Liquid Chromatography-tandem Mass Spectrometry Analysis Research Articles

Avoiding Interferences in Advance: Cyclodextrin Polymers to Enhance Selectivity in Extraction of Organic Micropollutants for Carbon Isotope Analysis

Compound-specific isotope analysis (CSIA) of organic water contaminants can provide important information about their sources and fate in the environment. Analyte enrichment from water remains nonetheless a critical yet inevitable step before measurement. Commercially available solid-phase extraction (SPE) sorbents are inherently nonselective leading to co-extraction of concurrent dissolved organic matter (DOM) and in turn to analytical interferences, especially for low-occurring contaminants. Here, we (i) increased extraction selectivity by synthesizing cyclodextrin polymers (α-, β-, γ-CDP) as SPE sorbents, (ii) assessed their applicability to carbon isotope analysis for a selection of pesticides, and (iii) compared them with commonly used commercial sorbents. Extraction with β-CDP significantly reduced backgrounds in gas chromatography-isotope ratio mass spectrometry (GC-IRMS) and enhanced sensitivity by a factor of 7.5, which was further confirmed by lower carbon-normalized CDOM/Canalyte ratios in corresponding extracts as derived from dissolved organic carbon (DOC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Gibbs free energies of adsorption demonstrated weak competition between DOM and analyte on the three CDPs. No isotopic fractionation (Δδ13C within ± 0.3‰) was observed for the investigated pesticides after using β-CDP as an SPE sorbent covering a range of concentrations (5-500 μg L-1), flow velocities (5-40 cm min-1), and sorbent regeneration (up to six times). The present study highlights the benefit of selecting innovative extraction sorbents to avoid interferences in advance. This strategy in combination with existing cleanup approaches offers new prospects for CSIA at field concentrations of tens to hundreds of nanograms per liter.

Reduction in gefitinib resistance mediated by Yi-Fei San-Jie pill in non-small cell lung cancer through regulation of tyrosine metabolism, cell cycle, and the MET/EGFR signaling pathway

Ethnopharmacological relevanceThe Chinese herbal prescription Yi-Fei San-Jie pill (YFSJ) has been used for adjuvant treatment in patients with lung cancer for a long time. Aim of the studyReports have indicated that the combination of gefitinib (Gef) with YFSJ inhibits the proliferation of EGFR-TKI-resistant cell lines by enhancing cellular apoptosis and autophagy in non-small cell lung cancer (NSCLC). However, the molecular mechanisms underlying the effect of YFSJ on EGFR-TKI resistance and related metabolic pathways remain to be explored. Materials and methodsIn our report, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), metabolomics, network pharmacology, bioinformatics, and biological analysis methods were used to investigate the mechanism. ResultsThe UPLC-MS/MS data identified 42 active compounds of YFSJ extracts. YFSJ extracts can enhance the antitumor efficacy of Gef without hepatic and renal toxicity in vivo. The analysis of the metabolomics pathway enrichment revealed that YFSJ mainly affected the tyrosine metabolism pathway in rat models. Moreover, YFSJ has been shown to reverse Gef resistance and improve the effects of Gef on the cellular viability, migration capacity, and cell cycle arrest of NSCLC cell lines with EGFR mutations. The results of network pharmacology and molecular docking analyses revealed that tyrosine metabolism-related active compounds of YFSJ affect EGFR-TKIs resistance in NSCLC by targeting cell cycle and the MET/EGFR signaling pathway; these findings were validated by western blotting and immunohistochemistry. ConclusionsYFSJ inhibits NSCLC by inducing cell cycle arrest in the G1/S phase to suppress tumor growth, cell viability, and cell migration through synergistic effects with Gef via the tyrosine metabolic pathway and the EGFR/MET signaling pathway. To summarize, the findings of the current study indicate that YFSJ is a prospective complementary treatment for Gef-resistant NSCLC.

