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In order to understand the mechanism of bacterial drug resistance under the stress of quinolone antibiotics, this study took Aeromonas hydrophila as the research object, quantitative proteomics was used to compare the protein expression differences in A. hydrophila with and without enrofloxacin treatment. The results showed that total 446 differentially expressed proteins were identified by mass spectrometry, including 233 up-regulated proteins and 213 down-regulated proteins. Bioinformatics analysis showed that A. hydrophila survival may be promoted by up-regulating the expressions of most DNA repair and sulfur metabolism related proteins. Through the growth curve determination, cysteine combined with enrofloxacin can better inhibit the growth of bacteria. Meanwhile, qPCR was used to verify the expression levels of sulfur metabolism-related genes at the mRNA level, and it was uncovered that the differential expression levels of most genes at the transcription level were consistent with the protein level. These results indicate that sulfur metabolism plays an important role in the ENR stress of A. hydrophila. [ABSTRACT FROM AUTHOR]

Background: Terminalia ivorensis (TI) is used in West African ethnomedicine for the treatment of conditions including ulcers, malaria and wounds. Despite its widespread use, the phytochemical profile of TI remains largely undetermined. This research investigated the effects of extraction method, season, and storage conditions on the phytochemical composition of TI to contribute towards understanding the potential benefits. Methods: TI bark was collected in September 2014, September 2018 and February 2018 during the rainy or dry seasons in Eastern Region, Ghana. Samples were extracted sequentially with organic solvents (petroleum ether, chloroform, ethyl acetate and ethanol) or using water (traditional). Metabolites were identified by liquid chromatography–mass spectrometry/mass spectrometry and compared statistically by ANOVA. Results: A total of 82 different phytochemicals were identified across all samples. A greater yield of the major phytochemicals (44%, p < 0.05) was obtained by water as compared with organic extraction. There was also a higher concentration of metabolites present in cold (63%, p < 0.05) compared with hot water extraction. A significantly (p < 0.05) higher number of phytochemicals were identified from TI collected in the dry (85%) compared to the rainy season (69%). TI bark stored for four years retained 84% of the major phytochemicals. Conclusion: This work provides important information on composition and how this is modified by growing conditions, storage and method of extraction informing progress on the development of TI as a prophylactic formulation or medicine. [ABSTRACT FROM AUTHOR]

In this study, two hundred and thirteen liver samples were randomly collected from different ages and both sexes of cattle carcasses in El-Basatin abattoir, Cairo governorate, Egypt. One hundred ninety-seven samples out of the examined specimens were free from any bacterial infections; but showed enlargement and pallor. Thirty-nine specimens representing about 20% of the bacteria-free specimens were examined to detect the Aflatoxins and its metabolites using thin layer chromatography (TLC) and LC MS/MS. All Pathological appraisals were recorded and the genotoxicity of the studied mycotoxin was detected using single cell gel electrophoresis (Comet). Our study revealed that there were prominent deleterious effects of Aflatoxin M1 on the hepatic tissue. The detection of aflatoxins' metabolites in the hepatic tissues is of great importance on the health of human consuming organ meat; and on the other hand, it may affect the farm animals' performance. [ABSTRACT FROM AUTHOR]

• This is a sensitive method (1 ng/ml). • This method generates the shortest run time <1 min (rapid analysis). • This is the only published method that utilized Thiorphan_D7 as an internal standard. • There is an important application to prove the reliability of the new method, which is bioequivalence study in Jordanian subjects. Racecadotril, an anti-secretory medication, has been used as an adjuvant in an oral rehydration therapy for children experiencing severe diarrhea. Racecadotril is quickly converted to thiorphan, an active metabolite, after oral treatment, which mediates all subsequent activities. An efficient and rapid liquid chromatography-tandem mass spectrometry method was developed and fully validated to measure thiorphan in human plasma, using thiorphan-d7 as an internal standard. The extraction method used was protein precipitation while chromatographic separation was achieved using InertSil CN-3 (50 × 2.1 mm, 5 µm column). The assay was linear over the concentration range of 1–200 ng/ml with correlation coefficients of ≥0.9991. The intra- and inter-day precisions were less than 10.0 % for all concentrations investigated. 0.02 % aqueous formic acid and methanol (30:70 v: v) were used as mobile phases, with an analysis time of less than 1 min. This method proved stable under several conditions. The developed method worked well in a three-period pharmacokinetic bioequivalence study after a single oral administration of 100 mg racecadotril to 15 healthy Jordanian volunteers under fasting conditions. [ABSTRACT FROM AUTHOR]

