Chemistry-specific responses due to rice-microbe interactions in the rhizosphere to counteract mefenacet stress.

The widespread use of herbicides has raised considerable concern with regard to their harmful consequences on plant growth, crop yield and the soil ecological environment. It has been well documented that colonization of rhizobacteria in the plant root system has a positive effect on activation of plant defenses to protect the plant from damage. Using the platform of high-throughput analysis with tandem mass spectrometry and Illumina sequencing, we identified the specific activated rhizobacteria, the key growth stimulating substances and the metabolic pathways involved in seedling stage tolerance to mefenacet stress in rice. The relative abundance of beneficial rhizospheremicrobes such as Acidobacteria and Firmicutes increased with mefenacet treatment, indicating that the rhizosphere recruited some beneficial microbes to resist mefenacet stress. Mefenacet treatment induced alterations in several interlinked metabolic pathways, many of which were related to activation of defense response signaling, especially the indole-3-pyruvate pathway. Indole-3-acetaldehyde and indole-3-ethanol from this pathway may act as flexible storage pools for indole-3-acetic acid (IAA). Our findings also suggest that a significant increase of IAA produced by the enrichment of beneficial rhizospheremicrobes, for example genus Bacillus, alleviated the dwarfing phenomenon observed in hydroponic medium following mefenacet exposure, which may be a key signaling molecule primarily for phytostimulation and phytotolerance in microbe-plant interactions.

Simultaneous determination of three organotin pesticides in fruits and vegetables by high-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: The presence of organotins in the environment affects food safety, making it important to monitor the levels of organotin pesticides (OTPs) in fruit and vegetable samples. METHODS: In the present study, a simple and low cost method for simultaneous determination of three OTPs (azocyclotin, fenbutatin oxide and triphenyltin hydroxide) in vegetable and fruit samples was developed and validated, based on solid-phase extraction and liquid chromatography/tandem mass spectrometry. RESULTS: Extraction with acetonitrile containing 0.2% formic acid positively affected the recoveries of the three OTPs. Moreover, the simultaneous purification of the three OTPs was the most efficient using mixed-mode cation-exchange cartridges and 5.0% ammonium hydroxide in methanol as eluent, and, in this case, mild matrix effects (-9.3% to 21.6%) were obtained for the three OTPs monitored. The developed method reached limits of quantification of 1 µg kg-1 , and linearity was satisfactory, with correlation coefficients >0.995. A fortification study showed that when spiked at 1.0-50.0 µg kg-1 the mean trueness values were from 72.3 to 110.0% in all matrices (three vegetables and three fruits). The intra-day precision was <14.1%, and the inter-day precision (n = 11) was <18.2%. CONCLUSIONS: The proposed method was successfully applied to the simultaneous analysis of three OTPs in vegetables and fruits.

Dissipation of two field-incurred pesticides and three degradation products in rice (Oryza sativa L.) from harvest to dining table.

BACKGROUND: High levels of harmful pesticide residues in rice can cause undesirable side effects and are a source of great concern to consumers. Reduction of pesticide residues to provide rice security has thus became an urgent problem. RESULTS: In this study, the effects of commercial and home processing on removal of chlorpyrifos and carbosulfan residues from rice, and the formation of metabolites during processing, were studied. The results showed that 3,5,6-trichloro-2-pyridinol (0.87 mg kg-1 ) and carbofuran (0.43 mg kg-1 ) were the predominant components detected in paddy rice. All detected residues were primarily deposited on the rice hull and bran. Washing twice followed by high-pressure cooking was able to further decrease residues in polished rice with the processing factor value <0.25. Following application of pesticides at the recommended rate and twice the recommended rate, with a preharvest interval of 28 days, changes in residues from harvest to dining table based on efficient processing techniques were investigated. The final residues dropped to below maximum residue levels after washing twice followed by high-pressure cooking. CONCLUSION: This simple cooking process thus reduces the risk of dietary exposure, and it is recommended that it is adopted by all consumers. © 2019 Society of Chemical Industry.