Effect of Jiawei Tongqiao Huoxue decoction in basilar artery dolichoectasia mice through yes-associated protein/transcriptional co-activator with PDZ-binding motif pathway

Ethnopharmacological relevanceThe Jiawei Tongqiao Huoxue decoction (JTHD), composed of Acorus calamus var. angustatus Besser, Paeonia lactiflora Pall., Conioselinum anthriscoides ‘Chuanxiong’, Prunus persica (L.) Batsch, Ziziphus jujuba Mill., Carthamus tinctorius L., Pueraria montana var. lobata (Willd.) Maesen & S.M.Almeida ex Sanjappa & Predeep, Zingiber officinale Roscoe, Leiurus quinquestriatus, and Moschus berezovskii Flerov, was developed based on Tongqiao Huoxue decoction in Wang Qingren's “Yilin Gaicuo” in the Qing Dynasty. It has the effect of improving not only the blood flow velocity of vertebral and basilar arteries but also the blood flow parameters and wall shear stress. Especially in recent years, the potential efficacy of traditional Chinese medicine (TCM) for the treatment of basilar artery dolichoectasia (BAD) has attracted great attention as there are still no specific remedies for this disease. However, its molecular mechanism has not been elucidated. To identify the potential mechanisms of JTHD will help to intervene BAD and provide a reference for its clinical application. Aim of the studyThis study aims to establish a mouse model of BAD and explore the mechanism of JTHD regulating yes-associated protein/transcriptional co-activator with PDZ-binding motif (YAP/TAZ) pathway for attenuating BAD mice development. Materials and methodsSixty post-modeling C57/BL6 female mice were randomly divided into sham-operated, model, atorvastatin calcium tablet, low-dose JTHD, and high-dose JTHD groups. After 14 days of modeling, the pharmacological intervention was given for 2 months. Then, JTHD was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS). ELISA was utilized to detect the changes in vascular endothelial growth factor (VEGF) and lipoprotein a (Lp-a) in serum. EVG staining was conducted to observe the pathological changes of blood vessels. TUNEL method was employed to detect the apoptosis rate of vascular smooth muscle cells (VSMCs). Micro-CT and ImagePro Plus software were used to observe and calculate the tortuosity index, lengthening index, percentage increase in vessel diameter, and tortuosity of the basilar artery vessels in mice. Western blot analysis was performed to detect the expression levels of YAP and TAZ proteins in the vascular tissues of mice. ResultsMany effective compounds such as choline, tryptophan, and leucine with anti-inflammation and vascular remodeling were identified in the Chinese medicine formula by LC-MS analysis. The serum levels of VEGF in the model mice decreased significantly while the levels of Lp-a increased obviously compared with those in the sham-operated group. The intima-media of the basilar artery wall showed severe disruption of the internal elastic layer, atrophy of the muscular layer, and hyaline changes of the connective tissue. Apoptosis of VSMCs added. Dilatation, elongation, and tortuosity of the basilar artery became notable, and tortuosity index, lengthening index, percentage increase in vessel diameter, and bending angle remarkably improved. The expression levels of YAP and TAZ protein in blood vessels elevated conspicuously (P < 0.05, P < 0.01). JTHD group markedly reduced the lengthening, bending angle, percentage increase in vessel diameter, and tortuosity index of basilar artery compared with the model group after 2 months of pharmacological intervention. The group also decreased the secretion of Lp-a and increased the content of VEGF. It inhibited the destruction of the internal elastic layer, muscular atrophy, and hyaline degeneration of connective tissue in basilar artery wall. The apoptosis of VSMCs was decreased, and the expression levels of YAP and TAZ proteins were abated (P < 0.05, P < 0.01). ConclusionsThe mechanism of inhibition of basilar artery elongation, dilation, and tortuosity by JTHD, which has various anti-BAD effective compound components, may be related to the reduction in VSMCs apoptosis and downregulation of YAP/TAZ pathway expression.

Liquid Chromatography–Tandem Mass Spectrometry Analysis of Aminoglycosides in Foods Using an Ethylene-Bridged Hybrid Zwitterionic Stationary Phase and Hydrophilic–Lipophilic-Balanced Solid-Phase Extraction Cartridges

This work aimed to develop an analytical method for the screening of multiple aminoglycoside residues in foods of animal origin using an ethylene-bridged hybrid (BEH) particle-based sulfoalkylbetaine stationary phase. The effects of chromatographic conditions on the separation of 17 aminoglycosides have been systematically investigated. Sample preparation and mass spectrometry detection have also been investigated and optimized. In contrast to high buffer concentrations in the mobile phase required for silica-based sulfoalkylbetaine stationary phases, a moderate buffer concentration (20 mM) provided the optimal separation of 17 aminoglycosides with the BEH sulfoalkylbetaine stationary phase. The developed method has been evaluated in milk, beef, pork, liver, and honey samples with good performance for retention, selectivity, sensitivity, linearity, precision, and accuracy. The majority of the limit of quantitation estimated with the matrix was less than 25 μg/kg. The overall accuracy across five matrices was in the range from 96 to 111%, with standard deviations of less than 19%.