Background: Vitamin D deficiency is universal among patients with chronic liver disease. Vitamin D may be involved in the regulation of immune function of chronic hepatitis B and related to disease progression. Methods: The study was a cross-sectional study. The level of vitamin 25(OH)D was detected in patients with chronic hepatitis B, hepatitis B cirrhosis, hepatitis B cancer, and healthy groups by isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS). At the same time, the clinical data, biochemical indexes, and T lymphocyte subsets were collected to study the relationship between vitamin 25(OH)D deficiency and clinical indexes of hepatitis B patients. Results: The prevalence of vitamin D deficiency (< 20 ng/mL) was higher in patients with liver cancer group (96.97%, 10.59 ± 3.06 ng/mL) and cirrhosis group (93.18%, 11.85 ± 2.66 ng/mL) than in the healthy group (76.92%, 16.38 ± 5.53 ng/mL) and chronic hepatitis B group (77.83%, 15.06 ± 4.91 ng/mL). There were significant differences in vitamin 25(OH)D levels between the cirrhosis groups and the healthy groups, the liver cancer groups and the healthy groups, the hepatitis B cirrhosis groups and the chronic hepatitis B groups, the liver cancer groups and the chronic hepatitis B groups (p < 0.05). There was no significant difference in vitamin 25 (OH)D level between liver cancer group and hepatitis B cirrhosis group, healthy group and chronic hepatitis B group (p > 0.05). Vitamin 25(OH)D level was correlated with age (r = -0.24, p = 0.015), lymphocyte (r = 0.24, p = 0.015), hemoglobin (r = 0.28, p = 0.005), platelet (r = 0.27, p = 0.006), PTA (r = 0.33, p = 0.001), albumin (r = 0.30, p = 0.002), prealbumin (r = 0.39, p = 0.001), cholinesterase (r = 0.29, p = 0.003), CD3+ (r = 0.20, p = 0.04), CD3+ CD8+ (r = 0.20, p = 0.04), CD45+ (r = 0.24, p = 0.017), but none correlated with liver function and HBV-DNA. Conclusions: Vitamin D deficiency existed in patients with hepatitis B, which was related to the clinical progress of hepatitis B and may be involved in the regulation of immune function in patients with chronic HBV infection. [ABSTRACT FROM AUTHOR]

Hypericum lanceolatum Lam. (H. lanceolatum) is a traditional medicinal plant from Reunion Island used for its pleiotropic effects mainly related to its antioxidant activity. The present work aimed to 1) determine the potential toxicity of the plant aqueous extract in vivo and 2) investigate its putative biological properties using several zebrafish models of oxidative stress, regeneration, estrogenicity, neurogenesis and metabolic disorders. First, we characterized the polyphenolic composition by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified chlorogenic acid isomers, quercetin and kaempferol derivatives as the major compounds. We then evaluated for the first time the toxicity of an aqueous extract of H. lanceolatum and determined a maximum non-toxic concentration (MNTC) in zebrafish eleutheroembryos from 0 to 96 hpf following OECD (Organization for Economic Cooperation and Development) guidelines. This MNTC test was also determined on hatched eleutheroembryos after 2 days of treatment (from 3 to 5 dpf). In our study, the anti-estrogenic effects of H. lanceolatum are supported by the data from the EASZY assay. In a tail amputation model, we showed that H. lanceolatum at its MNTC displays antioxidant properties, favors immune cell recruitment and tissue regeneration. Our results also highlighted its beneficial effects in metabolic disorders. Indeed, H. lanceolatum efficiently reduces lipid accumulation and body mass index in overfed larva- and adult-models, respectively. In addition, we show that H. lanceolatum did not improve fasting blood glucose levels in a hyperglycemic zebrafish model but surprisingly inhibited neurogenesis impairment observed in diabetic conditions. In conclusion, our study highlights the antioxidant, pro-regenerative, anti-lipid accumulation and pro-neurogenic effects of H. lanceolatum in vivo and supports the use of this traditional medicinal plant as a potential alternative in the prevention and/or treatment of metabolic disorders. [ABSTRACT FROM AUTHOR]