Correction to: Analysis of bronopol (2-bromo-2-nitropropan-1,3-diol) residues in rice (Oryza sativa L.) by SPE using Bond Elut Plexa and liquid chromatography-tandem mass spectrometry.

The authors would like to call the reader's attention to the fact that unfortunately during a recent cross-check of the experimental record, they found that the positions of intercept and slope were reversed in Table 1 in the original manuscript. The authors apologize for the mistake.

Analysis of bronopol (2-bromo-2-nitropropan-1, 3-diol) residues in rice (Oryza sativa L.) by SPE using Bond Elut Plexa and liquid chromatography-tandem mass spectrometry.

A novel method has been developed for the direct, sensitive, and rapid detection of bronopol in rice using a simple solid-phase extraction (SPE) procedure followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), with electrospray ionization (ESI). Bronopol was stable under acidic conditions, and an acidic environment was thus needed before sample loading to ensure the stability of bronopol. Rice extracts containing bronopol were pretreated using a hydrophilic-lipophilic balanced (Bond Elut Plexa) cartridge to reduce the matrix effect. An XDB-C18 column (150 mm × 2.1 mm, 3.5 µm) was used for chromatographic separations, with a mobile phase comprising methanol and aqueous ammonium formate (5 mM). The linearity of the method was satisfactory with regression coefficient (R 2) = 0.9992. The limit of quantification was 3.3 µg kg-1. Three spiked levels (25, 125 and 625 µg kg-1) were used to determine the recovery of bronopol, which was found to be 73.3-96.7%, with relative standard deviations (RSD) in the range 1.2-7.9%. The RSD for intra-day precision (n = 7) was 7.6% and the RSD for inter-day precision (n = 15) was 8.3%. The newly developed analytical method was successfully used to quantify bronopol in rice samples.

Thyroid endocrine disruption of acetochlor on zebrafish (Danio rerio) larvae.

The herbicide acetochlor is widely used and detected in the environment and biota, and has been suspected to disrupt the thyroid endocrine system, but underlying mechanisms have not yet been clarified. In the present study, zebrafish larvae (7 days post-fertilization) were exposed to a series concentration of acetochlor (0, 1, 3, 10, 30, 100 and 300 µg l(-1) ) within a 14-day window until 21 days post-fertilization. Thyroid hormones and mRNA expression profiles of genes involved in the hypothalamic-pituitary-thyroid (HPT) axis were analyzed. Exposure to the positive control, 3,5,3'-triiodothyronine (T3 ), altered the mRNA expression, suggesting that the HPT axis in the critical window of zebrafish responded to chemical exposure and could be used to evaluate the effects of chemicals on the thyroid endocrine system. The mRNA expressions of genes involved in thyroid hormone synthesis (tshß, slc5a5 and tpo) were upregulated significantly with acetochlor treatment, which might be responsible for the increased thyroxine concentrations. The downregulation of genes related to thyroid hormone metabolism (dio1 and ugt1ab) and transport (ttr) in zebrafish larvae exposed to acetochlor might further explain the increased thyroxine levels and decreased T3 levels. The mRNA expression of the thyroid hormone receptor (trα) was also upregulated upon acetochlor exposure. Results suggested that acetochlor altered mRNA expression of the HPT axis-related genes and changed the whole body thyroid hormone levels in zebrafish larvae. It demonstrated that acetochlor could cause endocrine disruption of the thyroid system by simulating the biological activity of T3 . Copyright © 2015 John Wiley & Sons, Ltd.

Full-length transcriptomics study of Ustiloxins-induced hepatocyte injury.