Preclinical biocompatibility and biosafety evaluation of a new foldable brown diaphragm intraocular lens: An in vitro and in vivo study.

A new foldable brown diaphragm intraocular lens (IOL) was preclinically evaluated in vitro and in vivo by comparing its biocompatibility and biosafety with those of a commercially available IOL. The new foldable iris-diaphragm IOL is composed of hydrophobic acrylic material, with a transparent optical zone and surrounding brown diaphragm. Cellular experiments evaluating lens epithelial cell morphology, adhesion, and migration were conducted to exclude cytotoxic effects. Twelve New Zealand rabbits underwent implantation of a brown diaphragm IOL in one eye, whilst an additional 12 had a commercially available foldable IOL implanted, followed by slit-lamp evaluations of inflammatory reactions and capsular opacification. Corneal endothelial cells density was measured before and after implantation. Aqueous humour samples were obtained weekly for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to investigate dye leakage from the brown-diaphragm IOL. Following 12 weeks of observation, haematoxylin and eosin staining of ocular tissue and scanning electron microscopy (SEM) of the IOL surface were performed. Results from in vivo experiments found no statistically significant differences between the two groups in terms of postoperative inflammation and capsular biocompatibility. No significant changes in corneal endothelial cell density were observed in either group before and after surgery. LC-MS/MS analysis showed that the target dye was not detected in aqueous humour samples. Histopathology of ocular sections and SEM imaging of IOL surfaces showed similar changes in both groups. The newly invented IOL showed good biocompatibility and biosafety. Combined with its foldability and peripheral shading, it could be a new choice for patients with iris defects.

Profiling GABA(A) Receptor Subunit Expression in the Hippocampus of PMDD Rat Models Based on TCM Theories.

γ-Aminobutyric acid type A receptors (GABAARs) play an important role in cognitive and emotional regulation and are related to the hippocampus. However, little is known regarding patterns of hippocampal GABAAR subunit expression in rat models of premenstrual dysphoric disorder (PMDD). This study investigated the above changes by establishing two PMDD rat models based on Traditional Chinese Medicine (TCM) theories, namely, PMDD liver-qi invasion syndrome (PMDD-LIS) and PMDD liver-qi depression syndrome (PMDD-LDS). Behavioral tests were used to detect depression and irritability emotion. Western blot analysis was used to investigate protein levels of GABAAR α1, α2, α4, α5, β2, β3, and δ subunits, whereas ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis was performed to determine gamma-aminobutyric acid (GABA) and glutamate (Glu) levels in the hippocampus across each group. Concurrently, behavioral data indicated that the PMDD-LDS and PMDD-LIS rat models had been successfully established. GABAAR α2, α5, β2, and δ subunit was significantly upregulated, whereas α4 was significantly downregulated (P < 0.05) in PMDD-LDS rat models relative to controls. On the other hand, GABAAR α1, α2, and β3 were significantly downregulated while α4 and β2 were significantly upregulated in PMDD-LIS rat models relative to the control group (P < 0.05). Moreover, GABA levels significantly decreased, while Glu and the ratio of glutamate to GABA increased in PMDD-LIS rat models (P < 0.05). Conversely, GABA and Glu levels significantly decreased, whereas the ratio of glutamate to GABA increased in PMDD-LIS rat models (P < 0.05). Conclusively, our results revealed differential expression of GABAAR α1, α2, α4, α5, β2, β3, and δ subunits between PMDD-LIS and PMDD-LDS rat models, suggesting that they may be biomarkers in the pathogenesis of PMDD.

Diazo probe-based chemical isotope labeling assisted liquid chromatography-tandem mass spectrometry analysis for sensitive determination of amino acids in biofluids