Hypericum lanceolatum Lam. (H. lanceolatum) is a traditional medicinal plant from Reunion Island used for its pleiotropic effects mainly related to its antioxidant activity. The present work aimed to 1) determine the potential toxicity of the plant aqueous extract in vivo and 2) investigate its putative biological properties using several zebrafish models of oxidative stress, regeneration, estrogenicity, neurogenesis and metabolic disorders. First, we characterized the polyphenolic composition by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified chlorogenic acid isomers, quercetin and kaempferol derivatives as the major compounds. We then evaluated for the first time the toxicity of an aqueous extract of H. lanceolatum and determined a maximum non-toxic concentration (MNTC) in zebrafish eleutheroembryos from 0 to 96 hpf following OECD (Organization for Economic Cooperation and Development) guidelines. This MNTC test was also determined on hatched eleutheroembryos after 2 days of treatment (from 3 to 5 dpf). In our study, the anti-estrogenic effects of H. lanceolatum are supported by the data from the EASZY assay. In a tail amputation model, we showed that H. lanceolatum at its MNTC displays antioxidant properties, favors immune cell recruitment and tissue regeneration. Our results also highlighted its beneficial effects in metabolic disorders. Indeed, H. lanceolatum efficiently reduces lipid accumulation and body mass index in overfed larva- and adult-models, respectively. In addition, we show that H. lanceolatum did not improve fasting blood glucose levels in a hyperglycemic zebrafish model but surprisingly inhibited neurogenesis impairment observed in diabetic conditions. In conclusion, our study highlights the antioxidant, pro-regenerative, anti-lipid accumulation and pro-neurogenic effects of H. lanceolatum in vivo and supports the use of this traditional medicinal plant as a potential alternative in the prevention and/or treatment of metabolic disorders.

21-Hydroxylase deficiency (21OHD) is a rare genetic disorder in which saltwasting syndrome occurs in 75% of cases, due to inability to synthesize cortisol and aldosterone. Recent mass spectrometry progress allowed identification of 21-deoxysteroids, i.e., 17-hydroxyprogesterone (17OHP), 21-deoxycortisol (21DF), and 21-deoxycorticosterone (21DB). We hypothesized that they may interfere with mineralocorticoid signaling and fludrocortisone therapy in patients with congenital adrenal hyperplasia (CAH) without effective glucocorticoid replacement and ACTH suppression. Our goal was to quantify circulating 21-deoxysteroids in a pediatric cohort with CAH related to 21OHD and to examine their impact on mineralocorticoid receptor (MR) activation. Twenty-nine patients with salt-wasting phenotype were classified in two groups according to their therapeutic control. During routine follow-up, 17OHP, 21DF, 21DB, and cortisol levels were quantified by liquid chromatography with tandem mass spectrometry before hydrocortisone intake and 1 and 2.5 h following treatment administration. Luciferase reporter gene assays were performed on transfected HEK293T cells while in silico modeling examined structural interactions between these steroids within ligandbinding domain of MR. Plasma 17OHP, 21DF, and 21DB accumulate in uncontrolled patients reaching micromolar concentrations even after hydrocortisone intake. 21DF and 21DB act as partial MR agonists with antagonist features similar to 17OHP, consistent with altered anchoring to Asn770 and unfavorable contact with Ala773 in ligand-binding pocket of MR. Our results demonstrate a complex interaction between all accumulating 21-deoxysteroids in uncontrolled 21OHD patients and mineralocorticoid signaling and suggest that appropriate steroid profiling should optimize management and follow-up of such patients, as keeping those steroids to low plasma levels should attest therapeutic efficacy and prevent interference with MR signaling. [ABSTRACT FROM AUTHOR]