Ustiloxins is a mycotoxin produced by the metabolism of Rice false smut. Studies have shown that Ustiloxins may be toxic to animals, but there is still a lack of toxicological evidence. The liver, as the main organ for the biotransformation of foreign chemicals, may be the direct target organ of Ustiloxins toxicity. In this study, we found that cell viability decreased in a dose- and time-dependent manner when BNL CL.2 cells were treated with different concentrations of Ustiloxins (0, 5, 10, 20, 30, 40, 60, 80, 100, 150 and 200 µg/mL) for 24 and 48 h. In addition, scanning electron microscope observation showed that the cell membrane of the experimental group was damaged, with the appearance of apoptotic bodies. Moreover, the ROS and GSH levels were significantly increased in cells exposed to Ustiloxins. We analyzed the key action targets of Ustiloxins on hepatocyte injury using full-length transcriptomics. A total of 1099 differentially expressed genes were screened, of which 473 genes were up-regulated, and 626 genes were down-regulated. Besides, we also found that the expression of MCM7 and CDC45 in BNL CL.2 cells treated with Ustiloxins decreased, and the expression of CCl-2, CYP1b1, CYP4f13, and GSTM1 increased according to qRT-PCR. Ustiloxins might change CYP450 and GST-related genes, affect DNA replication and cell cycle, and lead to oxidative stress and liver cell injury.

Phyllosphere microbiome induces host metabolic defence against rice false-smut disease.

Mutualistic interactions between host plants and their microbiota have the potential to provide disease resistance. Most research has focused on the rhizosphere, but it is unclear how the microbiome associated with the aerial surface of plants protects against infection. Here we identify a metabolic defence underlying the mutualistic interaction between the panicle and the resident microbiota in rice to defend against a globally prevalent phytopathogen, Ustilaginoidea virens, which causes false-smut disease. Analysis of the 16S ribosomal RNA gene and internal transcribed spacer sequencing data identified keystone microbial taxa enriched in the disease-suppressive panicle, in particular Lactobacillus spp. and Aspergillus spp. Integration of these data with primary metabolism profiling, host genome editing and microbial isolate transplantation experiments revealed that plants with these taxa could resist U. virens infection in a host branched-chain amino acid (BCAA)-dependent manner. Leucine, a predominant BCAA, suppressed U. virens pathogenicity by inducing apoptosis-like cell death through H2O2 overproduction. Additionally, preliminary field experiments showed that leucine could be used in combination with chemical fungicides with a 50% reduction in dose but similar efficacy to higher fungicide concentrations. These findings may facilitate protection of crops from panicle diseases prevalent at a global scale.

[Determination of three oryzanols in rice by mixed-mode solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry].

Rice is a major dietary staple in many communities owing to its high nutritional value and characteristic aroma. Oryzanol, a mixture of ferulic acid esters of triterpene alcohols and phytosterols, is a major group of phytochemicals found in rice. 24-Methylenecycloartanyl ferulate (24MCA-FA), cycloartenyl ferulate (CA-FA), and campestanyl ferulate (Camp-FA) have been identified as the primary components of oryzanol. At present, for the quantification of oryzanol in rice and rice products, UV spectroscopy or high performance liquid chromatography (HPLC) is widely employed. However, these methods cannot differentiate individual oryzanols, resulting in higher measured values. To extract oryzanol, methods including liquid-liquid extraction, acidulation extraction, and direct solvent extraction have been typically employed, as they do not require specific extraction instrumentation. However, there has been no systematic study on the direct solvent extraction and purification conditions of oryzanol in rice. In this study, a rapid and accurate analytical method based on HPLC-MS/MS and mixed-mode anion exchange (MAX) solid-phase extraction was established to determine the content of three oryzanols (24MCA-FA, CA-FA, and Camp-FA) in rice. The MS parameters, such as the collision energy of three ion pairs of each oryzanol, were optimized. Further, the chromatographic separation conditions and response intensities of the oryzanols in different mobile phases were compared. The effects of different pretreatment conditions on the extraction efficiency of the three oryzanols in rice samples and different purification conditions on their recovery were investigated. Combined with the external standard method, the three oryzanols in rice were successfully quantified. The results showed that the baseline separation and highest response for the three oryzanols were achieved using the Agilent Eclipse XDB-C8 chromatographic column (150 mm×2.1 mm, 3.5 µm) when methanol∶ acetonitrile in a 1∶1 ratio (v/v) and an aqueous solution of 5 mmol/L ammonium acetate were used as the mobile phases for gradient elution. The extraction rate of the three oryzanols was highest when using 2.5 g of the sample, adding 20 mL of methanol, soaking for 12 h, ultrasonicating at a temperature of 40 ℃ for 20 min, and centrifuging the extracted solutions at 4500 r/min for 10 min. The samples were purified by MAX, and the sample matrix effect was found to be lesser than 1.6%-10.8%. Under the optimum conditions, the calibration curves of the three oryzanols showed good linearity (correlation coefficients r2≥0.9983) within their respective linear ranges. The limits of detection were in the range of 0.5-1.0 µg/L, and limits of quantification were in the range of 2.0-3.5 µg/L. Accuracy and precision experiments were performed on rice samples spiked at three levels (2, 5, and 10 times the background concentration), with three replicates. The average recoveries of the three oryzanols ranged from 86.1% to 110.6%, and the relative standard deviations (RSDs) were between 0.9% and 3.2%. The method showed good performance when applied to the analysis of real samples. In conclusion, the developed method can determine the content of the three oryzanols in rice quickly and accurately, and can be used for the subsequent measurement of oryzanol compounds in rice.