Amino acids are important biomolecules and contribute to essential biological processes. Liquid chromatography tandem mass spectrometry (LC–MS) now is a powerful tool for the analysis of amino acid metabolites; however, the structural similarity and polarity of amino acids can lead to the poor chromatographic retention and low detection sensitivities. In this study, we used a pair of light and heavy isotopomers of diazo probes, d0/d5-2-(diazomethyl)-N-methyl-N-phenyl-benzamide (2-DMBA/d5 -2-DMBA) to label amino acids. The paired MS probes of 2-DMBA and d5 -2-DMBA carry diazo groups that can efficiently and specifically react with the carboxyl group on free amino acid metabolites under mild conditions. Benefiting from the transfer of the 2-DMBA/d5 -2-DMBA to carboxyl group on amino acids, the ionization efficiencies of amino acids presented great enhancement during LC–MS analysis. The results suggested that the detection sensitivities of 17 amino acids increased by 9-133-fold upon 2-DMBA labeling, and the obtained limits of detection (LODs) of amino acids on-column ranged from 0.011 fmol-0.057 fmol. With the application of the developed method, we successfully achieved the sensitive and accurate detection of the 17 amino acids in microliter level of serum sample. Moreover, the contents of most amino acids were different in the serum from normal and B16F10-tumour mice, demonstrating that endogenous amino acids may play important roles in the regulation of tumors development. This developed method of chemical labeling of amino acids with diazo probes assisted LC–MS analysis provides a potentially valuable tool to investigate the relationships between amino acids metabolism and diseases.

Pharmacokinetics of Lixisenatide, a GLP-1 Receptor Agonist, Determined by a Novel Liquid Chromatography–Tandem Mass Spectrometry Analysis in Rats

Because of its greater binding affinity and longer half-life than native glucagon-like peptide-1 (GLP-1), the GLP-1 receptor agonist lixisenatide is commonly used to treat type 2 diabetes mellitus. This study aimed to establish a simple and robust liquid chromatography–tandem mass spectrometry (LC–MS/MS) approach for lixisenatide for in vivo pharmacokinetic investigation. Methanol-based protein precipitation with formic acid was exploited for plasma sample extraction, using esomeprazole as the internal standard. Gradient elution with 0.1% formic acid in distilled water and acetonitrile was utilized for chromatographic separation. Mass spectrometry was used to monitor the MRM transition at m/z 810.8 → 129.2 for lixisenatide. In rat plasma, lixisenatide had a lower limit of quantification of 10 ng/mL. The LC–MS/MS was applied to describe the pharmacokinetics of lixisenatide in rats following intravenous and subcutaneous dosing. The average half-life of lixisenatide was 0.37 ± 0.06 h after intravenous injection. The estimated subcutaneous bioavailability of lixisenatide was 2.17%. This LC–MS/MS analysis might be relevant in future research to create novel dosage formulations of lixisenatide and other GLP-1 receptor agonists with optimal therapeutic effectiveness.

4D label-free proteomic analysis of vitreous from patients with rhegmatogenous retinal detachment.

To identify metabolites, proteins, and related pathways involved in the etiology of rhegmatogenous retinal detachment (RRD) for use as biomarkers in diagnosing and treating RRD. Vitreous specimens were collected and liquid chromatography-tandem mass spectrometry analysis was performed using the four-dimensional label-free technique. Statistically significant differentially expressed proteins, gene ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway representations, and protein interactions were analyzed. Nine specimens were subjected to proteomic analysis. In total, 161 proteins were identified as differentially expressed proteins (DEPs), including 53 upregulated proteins and 108 downregulated proteins. GO functional analysis revealed that some DEPs were enriched in neuron-related terms and membrane protein terms. Moreover, KEGG analysis indicated that the cell adhesion molecule metabolic pathway was associated with the greatest number of DEPs. Finally, the evaluation of protein-protein interaction network revealed that DEPs were clustered in neuronal adhesion, apoptosis, inflammation and immune responses, correct protein folding, and glycolysis. Proteomic profiling is useful for the exploration of molecular mechanisms that underlie RRD. This study reveals increased expression levels of proteins related to heat shock protein content, glycolysis, and inflammatory responses in RRD. Knowledge regarding biomarkers of RRD pathogenesis may help to prevent the occurrence of RRD in the future.

Iodate respiration by Azoarcus sp. DN11 and its potential use for removal of radioiodine from contaminated aquifers.

Azoarcus sp. DN11 was previously isolated from gasoline-contaminated groundwater as an anaerobic benzene-degrading bacterium. Genome analysis of strain DN11 revealed that it contained a putative idr gene cluster (idrABP1P2 ), which was recently found to be involved in bacterial iodate (IO3 -) respiration. In this study, we determined if strain DN11 performed iodate respiration and assessed its potential use to remove and sequester radioactive iodine (129I) from subsurface contaminated aquifers. Strain DN11 coupled acetate oxidation to iodate reduction and grew anaerobically with iodate as the sole electron acceptor. The respiratory iodate reductase (Idr) activity of strain DN11 was visualized on non-denaturing gel electrophoresis, and liquid chromatography-tandem mass spectrometry analysis of the active band suggested the involvement of IdrA, IdrP1, and IdrP2 in iodate respiration. The transcriptomic analysis also showed that idrA, idrP1 , and idrP2 expression was upregulated under iodate-respiring conditions. After the growth of strain DN11 on iodate, silver-impregnated zeolite was added to the spent medium to remove iodide from the aqueous phase. In the presence of 200 μM iodate as the electron acceptor, more than 98% of iodine was successfully removed from the aqueous phase. These results suggest that strain DN11 is potentially helpful for bioaugmentation of 129I-contaminated subsurface aquifers.