Abstract Objective Thyroglobulin (Tg) measurements assess recurrence in post-thyroidectomy thyroid cancer patients. Tg measurements by enzyme immunoassays (EIA) can be falsely elevated by interference from Tg autoantibodies (TgAb). Radioimmunoassay (RIA) is less susceptible to TgAb interference and has been the standard-of-care test for TgAb positive patients. Recently developed liquid chromatography tandem mass spectrometry (LC–MS/MS) methods may eliminate TgAb interference. We assessed the performance of Tg measurements by EIA, RIA and LC–MS/MS to evaluate TgAb interference differences. Results We measured TgAb and Tg in 50 plasma samples from 40 patients in whom Tg measurement was part of their routine follow-up and 10 healthy volunteers. Discrepancy between EIA and both LC–MS/MS and RIA was observed at low Tg concentrations (≤ 7.55 ng/mL) in TgAb positive specimens (LC–MS/MS = 1.9 * EIA − 0.03, r = 0.68). RIA and LC–MS/MS Tg measurements in TgAb positive specimens with low Tg concentrations had improved correlation but demonstrated bias (LC MS/MS = 0.6 * RIA − 1.4, r = 0.90). Disagreement between methods may be attributed to LC–MS/MS reported Tg concentrations as undetectable compared to RIA. It seems likely that most discrepant cases are falsely elevated in RIA due to TgAb interference, however, some cases appear below the detection limit of LC–MS/MS; implementation of LC–MS/MS by clinicians will require lower detection limits.

Many plants that are commonly used in folk medicine have multidirectional biological properties confirmed by scientific research. One of them is Aerva lanata (L.) Juss. (F. Amaranthaceae). It is widely used, but there are very few scientific data about its chemical composition and pharmacological activity. The aim of the present study was to investigate the chemical composition of phenolic acid (PA)-rich fractions isolated from methanolic extracts of A. lanata (L.) Juss. herb using the liquid/liquid extraction method and their potential antioxidant, anti-inflammatory, and anti-diabetic properties. The free PA fraction (FA), the PA fraction (FB) released after acid hydrolysis, and the PA fraction (FC) obtained after alkaline hydrolysis were analysed using liquid chromatography/electrospray ionization triple quadrupole mass spectrometry (LC-ESI-MS/MS). The phenolic profile of each sample showed a high concentration of PAs and their presence in A. lanata (L.) Juss. herb mainly in bound states. Thirteen compounds were detected and quantified in all samples, including some PAs that had not been previously detected in this plant species. Bioactivity assays of all fractions revealed high 2,2-diphenyl-1-picrylhydrazyl (DPPH•) (2.85 mM Trolox equivalents (TE)/g) and 2,2-azino-bis-3(ethylbenzthiazoline-6-sulphonic acid) (ABTS•+) (2.88 mM TE/g) scavenging activity. Fraction FB definitely exhibited not only the highest antiradical activity but also the strongest xanthine oxidase (XO) (EC50 = 1.77 mg/mL) and lipoxygenase (LOX)(EC50 = 1.88 mg/mL) inhibitory potential. The fraction had the best anti-diabetic properties, i.e., mild inhibition of α-amylase (EC50 = 7.46 mg/mL) and strong inhibition of α-glucosidase (EC50 = 0.30 mg/mL). The activities of all analysed samples were strongly related to the presence of PA compounds and the total PA content.

The purpose of this study was to see if the microalgae species Chlorella miniata possess an antioxidant and antiinflammatorypotential and to see if there is a correlation between the cultivation conditions and these potentials. For this purpose, cultivation of the algae under different growth conditions had been carried out; afterwards the soxhletextraction of the produced biomass had been completed. Lastly the assays of Trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant capacity (FARP), total antioxidant capacity (TAO), xanthine oxidase inhibition, and hyaluronidase inhibition were studied using extracts of microalgae obtained from the different cultivation parameters. It's important to mention that this was the first study using these assays on species of Chlorella miniata and the first that utilized the cultivation parameters on microalgae. The results were, 22,91 mg/g algae TEAC, 776,70 mg/g algae gallic acid equivalent of FRAP and 37,07% xanthine oxidase inhibition with a concentration of 10,5 mg algae per ml ethanol, 65,39% of hyaluronidase inhibition with a concentration of 0,9 mg algae per ml ethanol. The effects of the cultivation parameters on these assays and the Lc MS/MS analyses for various phenolic compounds in the extracts were also a part of the study. [ABSTRACT FROM AUTHOR]