[Simultaneous determination of three allergic proteins in rice and products by high performance liquid chromatography-tandem mass spectrometry combined with stable isotope-labeled peptides].

Rice is an important cereal that is consumed as both an energy and protein source by a large proportion of the population worldwide. However, clinical studies have found that rice grains are responsible for cases of severe asthma, eczema, and atopic dermatitis in some adult patients. Several allergenic proteins have been identified and biochemically and immunochemically characterized from rice grains. These include α-amylase/trypsin inhibitors, glyoxalase Ⅰ, and α-globulin. In this study, we proposed an approach for the simultaneous quantification of three allergenic proteins in rice and its products, based on a stable isotope-labeled signature peptide standard and liquid chromatography-tandem mass spectrometry. Samples of rice and products were extracted by a salt solution, hydrolyzed by Lys-C and Trypsin, and purified by C18-SD. The linear ion trap-high resolution mass spectrometry (LTQ-Orbitrap) and Protein Discovery software were used to acquire and identify allergenic proteins in rice samples. In present study, three proteins including seed allergenic protein RAG2, glyoxalase Ⅰ, and 19 kDa globulin were identified. To establish a stable quantitative detection method, the signature peptides selected from the identified enzymatic hydrolysis peptides must have greater abundance and higher specificity as characteristic peptides. Three corresponding signature peptides in rice were screened based on the principles of previous study, and were validated through comparisons of the basic local alignment search tool (BLAST) with the NCBI and UniProt databases. The three signature peptides were successively eluted by liquid chromatography and separated on a Poroshell column. They were then detected by positive electrospray ionization (ESI+) in multiple reaction monitoring mode and quantified by an isotope dilution method. To achieve an improvement in the detection sensitivity and specificity, mass spectrometry parameters, such as the collision energy of three ion pairs of each peptide, were optimized. Three recombinant allergenic proteins and the winged stable isotope-labeled signature peptide standard were synthesized. These were then used to compare the effects of different enzymatic conditions, including hydrolysis solvents containing sodium dodecyl sulfate (SDS) with different contents, as well as the enzymes and their amounts, on the digestion efficiency. The data showed that the digestion efficiency of the three proteins could be improved to 65.7%-97.3% when 1 g/L of the SDS-containing hydrolysis solvent, and the combined digestion strategy of Lys-C and Trypsin, were adopted in the enzymatic process. These results indicate the following inferences: a small amount of SDS (1 g/L) in the enzymatic hydrolysis system is beneficial to complete protein denaturation, a Lys-C and Trypsin combined digestion strategy can complement the shortcomings of the two enzymes and improve the digestion efficiency, and the recoveries of the three proteins was not significantly increased by increasing the amount of enzyme when the ratio of protein to enzyme reached more than 20∶1. The method displayed good linearity in the range of 1-200 nmol/L with the correlation coefficients greater than 0.9972. The limits of detection and limits of quantification of the three proteins were 3 mg/kg and 10 mg/kg, respectively. The average recoveries of the three proteins spiked at three levels in different matrices ranging between 80.6%-103.7%, with the intra-day and inter-day precision less than 11.5%. Due to its high stability, excellent sensitivity, and simple operation, this method presents a wide range of application prospects in the analysis of the three allergenic proteins in different rice and rice food products.

