGCG at 5, 10 and 20 µM significantly promoted cumulus cell expansion, and EGCG at 10 µM increased the oocyte maturation rate. EGCG (10 µM) treatment reduced the ROS and MDA levels, while increased the antioxidant capacity and GSH concentrations in the mature oocytes. The qRT-PCR results showed that EGCG treatment up-regulated the mRNA expression of catalase, glutathione peroxidase and superoxide dismutase in the mature oocytes. In addition, EGCG treatment also decreased the mRNA expression levels of Bax and caspase-3 and increased the Bcl-2 mRNA expression level in the mature oocytes. In addition, the cleavage rate and blastocyst rate of oocytes treated with 10 µM EGCG during IVM and IVF were significantly higher than those of the control group. CONCLUSION: Our results suggest that EGCG promotes the in vitro maturation and embryo development following IVF of porcine oocytes. The protective effects of EGCG on the oocytes may be associated with its antioxidant and anti-apoptosis properties.

Analysis of Erythroxylum coca Leaves by Imaging Mass Spectrometry (MALDI-FT-ICR IMS).

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) can determine the chemical identity and spatial distribution of several molecules in a single analysis, conserving its natural histology. However, there are no specific studies on the spatial distribution of alkaloids in Erythroxylum coca leaves by MALDI IMS, preserving the histology of the monitored compounds. Therefore, in this work, positive-ion mode MALDI Fourier-transform ion cyclotron resonance imaging mass spectrometry (MALDI(+)FT-ICR IMS) was applied to identify and analyze the distribution of alkaloids on the surface of coca leaves, evaluating the ionization efficiency of three matrices (α-cyano-4-hydroxycinnamic acid (CHCA), 2-mercaptobenzothiazole (MBT), and 2,5-dihydroxybenzoic acid (DHB)). The last was chosen as the best matrix in this study, and it was studied in five concentrations (0.5, 1.0, 2.0, 4.0, and 8.0 mg·mL-1), where 2 mg·mL-1 was the most efficient. The washing of coca leaves with the organic solvents (acetonitrile, methanol, toluene, and dichloromethane) tested did not improve the performance of the ionization process. Finally, a tissue section, 50 µm thick, was used to study the inner part of the leaf tissue, where alkaloids and flavonoid molecules were detected.

An improved staining method of cell cycle analysis with Sybr Green I for fungi: Cryptococcus neoformans and Saccharomyces cerevisiae.

Cryptococcus neoformans is a pathogenic fungus which causes millions of deaths and infections, especially threatening immunocompromised individuals. During the development of new drugs, the ubiquitination has been found to play an important role in the regulation of the virulence and cell cycle of this fungus. Based on this mechanism, ubiquitination-related mutant strains exhibiting cell cycle arrest have been established for drug development for the fungus. However, flow cytometry detection of the cell cycle in fungi is generally difficult because the thick cell wall and capsule of fungi generally contribute to a nonspecific signal of cytometry. In this study, an improved method, derived from Saccharomyces cerevisiae assays, is developed to specifically stain C. neoformans, in whose cell cycle the G1 and G2 peaks are separated enough to be allowed for cell cycle analysis. As a result, the improved method facilitates the detection of the alterations in the cell cycle of C. neoformans with a mutation that results in cell cycle arrest, which distinctly delays the cell division of C. neoformans. Thus, the improved method reported here provides detailed technical information regarding assays on C. neoformans and, more importantly, offers a solution for assessing the cell cycle in other fungi in the future. Abbreviation: PI: propidium iodide.

Dimeric G-Quadruplex: An Effective Nucleic Acid Scaffold for Lighting Up Thioflavin T.

As a recently identified higher-order quadruplex (G4) structure, the G4 dimer possesses unique structure and biological functions. In this work, we found accidentally that two tandem PW17 (one known G4-forming DNA) sequences can fold into a stable G4 dimer, and the G4 dimer can enhance dramatically the fluorescence intensity of thioflavin T (ThT). The G4 dimer/ThT fluorescence intensity is about ninefold that of the corresponding G4 monomer/ThT. Meanwhile, compared with the common G4/ThT system, G4 dimer/ThT exhibited more stable fluorescence emission in the media with various concentrations of Na+ and K+. On the basis of these findings, G4 dimer/ThT was used as a fluorescence indicator to construct one arched DNA probe for label-free detection of DNA. By incorporating a G4 dimer sequence in amplified products, we further designed one rolling circle amplification-based biosensing strategy to show the utility of this G4 dimer/ThT fluorescence indicator. This study demonstrates that dimeric G4 is an effective nucleic acid scaffold for lighting up ThT, showing promising applications in a label-free bioassay.

A Heparin based dual ratiometric sensor for Thrombin.

Thrombin is an important enzyme that plays a pivotal role in the blood clotting pathways. An imbalance in the activity of this enzyme is clinically known to be associated with various diseases, such as thrombosis, inflammation, atherosclerosis, and haemophilia, suggesting the need to devise sensors for Thrombin detection. However, the majority of the fluorescence-based Thrombin assays rely on fluorescence labelling assays or Thrombin specific recognition biomolecules, such as, aptamers or antibody which requires sophisticated techniques and makes it very expensive. Herein, we report a simple, selective, sensitive and label-free fluorescence detection scheme for Thrombin which is based on the interaction between Thrombin and a fluorescent complex of Heparin with a molecular rotor dye, Thioflavin-T. The detection scheme exploits selective interaction between cationic Thrombin and anionic Heparin to modulate the monomer-aggregate equilibrium of the Thioflavin-T-Heparin system. Importantly, the present system offers a ratiometric response that has the ability for robust quantification of Thrombin concentration even in complex medium. The involvement of all commercially available components is a crucial advantage of this detection scheme. Further, the detection scheme also shows reasonable response in diluted serum matrix.

Accurate and sensitive detection of Catechol-O-methyltransferase activity by liquid chromatography with fluorescence detection.

Catechol-O-methyltransferase (COMT) is a major drug metabolizing enzyme in humans. COMT expression is directedly associated with various mental diseases and cancers due to its essential role in catalyzing metabolic inactivation of endogenous catecholamines and catechol estrogens. However, a practical method to precisely measure COMT activities in biological samples is lacking. In the current study, we established a liquid chromatography-fluorescence detection (LC-FD) method based on fluorometric detection of the methylated product of 3-BTD, a fluorescent probe for COMT, to sensitively quantify COMT activities in human erythrocytes and cell homogenates. Assay validation of the established LC-FD based method was conducted for selectivity and sensitivity, range of linearity, precision and accuracy, recovery, biological matrices effect and stability. The limit of quantification for 3-BTMD (the methylated product of 3-BTD by COMT) of this method was 0.0083 nM, which is nearly 10-fold lower than that for previously published methods. The method was precise with intra- and inter-day relative standard deviation (RSD) lower than 5%. In addition, this method showed an excellent anti-interference ability with no effects of the endogenous substances on the fluorometric detection of 3-BTMD. The practical use of this method was established by its successful application for the measurement of COMT activities in individual human erythrocytes (n = 13), and in cell homogenates generated from four different human cell lines. Our results suggest that this method will be of great value in accurately determining the native activity of COMT in biological samples, which is beneficial for a complete understand of the role of COMT both in physiological and pathological conditions.