Objective: Thyroglobulin (Tg) measurements assess recurrence in post-thyroidectomy thyroid cancer patients. Tg measurements by enzyme immunoassays (EIA) can be falsely elevated by interference from Tg autoantibodies (TgAb). Radioimmunoassay (RIA) is less susceptible to TgAb interference and has been the standard-of-care test for TgAb positive patients. Recently developed liquid chromatography tandem mass spectrometry (LC-MS/MS) methods may eliminate TgAb interference. We assessed the performance of Tg measurements by EIA, RIA and LC-MS/MS to evaluate TgAb interference differences. Results: We measured TgAb and Tg in 50 plasma samples from 40 patients in whom Tg measurement was part of their routine follow-up and 10 healthy volunteers. Discrepancy between EIA and both LC-MS/MS and RIA was observed at low Tg concentrations (= 7.55 ng/mL) in TgAb positive specimens (LC-MS/MS = 1.9 * EIA - 0.03, r = 0.68). RIA and LC-MS/MS Tg measurements in TgAb positive specimens with low Tg concentrations had improved correlation but demonstrated bias (LC MS/MS = 0.6 * RIA - 1.4, r = 0.90). Disagreement between methods may be attributed to LC-MS/MS reported Tg concentrations as undetectable compared to RIA. It seems likely that most discrepant cases are falsely elevated in RIA due to TgAb interference, however, some cases appear below the detection limit of LC-MS/MS; implementation of LC-MS/MS by clinicians will require lower detection limits. [ABSTRACT FROM AUTHOR]

Small molecules containing carboxylic acid functional groups are ubiquitous throughout biology, playing vital roles in biological chemistry ranging from energy metabolism to cellular signaling. This paper describes a new derivatization reagent, 4-bromo- N-methylbenzylamine, which was selected for its potential to derivatize mono-, di- and tri-carboxylic acids, such as the intermediates of the tricarboxylic acid (TCA) cycle. This derivatization procedure facilitated the use of positive electrospray ionization (ESI) tandem mass spectrometry (MS/MS) detection of derivatized species allowing for clear identification thanks to the easily recognizable isotope pattern of the incorporated bromine. A liquid chromatography (LC)-MS/MS method was developed which provided limits of detection between 0.2 and 44 μg L in under 6 min, depending on the analyte and total analysis time. This method was successfully applied in both in vitro and in vivo models. [ABSTRACT FROM AUTHOR]

The Mediterranean marine sponge Spongia officinalis has been reported as a rich source of secondary metabolites and also as a bioindicator of water quality given its capacity to concentrate trace metals. In this study, we evaluated the chemical diversity within 30 S. officinalis samples collected over three years at two sites differentially impacted by anthropogenic pollutants located near Marseille (South of France). Untargeted liquid chromatography—mass spectrometry (LC–MS) metabolomic profiling (C18 LC, ESI-Q-TOF MS) combined with XCMS Online data processing and multivariate statistical analysis revealed 297 peaks assigned to at least 86 compounds. The spatio-temporal metabolite variability was mainly attributed to variations in relative content of furanoterpene derivatives. This family was further characterized through LC–MS/MS analyses in positive and negative ion modes combined with molecular networking, together with a comprehensive NMR study of isolated representatives such as demethylfurospongin-4 and furospongin-1. The MS/MS and NMR spectroscopic data led to the identification of a new furanosesterterpene, furofficin (2), as well as two derivatives with a glycinyl lactam moiety, spongialactam A (12a) and B (12b). This study illustrates the potential of untargeted LC–MS metabolomics and molecular networking to discover new natural compounds even in an extensively studied organism such as S. officinalis. It also highlights the effect of anthropogenic pollution on the chemical profiles within the sponge. [ABSTRACT FROM AUTHOR]