Modulation of steroid metabolism and xenobiotic biotransformation responses in zebrafish (Danio rerio) exposed to triadimefon.

The widely used fungicide triadimefon (TDF) has been detected in aquatic environments, and appears to disrupt steroid homeostasis; however, the toxic effects on fish reproduction triggered by TDF via the key receptor signaling pathways remain largely unknown. The present study showed that TDF (0.069, 0.138, 0.690 mg/L) exposure not only caused disordered germ cell maturation, but also decreased spawned egg production. In order to better understand this reproductive inhibition, we investigated the effects of TDF based on quantitative PCR, Western blot and mass spectrometry methodology in zebrafish. Due to the preferential accumulation of TDF in the liver, a general pattern of up-regulation of genes involved in biotransformation pathway was observed. A significant increase in abcb4 expression appeared to be responsible for TDF excretion. TDF-induced receptors (AhR2 and PXR) changed many genes involved in steroid metabolism, and subsequent disruptions in steroid homeostasis, which might be the key biological pathway in TDF reproductive toxicity. However, due to the different metabolic demands, the transcript profiles involved in steroid metabolism in zebrafish exhibited a sex-specific expression pattern. For example, the increase in gene expression of ahr2 was accompanied by a reduction in the rate of E2 biosynthesis resulting from the diminished cyp19a1a expression, and in turn led to down-regulation of esr1 and vtg1 in the liver, supporting the anti-estrogenic effect of TDF in male fish. In contrast, the increase in E2 production was accompanied by an increase in Esr1 protein expression caused by TDF and paralleled the increase in ahrr1 expression, suggesting that TDF may induce estrogenic activity through AhR-ER interactions in females. In addition, over-induction of cyp3a65 activity mediated through pxr, which helped to accelerate the transformation from TDF to triadimenol in the liver, appeared to elevate T metabolite rate in females. The down-regulation of fshß transcript in males further suggested that TDF might adversely affect normal gametogenesis and induce reproductive toxicity.

Absolute Quantification of Allergen Glb33 in Rice by Liquid Chromatography-Mass Spectrometry using Two Isotope-Labeled Standard Peptides.

Allergen Glb33 is an important allergen in rice that can cause allergic reactions such as asthma and atopic dermatitis. However, knowledge of the content in rice is sparse. In the present work, an absolute protein quantification method was established for allergen Glb33 in rice samples using liquid chromatography-tandem mass spectrometry. After extraction of allergen Glb33 from rice grains using salt solution, the isotope-labeled peptide internal standard was added to the extract, followed by enzymatic digestion with trypsin. The signature peptide and its isotope-labeled analogue from the tryptic hydrolysates of allergen Glb33 and the internal standard were detected by liquid chromatography-tandem mass spectrometry. The quantitative bias caused by tryptic efficiency and matrix effect was corrected by using two isotope-labeled standard peptides. The method exhibited good linearity in the range of 1-200 nM, with coefficients of determination of R2 > 0.998. A high sensitivity was observed, with a limit of quantification of 0.97 nM. Mean recoveries obtained from different rice matrices ranged from 82.7%-98.1% with precision <8.5% in intraday trials ( n = 6), while mean recoveries were from 75.1%-107.4% with precision <14.6% in interday trials ( n = 14). The developed method was successfully applied to the analysis of allergen Glb33 in 24 different rice cultivars.

Analysis of four sulfonylurea herbicides in cereals using modified Quick, Easy, Cheap, Effective, Rugged, and Safe sample preparation method coupled with liquid chromatography-tandem mass spectrometry.