Water-related atmospheric agents and solubility: two parameters of validation in toxicological screening on clothing worn by skeletal remains

In forensic toxicology, when conventional matrices are no longer available, alternative matrices can be used to assess toxicological investigations. Clothes worn by skeletal remains may be a good unconventional matrix for toxicological analyses considering that they have absorbed decomposition fluids and blood from a body. We hypothesized a scenario in which a skeleton, wearing clothes, was discovered in an open environment. From this starting point, an experimental study was developed on different textiles (cotton, wool, and polyester) to evaluate whether water-related atmospheric agents and molecule solubility can largely influence the detection of molecules of toxicological interest on this specific matrix, together with the characteristics of different garments chosen. The experimental study was performed on blood spots, previously spiked with 6-monoacetylmorphine and morphine, accurately placed on different textiles and washed with different quantities of deionized water adjusted at pH 5.6 with formic acid to simulate different rainfall conditions. Toxicological analyses were performed via Solid-Phase Extraction and High-Performance Liquid Chromatography—Tandem Mass Spectrometry analyses (Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer). From the experimental study morphine could not be detected on 100% cotton and 100% wool fabric after the passing of 500 mL of deionized water and in 100% synthetic polyester textile after washing with 250 mL of deionized water. In conclusion, when toxicological analyses are carried out on unconventional matrices as textiles worn by corpses exposed to different environmental conditions, it is of great importance, in using such substrates as evidence for the presence of molecules of toxicological interest, to evaluate chemical-physical characteristics of each analyte under investigation in order to correctly interpret the toxicological data obtained.

Relationship between plasma glutamate and cardiovascular disease risk in Chinese patients with type 2 diabetes mellitus by gender.

This study aimed to assess the association between plasma glutamate (Glu) and the risk of cardiovascular disease (CVD) in patients with type 2 diabetes mellitus (T2DM) and whether this association differs by gender. We retrieved clinical information on 1032 consecutive patients with T2DM from a same tertiary care center from May 2015 to August 2016. Glu was quantified by liquid chromatography-tandem mass spectrometry analysis. Glu was converted into a categorical variable based on the median concentration in the whole population, while logistic regression was used to obtain the odds ratio (OR) and 95% confidence interval (CI), and the correlation between Glu and various biochemical indices was analyzed. We found that Glu was positively associated with the risk of CVD in patients with T2DM. This correlation was more significant in women. In T2DM patients, the higher the age, body mass index (BMI), weight and systolic blood pressure (SBP), the lower the glycosylated hemoglobin (HbA1C) concentration and the higher the Glu. In female patients, the correlation between age, weight, BMI, SBP, and plasma Triglycerides (TG), and Glu was also statistically significant. In conclusion, female T2DM patients with high levels of Glu have a higher risk of developing CVD.

Proteomic Analyses of Autologous Chondrocyte Implantation Plasma Highlight Cartilage Acidic Protein 1 as a Candidate for Preclinical Screening