The holocellulase from Aspergillus niger SH3 was characterized and found to contain 125 proteins including cellulases (26), hemicellulases (21), chitinases (10), esterases (6), amylases (4) and hypothetical protein (32). The crude enzyme was immobilized on five different nanoparticles (NPs) via physical adsorption and covalent coupling methods. The enzyme-nanoparticle complexes (ENC) were screened for protein binding, enzymatic activities and immobilization efficiency. Magnetic enzyme-nanoparticle complexes (MENC) showed higher immobilization efficiency (60–80%) for most of the enzymes. MENC also showed better catalytic efficiencies in term of higher V max and lower K m than free enzyme. Saccharification yields from alkali treated paddy straw were higher (375.39 mg/gds) for covalently immobilized MENC than free enzyme (339.99 mg/gds). The immobilized enzyme was used for two cycles of saccharification with 55% enzyme recovery. Hence, this study for the first time demonstrated the immobilization of indigenous enzyme and its utilization for saccharification of paddy straw. [ABSTRACT FROM AUTHOR]

A matrix removal procedure with ion-exchange resin prior to analysis for 18 fluorinated benzoic acids (FBAs) tracers in saline (>25% salt) reservoir water was optimized. The elimination of >98% of salt and the simultaneous matrix sample cleanup allowed the direct analysis using the supernatant by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This resulted in a gain in detection limits for most of the tracers in comparison with the reference method (direct analysis after minimum required dilution). The limits of detection (LODs) were in the range of 0.01-0.15 ng/ml and compared to other studies the developed method provided comparable limits of detection and advantage of simplified and shorter sample preparation. The presented method offers a considerable gain in simplicity and analysis time. Recoveries for all the tracers reached 80-100%, except for 2-FBA and 2,6-dFBA for which they were ca. 60%. The low recoveries were corrected by the use of five isotopically labeled internal standards. The method was validated by the analysis of spiked samples and by an independent comparison of the results with those obtained by solid-phase extraction LC-MS/MS method. [ABSTRACT FROM AUTHOR]

All activities aim to eliminate genotoxic impurities or/and protect the medicinal compounds against degradation. There is a need to monitor impurities from all classification groups, so that ivabradine forced degradation studies were performed. Ivabradine proved to be a quite durable active substance, but still new and not known particularly. Increasing temperature, acid, base, oxidation reagent and light cause its degradation. Thirteen degradation products were determined with the usage of HPLC equipped with Q-TOF-MS detector. Six of them, which the most probable structures managed to establish, were selected to ADME/Tox calculations for prediction studies of their pharmacological and toxicological properties. Target prediction algorithm was applied on the basis of Hyperpolarization-activated cyclic nucleotide-gated cation channels as well as more general parameters like logP and aqueous solubility. Ames test and five cytochromes activities were calculated for toxicity assessment of selected degradation products. Pharmacological activity of photodegradation product (UV4) that is known as active metabolite was qualified and identified. Two other degradation compounds (Ox1 and N1) which were formed during degradation process were found to be pharmacologically active with high probability.

In this paper, a strategy for structured monitoring of surface modifications to control protein adsorption to membrane structures is presented. The already established on-surface enzymatic digestion (oSED) method combined with nano-liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis was employed for the analysis of proteins in ventricular cerebrospinal fluid (vCSF) from neurointensive care patients. Protein adsorption was studied by in-situ sampling in a temporally resolved manner on both immobilized native and Pluronic-deactivated membranes. Deactivation was significantly reducing the protein adsorption but it also induced novel selective properties of the surface. The proposed versatile strategy will facilitate protein-biomaterial, protein-polymer, protein-protein interaction studies in the future.