A modified Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) sample preparation method, coupled with liquid chromatography-electrospray ionization tandem mass spectrometry, has been developed for the simultaneous analysis of four commonly used sulfonylurea herbicides, ethoxysulfuron, halosulfuron-methyl, mesosulfuron-methyl and orthosulfamuron, in rice, maize, wheat and soybean. Adsorption of the analytes onto the primary-secondary amine used in the clean-up step was avoided by using 1% formic acid in acetonitrile as the extraction solvent to maintain the acidic herbicides in a non-ionized state. Trueness studies were carried out at three levels (2.5, 25 and 250 µg/kg for ethoxysulfuron and 5, 50 and 500 µg/kg for the other three analytes). Promising trueness (70.2%-119.8%) was achieved for all herbicides in all matrices after clean-up, with relative standard deviations (RSDr) < 18.6%. Satisfactory matrix effects (-19.7% to 14.8%) were also obtained. Good linearity of the calibration curves was achieved, with determination coefficients (r) ≥ 0.9956, when the concentration of ethoxysulfuron was in the range 0.5-50 µg/L and that of the other three analytes was in the range 1.0-500 µg/L. The RSDwR for within-laboratory reproducibility was 5.7%. The validated method was successfully used to analyze real samples.

[Two-dimensional liquid chromatography separation and high resolution mass spectrometry analysis for proteome of rice leaves based on different extraction methods].

A two-dimensional liquid chromatography (2D LC) system was developed to separate proteins from rice leaves, which was extracted by phenol method, followed by the analysis with linear trap quadrupole orbitrap mass spectrometry (LTQ/Orbitrap MS). After proteins were extracted with phenol method, the enzymolytic peptides were separated by offline two-dimensional RP-RP system and detected by LTQ/Orbitrap MS, yielding 2712 proteins. Liquid chromatography separation system (1D LC and 2D LC) and protein extraction methods (phenol method, sodium dodecyl sulfate method (SDS method) and trichloroacetic acid/acetone method (TCA/acetone method)) were compared. Proteins identified by 2D LC were 2712, 2415 and 1914 with the above three extraction methods, respectively. The proteins were 2.7-fold, 2.5-fold and 1.9-fold the number of proteins identified by 1D LC respectively. And in terms of 2D LC, the proteins identified by phenol method were 297 and 798 more than SDS method and TCA/acetone method, respectively. Some proteins with extreme properties, such as very acidic or basic protein and high relative molecular mass proteins, were only identified in phenol method. Furthermore, proteins, which were extracted by different extraction methods and separated by 2D LC, were classified according to biological functions. It was found that protein functions by the three extraction methods were complementary. However, phenol method had the most variety of functions. The method provides technological support for rice proteomics and reference for research techniques of other crop proteomics.

[Determination of pyriminobac-methyl and bispyribac-sodium residues in rice by liquid chromatography-tandem mass spectrometry based on QuEChERS].

A method was developed for the determination of pyriminobac-methyl and bispyribac-sodium residues in rice by liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with modified QuEChERS. The samples were extracted with acidified acetonitrile, and then purified by octadecylsilane bonded silica (C18) adsorbents. The analytes were separated on a ZORBAX SB C18 column through a gradient elution using 0.1% (v/v) aqueous formic acid aqueous containing 5 mmol/L ammonium acetate and acetonitrile as mobile phases. Positive electrospray ionization (ESI+) was used. Qualitative work was performed using selected dynamic multiple reaction monitoring (dynamic MRM) mode. Quantization was performed using external standard method. The results showed good linearities of pyriminobac-methyl and bispyribac-sodium with correlation coefficients (r2) not less than 0.996. The limits of detection (LODs) of the method were 0.8 µ g/kg for pyriminobac-methyl, and 3 µ g/kg for bispyribac-sodium. The mean spiked recoveries of pyriminobac-methyl and bispyribac-sodium at three spiked levels were 76.6%-85.6% and 73.0%-86.7%, respectively, and the relative standard deviations (RSDs) of pyriminobac-methyl and bispyribac-sodium were 0.9%-3.4% and 1.2%-5.5%, respectively. This method is simple, rapid, sensitive, and suitable for the simultaneous determination of pyriminobac-methyl and bispyribac-sodium in rice.