Background: Stratification is required to ensure that only patients likely to benefit receive autologous chondrocyte implantation (ACI). It would be advantageous to identify biomarkers to predict ACI outcome that are measurable in blood, avoiding the need for an invasive synovial fluid harvest. Purpose: To assess if proteomic analyses can be used to identify novel candidate blood biomarkers in individuals who respond well or poorly to ACI. Study Design: Controlled laboratory study. Methods: Isobaric tagging for relative and absolute quantitation (iTRAQ) mass spectrometry was used to assess the proteome in plasma pooled from ACI responders (mean Lysholm improvement after ACI, 33; n = 10) or nonresponders (mean, −13; n = 10), collected at the time of surgery for cartilage harvest (stage 1) or implantation of culture-expanded chondrocytes (stage 2). An alternative proteomic method, label-free quantitation liquid chromatography–tandem mass spectrometry, was used to analyze plasma samples (majority matched to iTRAQ) individually. Differentially abundant proteins (±2.0-fold) were analyzed from both proteomic data sets, and markers of interest identified via pooled iTRAQ were validated via immunoassay of individual samples. Results: Protein differences could be detected in the plasma preoperatively between ACI responders and nonresponders (16 proteins; ≥±2.0-fold change; P < .05) using iTRAQ proteomics. The most pronounced plasma proteome shift was evident in response to stage 1 surgery in ACI nonresponders, with 48 proteins being differentially abundant between the procedures. Label-free quantitation liquid chromatography–tandem mass spectrometry analysis of these same plasma samples (nonpooled) resulted in very few proteins being identified that were significantly differentially abundant. However, this work highlighted cartilage acidic protein 1 as being increased preoperatively in nonresponders as compared with responders. Conclusions: This study is the first to use proteomic techniques to profile the plasma of individuals treated with ACI. Despite iTRAQ analysis of pooled plasmas indicating that there are differences in the plasma proteome between responders and nonresponders to ACI, these findings were not replicated when assessed using an alternative nonpooled technique. This study highlights some of the difficulties in profiling the plasma proteome in an attempt to identify novel biomarkers. Regardless, cartilage acidic protein 1 has been identified as a protein candidate, which is detectable in plasma and can predict outcome to ACI before treatment. Clinical Relevance: Candidate plasma protein biomarkers identified in this study have the potential to help determine which patients will be best suited to treatment with ACI.

Abstract 2028: Using bioinformatic analysis of proteomic data to probe novel drug mechanism of action

Abstract Introduction: Metastasis is a major cause of death for patients with solid tumors. Our group has discovered a selective inhibitor of cell motility, KBU2046, that blocks the activation of Raf1 by inhibiting the phosphorylation of ser338 on its activation motif (Nature Communications 2018). To better understand the effect of KBU2046 on human prostate cancer (PCa) cells, we undertook a proteomic analysis. Experimental Procedures: After treatment of PC3 cells with KBU2046 or vehicle (N=3/group), protein expression of whole cell lysate was quantified by Liquid Chromatography/Tandem Mass Spectrometry analysis (LC-MS/MS). In separate experiments (N=4), membrane fractions were isolated, and LC-MS/MS was performed after Tandem Mass Tag (TMT) labeling. Resultant data were analyzed by several complementary bioinformatics tools, including: Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO) term analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, Ingenuity Pathway Analysis (IPA), and protein-protein interaction (PPI) network analysis to characterize the proteins that were significantly differentially expressed upon KBU2046 treatment. Results: Independent bioinformatic analyses of differentially expressed proteins affected by KBU2046 treatment identify significantly enriched processes related to cell motility, inclusive of effects on structural machinery. This constitutes orthogonal confirmation of KBU2046’s known effects on cell motility. Of importance, major effects on cellular energy-generating processes were also identified. This is consistent with prior studies that found changes in motility induce obligatorye changes in cellular energy requirements. Our focused analysis of membrane fractions identified changes in membranous compartments, providing a relevant positive control for bioinformatic analysis, which we confirmed by Western blot. Analytics also pointed to a major role for adhesion proteins in response to KBU2046, consistent with our prior identification of primary effects on cell migration. Conclusions: These findings provide orthogonal mechanistically relevant support for a novel acting agent. They highlight the ability of proteomics and bioinformatic tools to efficiently probe the mechanism of action and global impact on PCa in response to treatment. Citation Format: Weining Chen, Nicholas Woods, Fangfang Qiao, Henry Chun Hin Law, Sankarasubramanian Jagadesan, Chittibabu Guda, Raymond C. Bergan. Using bioinformatic analysis of proteomic data to probe novel drug mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2028.