Chronic post-surgical pain (CPSP) is a normal and significant symptom in clinical surgery, such as breast operation, biliary tract operation, cesarean operation, uterectomy and thoracic operation. Severe chronic post-surgical pain could increase post-surgical complications, including myocardial ischemia, respiratory insufficiency, pneumonia and thromboembolism. However, the underlying mechanism is still unknown. Herein, a rat CPSP model was produced via thoracotomy. After surgery, in an initial study, 5 out of 12 rats after surgery showed a significant decrease in mechanical withdrawal threshold and/or increase in the number of acetone-evoked responses, and therefore classified as the CPSP group. The remaining seven animals were classified as non-CPSP. Subsequently, open-chest operation was performed on another 30 rats and divided into CPSP and non-CPSP groups after 21-day observation. Protein expression levels in the dorsal spinal cord tissue were determined by 12.5 % SDS-PAGE. Finally, differently expressed proteins were identified by LC MS/MS and analyzed by MASCOT software, followed by Gene Ontology cluster analysis using PANTHER software. Compared with the non-CPSP group, 24 proteins were only expressed in the CPSP group and another 23 proteins expressed differentially between CPSP and non-CPSP group. Western blot further confirmed that the expression of glutaminase 1 (GLS1) was significantly higher in the CPSP than in the non-CPSP group. This study provided a new strategy to identify the spinal proteins, which may contribute to the development of chronic pain using differential proteomics, and suggested that GLS1 may serve as a potential biomarker for CPSP. [ABSTRACT FROM AUTHOR]

Arthropod venoms may be considered important sources of bioactive molecules; however, technical difficulties, such as venom extraction and homogeneity may impair the biochemical identification of new molecules. In this context, we have developed a method to maintain wasps in captivity that allows the collection of the venom, without use of chemical, mechanical or electrical stimuli. The crude venom was analyzed by RP-HPLC-ESIQ-ToF and 20 peptides were identified by de novo peptide sequencing, among them Eumenine-Mastoparan and a Ponericin-G2-simile peptide. [ABSTRACT FROM AUTHOR]

The intense use of pesticides against plant pests and diseases during cucumber growing in greenhouses may lead to high levels of pesticide residues in the final product. The concentration of pesticide residues in cucumber fruits can change during subsequent pickle processing steps and could be affected with washing, sterilising or keeping at low pH levels during fermentation1. This study was conducted to detect the residue levels of 2 acaricides, 3 insecticides and 6 fungicides in cucumbers grown at greenhouse during each step of pickling processing (washing, fermentation and pasteurisation). Pesticides were applied at their recommended field doses with a knapsack sprayer under greenhouse conditions on three independent plots 3 days before harvesting (Preharvest interval-PHI). After harvesting, raw materials were processed with two different processing techniques (canned and fermented) according to Compendium of Representative Processing Techniques Investigated in Regulatory Studies for Pesticides2. The extraction and cleaning up of the pesticides, namely, acetamiprid, ametoctradin, azoxystrobin, chlorantraniliprole, deltamethrin, difenoconazole, dimethomorph, etoxazole, fluopicolide, propamocarb and spiromesifen, were conducted by using QuEChERS method3. The residue levels were detected with LC MS/MS. The multi-residue analysis method was validated according to the SANTE guidelines4,5. The processing factor (pf) values obtained during canned pickle processing steps such as washing, pasteurisation and storage (21 days), were found as 0.25-1.10; 0.03-0.89; 0.01-0.68, respectively. The pf values of fermented pickle processing steps like washing, fermentation and pasteurisation, ranged with 0.39-0.98; 0.03-0.79; 0.01-0.85, respectively. During both types of pickle processes, pesticide residues remained lower than the Maximum Residue Levels. This study was funded by the collaboration of Bursa Uludag University and Perla Fruit Company with the grant number FKA-2021-531. [ABSTRACT FROM AUTHOR]

The paper presents the development of an advanced extraction and fast analytical LC MS/MS method for simultaneous analyses of reduced and oxidized glutathione (GSH and GSSG, respectively) in different animal tissues. The simultaneous determination of GSH and GSSG is crucial because the amount and ratio of both GSH and GSSG may be altered in response to oxidative stress, an important mechanism of toxicity. The method uses the derivatization of free thiol groups in GSH. Its performance was demonstrated for less explored tissues (lung, brain, and liver) in mouse. The combined extraction and analytical method has very low variability and good reproducibility, maximum coefficients of variance for within-run and between-run analyses under 8 %, and low limits of quantification; for GSH and GSSG, these were 0.2 nM (0.06 ng/mL) and 10 nM (6 ng/mL), respectively. The performance of the method was further demonstrated in a model experiment addressing changes in GSH and GSSG concentrations in lung of mice exposed to CdO nanoparticles during acute 72 h and chronic 13-week exposures. Inhalation exposure led to increased GSH concentrations in lung. GSSG levels were in general not affected; nonsignificant suppression occurred only after the longer 13-week period of exposure. The developed method for the sensitive detection of both GSH and GSSG in very low tissue mass enables these parameters to be studied in cases where only a little sample is available, i.e. in small organisms or in small amounts of tissue. [ABSTRACT FROM AUTHOR]