Quantitation of glutathione S-transferases in rice (Oryza sativa L.) roots exposed to cadmium by liquid chromatography-tandem mass spectrometry using isotope-labeled wing peptides as an internal standard.

BACKGROUND: Plant glutathione S-transferases (GSTs, EC 2.5.1.18) are multifunctional enzymes involved in heavy metal cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione to heavy metals. Previous studies demonstrated that individual rice GSTs were differentially induced by heavy metal exposure at the mRNA transcript level. However, little information is available concerning changes in protein concentration of rice GSTs under heavy metal stress. Because the correlation between changes in protein concentration and gene expression under abiotic stress is poor, direct determination of rice GSTs protein concentrations during cadmium (Cd) exposure is a more effective and reliable approach to explore possible mechanisms of rice Cd translocation and accumulation. RESULTS: This study established an optimized and advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics assay for quantification of OsGSTF14 and OsGSTU6 proteins in Cd-stressed rice roots. The tryptic signature peptides were chosen as surrogate analytes and winged peptides containing the isotope-labeled signature peptides were used as the internal standards. The signature peptides exhibited good linearity in the range of 0.6-60 and 0.3-30 nM, respectively. The limit of detection and limit of quantification were 4.5 and 14.5 µg/g for OsGSTF14, respectively, and 2.1 and 7.0 µg/g for OsGSTU6. The spiking recoveries rates at low, medium and high levels were in the range of 72.5-93.4%, with intra- and inter-day precisions of 5.5-9.1 and 4.2-10.2%, respectively. CONCLUSIONS: The assay successfully quantified the temporal and dose responses of OsGSTF14 and OsGSTU6 proteins in Cd-stressed rice roots, with good accuracy, precision and high-throughput. This assay will have significant application in developing quantification methods of other proteins in Cd-stressed rice, which may provide more insight into the mechanisms of Cd translocation and accumulation in rice.

Direct Determination of Six Cytokinin Nucleotide Monophosphates in Coconut Flesh by Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry.

Coconut contains many uncharacterized cytokinins that have important physiological effects in plants and humans. In this work, a method based on liquid chromatography-tandem mass spectrometry was developed for identification and quantification of six cytokinin nucleotide monophosphates in coconut flesh. Excellent separation was achieved using a low-coverage C18 bonded-phase column with an acidic mobile phase, which greatly improved the retention of target compounds. To enable high-throughput analysis, a single-step solid-phase extraction using mixed-mode anion-exchange cartridges was employed for sample preparation. This proved to be an effective method to minimize matrix effects and ensure high selectivity. The limits of detection varied from 0.06 to 0.3 ng/mL, and the limits of quantification ranged from 0.2 to 1.0 ng/mL. The linearity was statistically verified over 2 orders of magnitude, giving a coefficient of determination (R2) greater than 0.9981. The mean recoveries were from 81 to 108%; the intraday precision (n = 6) was less than 11%; and the interday precision (n = 11) was within 14%. The developed method was applied to the determination of cytokinin nucleotide monophosphates in coconut flesh samples, and four of them were successfully identified and quantified. The results showed that trans-zeatin riboside-5'-monophosphate was the dominant cytokinin, with a concentration of 2.7-34.2 ng/g, followed by N6-isopentenyladenosine-5'-monophosphate (≤12.9 ng/g), while the concentrations of cis-zeatin riboside-5'-monophosphate and dihydrozeatin riboside-5'-monophosphate were less than 2.2 and 4.9 ng/g, respectively.

Effects of azocyclotin on gene transcription and steroid metabolome of hypothalamic-pituitary-gonad axis, and their consequences on reproduction in zebrafish (Danio rerio).