Abstract 3738: A novel mechanism of STAT3 activation by genotoxic chemotherapy

Abstract CARP-1, a perinuclear phospho-protein, is a biphasic regulator of cell survival and apoptosis signaling induced by genotoxic drugs. We have previously found that apoptosis induced by genotoxic drugs involved elevated levels of CARP-1. Exposure to genotoxic drugs also induced CARP-1 interaction with NF-κB kinase subunit γ (aka, NEMO) to promote NF-κB activation and cell survival. To further elucidate chemotherapy-activated, CARP-1-dependent cell survival mechanisms, we UV cross-linked protein extracts from Adriamycin-treated HeLa cervical cancer cells with a CARP-1 (614-638) peptide, and conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the peptide-bound protein complexes. This experiment revealed STAT3 interaction with CARP-1 (614-638) peptide. Our mutagenesis and co-IP-WB experiments revealed CARP-1 (550-650) directly interacts with a 40 amino acid peptide located in the STAT3 DNA binding domain. Overexpression of mutant STAT3 with in-frame-deletion of CARP-1/CCAR1-binding epitope (CE) peptide (Gst-STAT3 (ΔCE) mutant), but not Gst-STAT3 (WT), failed to translocate to nucleus in Adriamycin-treated cells. Disruption of STAT3-CARP-1 interaction or expression of STAT3 (ΔCE) mutant abolished Adriamycin-induced STAT3 Y705 and S727 phosphorylation. Collectively our data demonstrate that CARP-1 interaction with STAT3 regulates genotoxic chemotherapy-activated STAT3 and its nuclear translocation to promote cancer cell survival and growth. Citation Format: Jaganathan Venkatesh, Magesh Muthu, Nuwan C. Acharige, Mary K. Pflum, Arun K. Rishi. A novel mechanism of STAT3 activation by genotoxic chemotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3738.

Isolation and characterization of a lipopeptide-producing Bacillus sp. strain JK08 with antagonistic activity against Vibrio parahaemolyticus.

The present study aimed to isolate a potential antagonist Bacillus sp. and evaluate its capacity for controlling pathogenic Vibrio parahaemolyticus in aquaculture. Strain JK08, which showed inhibitory activity against V. parahaemolyticus VP02r, was isolated from a Penaeus vannamei pond. Based on morphological, physiological, and biochemical characteristics and phylogenetic analysis, strain JK08 was identified as Bacillus sp. Through culture condition optimization, the maximal inhibition zone diameter (18.19±0.16 mm) was observed when strain JK08 was cultivated at a temperature of 30°C, pH of 7, and salinity of 20‰ in Luria-Bertani broth for 24 h. The inhibition zone against V. parahaemolyticus VP02r of strain JK08 (∼7 μg, in mass of crude antimicrobial substance, per tablet) was larger than those (14-18 mm in diameter) of several commercial antibiotics (10 μg per tablet) in the in vitro antagonism assay. Liquid chromatography-tandem mass spectrometry analysis results indicated the presence of three families of lipopeptides in the antimicrobial substance: surfactin (C12-C17), iturin A (C14-C17), and fengycin A (C14-C17) and B (C17), which might be the key components contributing to the antagonistic activity of strain JK08. Strain JK08, which is capable of producing antibacterial lipopeptides, shows effective antagonistic activity against V. parahaemolyticus VP02r, implying its promising potential for V. parahaemolyticus control in aquaculture.

Metamifop resistance in Echinochloa glabrescens via glutathione S-transferases-involved enhanced metabolism.

Echinochloa glabrescens Munro ex Hook. f. is one of the main Echinochloa spp. seriously invading Chinese rice fields and has evolved resistance to commonly used herbicides. Previously, an E. glabrescens population (LJ-02) with suspected resistance to the acetyl-CoA carboxylase (ACCase)-inhibiting herbicide metamifop was collected. This study aimed to determine its resistance status to metamifop and investigate the internal molecular mechanisms of resistance. Single-dose testing confirmed that the LJ-02 population had evolved resistance to metamifop. Gene sequencing and a relative expression assay of ACCase ruled out target-site based resistance to metamifop in LJ-02. Whole-plant bioassays revealed that, compared with the susceptible population XZ-01, LJ-02 was highly resistant to metamifop and exhibited cross-resistance to fenoxaprop-P-ethyl. Pretreatment with the known glutathione S-transferase (GST) inhibitor, 4-chloro-7-nitrobenzoxadiazole (NBD-Cl), largely reversed the resistance to metamifop by approximately 81%. Liquid chromatography-tandem mass spectrometry analysis indicated that the metabolic rates of one of the major metabolites of metamifop, N-(2-fluorophenyl)-2-hydroxy-N-methylpropionamide (HPFMA), were up to 383-fold faster in LJ-02 plants than in XZ-01 plants. There were higher basal and metamifop-inducible GST activities toward 1-chloro-2,4-dinitrobenzene (CDNB) in LJ-02 than in XZ-01. Six GST genes were metamifop-induced and overexpressed in the resistant LJ-02 population. This study reports, for the first time, the occurrence of metabolic metamifop resistance in E. glabrescens worldwide. The high-level metamifop resistance in the LJ-02 population may mainly involve specific isoforms of GSTs that endow high catalytic activity and strong substrate specificity. © 2023 Society of Chemical Industry.

