A method for the measurement of 24 hydroxylated polycyclic aromatic hydrocarbon metabolites (OH-PAHs) in urine has been developed. The method is based on enzymatic deconjugation, automated liquid-liquid extraction, and gas chromatography/isotope dilution high-resolution mass spectrometry after derivatization of the OH-PAHs to the trimethylsilylated derivatives. The metabolites included in the current method are formed from eight different parent compounds. The limits of detection were below 7 pg/mL when using a sample size of 2 mL of urine, except for 1- and 2-naphthols (18 and 12 pg/mL, respectively). The enzymatic deconjugation efficiency, verified by deconjugation of urine samples spiked with alpha-naphthyl beta-d-glucuronide sodium salt (1-NAP-GLU) and pyrene-1-sulfate potassium salt (1-PYR-SULF), was determined to be 97% for 1-NAP-GLU conjugate and 84% for 1-PYR-SULF. The overall coefficients of variance for six batches of quality control samples (n = 42), was 2.9-11%. Mean method recoveries of the 13C-labeled internal standards were 66-72%, except for 13C6-1-naphthol (46%). The throughput of this method has been determined to be 40 samples per day per analyst. This method is currently applied to epidemiological studies, such as the National Exposure and Nutrition Examination Survey (NHANES), to measure human exposure to PAHs.
Thirty-three bituminous coal samples were utilized to test the application of laser-induced breakdown spectroscopy technique for coal elemental concentration measurement in the air. The heterogeneity of the samples and the pyrolysis or combustion of coal during the laser-sample interaction processes were analyzed to be the main reason for large fluctuation of detected spectra and low calibration quality. Compared with the generally applied normalization with the whole spectral area, normalization with segmental spectral area was found to largely improve the measurement precision and accuracy. The concentrations of major element C in coal were determined by a novel partial least squares (PLS) model based on dominant factor. Dominant C concentration information was taken from the carbon characteristic line intensity since it contains the most-related information, even if not accurately. This dominant factor model was further improved by inducting non-linear relation by partially modeling the inter-element interference effect. The residuals were further corrected by PLS with the full spectrum information. With the physical-principle-based dominant factor to calculate the main quantitative information and to partially explicitly include the non-linear relation, the proposed PLS model avoids the overuse of unrelated noise to some extent and becomes more robust over a wider C concentration range. Results show that RMSEP in the proposed PLS model decreased to 4.47% from 5.52% for the conventional PLS with full spectrum input, while R(2) remained as high as 0.999, and RMSEC&P was reduced from 3.60% to 2.92%, showing the overall improvement of the proposed PLS model.
Twenty-nine marine bacterial strains were isolated from the sponge Hymeniacidon perleve at Nanji island, and antimicrobial screening showed that eight strains inhibited the growth of terrestrial microorganisms. The strain NJ6-3-1 with wide antimicrobial spectrum was identified as Pseudoalteromonas piscicida based on its 16S rRNA sequence analysis. The major antimicrobial metabolite, isolated through bioassay-guide fractionation of TLC bioautography overlay assay, was identified as norharman (a beta-carboline alkaloid) by EI-MS and NMR.
CE-based techniques with DAD and detection ESI-TOF-MS have been developed for the analysis of seven protoberberine alkaloids and one aporphinoid alkaloid in Huanglian (Rhizoma coptidis), a well-known traditional Chinese herbal medicine. One aqueous BGE and one nonaqueous BGE were developed for CE-DAD and CE-MS analyses, and the CE-ESI-TOF-MS conditions including nebulizer gas pressure, the sheath-liquid composition, its flow rate, etc. were optimized. Eight main alkaloids in R. coptidis could be separated with baseline resolution by CE-DAD with these two different BGEs, and identified by TOF-MS analysis. Moreover, three major alkaloids (berberine, palmatine, and jatrorrhizine) could be quantified accurately by CE-DAD and CE-MS with the BGE system consisting of 50:50 v/v water and ACN containing 50 mM ammonium acetate at pH 6.8. Both techniques provided similar LODs and could be applied with confidence within similar linear dynamic range. However, reproducibility and speed of analysis were better using CE-DAD. When the CE technique was compared with the RP-HPLC method, the CE-DAD and CE-MS methods provided greater efficiency and faster analysis speed, i.e., achieving baseline resolution for all the eight main basic compounds in less than 14 min. The CE method, as a viable alternative to HPLC, is suitable for use as a routine procedure for the rapid identification and quantification of basic compounds in herbal or natural product applications.
BackgroundHost RNA-dependent RNA polymerases (RDRs) 1 and 6 contribute to antiviral RNA silencing in plants. RDR6 is constitutively expressed and was previously shown to limit invasion of Nicotiana benthamiana meristem tissue by potato virus X and thereby inhibit disease development. RDR1 is inducible by salicylic acid (SA) and several other phytohormones. But although it contributes to basal resistance to tobacco mosaic virus (TMV) it is dispensable for SA-induced resistance in inoculated leaves. The laboratory accession of N. benthamiana is a natural rdr1 mutant and highly susceptible to TMV. However, TMV-induced symptoms are ameliorated in transgenic plants expressing Medicago truncatula RDR1.ResultsIn MtRDR1-transgenic N. benthamiana plants the spread of TMV expressing the green fluorescent protein (TMV.GFP) into upper, non-inoculated, leaves was not inhibited. However, in these plants exclusion of TMV.GFP from the apical meristem and adjacent stem tissue was greater than in control plants and this exclusion effect was enhanced by SA. TMV normally kills N. benthamiana plants but although MtRDR1-transgenic plants initially displayed virus-induced necrosis they subsequently recovered. Recovery from disease was markedly enhanced by SA treatment in MtRDR1-transgenic plants whereas in control plants SA delayed but did not prevent systemic necrosis and death. Following SA treatment of MtRDR1-transgenic plants, extractable RDR enzyme activity was increased and Western blot analysis of RDR extracts revealed a band cross-reacting with an antibody raised against MtRDR1. Expression of MtRDR1 in the transgenic N. benthamiana plants was driven by a constitutive 35S promoter derived from cauliflower mosaic virus, confirmed to be non-responsive to SA. This suggests that the effects of SA on MtRDR1 are exerted at a post-transcriptional level.ConclusionsMtRDR1 inhibits severe symptom development by limiting spread of virus into the growing tips of infected plants. Thus, RDR1 may act in a similar fashion to RDR6. MtRDR1 and SA acted additively to further promote recovery from disease symptoms in MtRDR1-transgenic plants. Thus it is possible that SA promotes MtRDR1 activity and/or stability through post-transcriptional effects.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0705-8) contains supplementary material, which is available to authorized users.
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