The widely used organotins have the potential to disrupt the endocrine system, but little is known of underlying mechanisms of azocyclotin toxicity in fish. The objective of the present study was to investigate the impact of azocyclotin on reproduction in zebrafish. Adult zebrafish were exposed to 0.09 and 0.45µg/L azocyclotin for 21days, and effects on steroid hormones and mRNA expression of the genes belonging to the hypothalamic-pituitary-gonad (HPG) axis were investigated. Mass spectrometry methodology was developed to profile steroids within the metabolome of the gonads. They were disrupted as a result of azocyclotin exposure. Alterations in the expression of key genes associated with reproductive endocrine pathways in the pituitary (lhß), gonad (cyp19a1a, cyp17a1 and 17ß-hsd3), and liver (vtg1, vtg2, cyp1a1, comt, ugt1a and gstp1) were correlated with significant reductions in estrogen in both sexes and increased testosterone in females. Azocyclotin-induced down-regulation of cyp19a1a in males suggested a reduction in the rate of estrogen biosynthesis, while up-regulation of hepatic cyp1a1 and comt in both sexes suggested an increase in estrogen biotransformation and clearance. Azocyclotin also induced change in the expression of 17ß-hsd3, suggesting increased bioavailability of 11-ketotestosterone (11-KT) in the blood. Furthermore, the down-regulation of lhß expression in the brains of azocyclotin-exposed fish was associated with inhibition of oocyte maturation in females and retarded spermatogenesis in males. As a histological finding, retarded development of the ovaries was found to be an important cause for decreased fecundity, with down-regulation of vtg suspected to be a likely underlying mechanism. Additionally, relatively high concentrations of azocyclotin in the gonads may have directly caused toxicity, thereby impairing gametogenesis and reproduction. Embryonic or larval abnormalities occurred in the F1 generation along with accumulated burdens of azocyclotin in F1 eggs, following parental exposure. Overall, our results indicate that exposure to azocyclotin can impair reproduction in fish, and induce toxicity related abnormalities in non-exposed offspring.

Analysis of ustiloxins in rice using polymer cation exchange cleanup followed by liquid chromatography-tandem mass spectrometry.

Ustiloxins are cyclopeptide mycotoxins produced by the pathogenic fungus Ustilaginoidea virens of rice false smut. Quantification of ustiloxins is essential to assess the food safety of rice infected by rice false smut disease. This paper describes a sensitive method for the simultaneous quantification of ustiloxins A, B, C, D and F in rice grains using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Since notable matrix enhancement effects (21%-78%) occurred for all of the target analytes (except for ustiloxin A), several solid phase extraction materials were tested for their ability to retain ustiloxins from aqueous solutions prior to the LC-MS/MS analysis, including C18 sorbents, polymer anion exchange sorbents resin (PAX), and polymer cation exchange resin (PCX). The PCX resin was adopted due to its higher extraction capability and selectivity for all targets compared to others, and in this case, almost no matrix effects (-5% to 8%) were observed for all of the ustiloxins monitored. The developed method reached limits of quantification of 0.2-2ngg-1, and linearity was statistically verified over two orders of magnitude with regression coefficients (R2)>0.991. The mean recoveries were from 85% to 109%, and the inter-day precisions (n=11) were less than 16%, with intra-day precisions (n=6) within 12%. Analysis of samples showed that ustiloxin A was the dominant species, with the content ranging from 5.5 to 273.8ngg-1, followed by ustiloxin B (≤88.7ngg-1), while concentrations of ustiloxins C, D and F were slightly lower (≤43.2ngg-1). To our knowledge, this is the first report on the determination and analysis of five ustiloxins simultaneously in a single analysis.

Fulvestrant-3 Boronic Acid (ZB716): An Orally Bioavailable Selective Estrogen Receptor Downregulator (SERD).

Orally bioavailable SERDs may offer greater systemic drug exposure, improved clinical efficacy, and more durable treatment outcome for patients with ER-positive endocrine-resistant breast cancer. We report the design and synthesis of a boronic acid modified fulvestrant (5, ZB716), which binds to ERα competitively (IC50 = 4.1 nM) and effectively downregulates ERα in both tamoxifen-sensitive and tamoxifen-resistant breast cancer cells. Furthermore, It has superior oral bioavailability (AUC = 2547.1 ng·h/mL) in mice, indicating its promising clinical utility as an oral SERD.