Flavonoid Synthesis-Related Genes Determine the Color of Flower Petals in Brassica napus L.

The color of rapeseed (Brassica napus L.) petal is usually yellow but can be milky-white to orange or pink. Thus, the petal color is a popular target in rapeseed breeding programs. In his study, metabolites and RNA were extracted from the yellow (Y), yellow/purple (YP), light purple (LP), and purple (P) rapeseed petals. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), RNA-Seq, and quantitative real-time (qRT-PCR) analyses were performed to analyze the expression correlation of differential metabolites and differential genes. A total of 223 metabolites were identified in the petals of the three purple and yellow rapeseed varieties by UPLC-MS/MS. A total of 20511 differentially expressed genes (DEGs) between P, LP, YP, versus Y plant petals were detected. This study focused on the co-regulation of 4898 differential genes in the three comparison groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation and quantitative RT-PCR analysis showed that the expression of BnaA10g23330D (BnF3'H) affects the synthesis of downstream peonidin and delphinidin and is a key gene regulating the purple color of petals in B. napus. L. The gene may play a key role in regulating rapeseed flower color; however, further studies are needed to verify this. These results deepen our understanding of the molecular mechanisms underlying petal color and provide the theoretical and practical basis for flower breeding targeting petal color.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of aldosterone in human serum and plasma.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) for aldosterone quantification in human serum and plasma is presented. The material used in this RMP was characterized by quantitative nuclear magnetic resonance (qNMR) to assure traceability to SI Units. For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis a two-dimensional heart cut LC approach, in combination with an optimal supported liquid extraction protocol, was established for the accurate analysis of aldosterone in human serum and plasma in order to minimize matrix effects and avoid the co-elution of interferences. Assay validation was performed according to current guidelines. Selectivity and specificity were assessed using spiked serum; potential matrix effects were examined by a post column infusion experiment and the comparison of standard line slopes. An extensive protocol over 5days was applied to determine precision, accuracy and trueness. Measurement uncertainty was evaluated according to the Guide to the Expression ofUncertainty in Measurement (GUM), for which three individual sample preparations were performed on at least two different days. The RMP allowed aldosterone quantification within the range of 20-1,200pg/mL without interference from structurally-related compounds and no evidence of matrix effects. Intermediate precision was≤4.7% and repeatability was 2.8-3.7% for all analyte concentrations. The bias ranged between-2.2 and 0.5% for all levels and matrices. Total measurement uncertainties for target value assignment (n=6) were found to be≤2.3%; expanded uncertainties were≤4.6% (k=2) for all levels. The RMP showed high analytical performance for aldosterone quantification in human serum and plasma. The traceability to SI units was established by qNMR content determination of aldosterone, which was utilized for direct calibration of the RMP. Thus, this candidate RMP is suitable for routine assay standardization and evaluation of clinical samples.

Isolation, purification, and mass spectrometry identification of enzyme involved in citrus flavor (+)-valencene biotransformation to (+)-nootkatone by Yarrowia lipolytica.

(+)-Nootkatone is a highly valuable sesquiterpene compound, which can be used as aromatics in food industry because of its grapefruit flavor and low sensory threshold. The unconventional yeast Yarrowia lipolytica has many unique physical and chemical properties, metabolic characteristics, and genetic structure, which has aroused the interest of researchers. Previous research showed that Yarrowia lipolytica possesses the ability of transforming the sesquiterpene (+)-valencene to (+)-nootkatone. The aim of this study was to isolate, purify and identify the enzyme involved in the (+)-valencene bioconversion to (+)-nootkatone of Yarrowia lipolytica. In this study, ultrasonic-assisted extraction, ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography were used to separate and purify the enzyme involved in the (+)-valencene bioconversion of Yarrowia lipolytica. The protein was identified as aldehyde dehydrogenase (ALDH) (gene0658) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The ALDH had the highest activity when the pH value was 6.0 and the temperature was 30 °C. The activity of ALDH was significantly stimulated by Fe2+ and inhibited by Ba2+ , Ca2+ and Mg2+ . This is the first time that ALDH was found to participate in (+)-valencene biotransformation by Yarrowia lipolytica. It may be involved in regulating the microbial transformation of (+)-valencene to (+)-nootkatone through redox characteristics. This study provided theoretical basis and reference for the biological synthesis of citrus flavor (+)-nootkatone. This article is protected by copyright. All rights reserved.