The mechanisms underlying resistance to challenge by gastrointestinal nematode parasites in sheep are complex. Using DIGE, we profiled ovine lymph proteins in lambs with host resistance (R), resilience (Ri) or susceptibility (S) to a daily trickle challenge with the nematode Trichostrongylus colubriformis . Efferent intestinal lymph was collected prior to infection (day 1) and on days 5 and 10 post-infection. Eight proteins identified by LC-MS/MS, showed differences relating to host genotype. Of these, Serpin A3-3 and Serpin A3-7 have not been reported previously in the lymph proteome. Three acute phase proteins showed significant differences relating to interactions between breeding line and parasite challenge, including complement C3β, C3α and haptoglobin (Hp) β. In the R lambs C3α was significantly up regulated ( P < 0.05) on day 10, while in the Ri lambs Hp β was significantly down regulated ( P < 0.05). In the S lambs, levels of C3β were up regulated and levels of Hp β down regulated (both P < 0.05) on day 10. Hence we demonstrate that acute phase inflammation proteins contribute to differences in the innate immune response of sheep to challenge by T. colubriformis . The findings may lead to the development of new approaches to combat nematode infestations in sheep production systems. Biological Significance Breeding lines of sheep with resistance (R), resilience (Ri) or susceptibility (S) to nematode infections provide an experimental model to examine the biological mechanisms underlying the ability of some sheep to expel worms and remain healthy without the use of an anthelmintic. Using proteomics we identified differences in the expression of acute phase lymph proteins in the R, Ri and S lambs. The results will assist the development of alternative control strategies to manage nematode infections in livestock. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the possible effect of pulse-modulated radiofrequency radiation (RFR) on rat skin hydroxyproline content, since skin is the first target of external electromagnetic fields. Skin hydroxyproline content was measured using liquid chromatography mass spectrometer method. Two months old male wistar rats were exposed to a 900 MHz pulse-modulated RFR at an average whole body specific absorption rate (SAR) of 1.35 W/kg for 20 min/day for 3 weeks. The radiofrequency (RF) signals were pulse modulated by rectangular pulses with a repetition frequency of 217 Hz and a duty cycle of 1:8 (pulse width 0.576 ms). A skin biopsy was taken at the upper part of the abdominal costa after the exposure. The data indicated that whole body exposure to a pulse-modulated RF radiation that is similar to that emitted by the global system for mobile communications (GSM) mobile phones caused a statistically significant increase in the skin hydroxyproline level ( p = 0.049, Mann-Whitney U test). Under our experimental conditions, at a SAR less than the International Commission on Non-Ionizing Radiation Protection safety limit recommendation, there was evidence that GSM signals could alter hydroxyproline concentration in the rat skin. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila is a widespread opportunistic pathogen of aquatic fishes in freshwater habitats. The current emergence of antimicrobial-resistant A. hydrophila has been reported in the world while the bacterial antibiotics adaptive mechanism remains poorly explored. In this study, using quantitative proteomics technology, the behavior of A. hydrophila was investigated by comparing the differentially expression proteins between with and without kanamycin (KAN) treatment. A total of 374 altered proteins including 184 increasing and 190 proteins decreasing abundances were quantified when responding to KAN stress. The bioinformatics analysis showed that stress related proteins were hub proteins that significantly increased to reduce the pressure from the misreading of mRNA caused by KAN. Moreover, several metallic pathways, such as oxidative phosphorylation and TCA cycle pathways may affect KAN resistance. Finally, eight selected genes were deleted and their antibiotics susceptibilities to kanamycin were valued, respectively. Results showed that OmpA II family protein A0KI26, and two-component system protein AtoC may involve in the KAN resistance in this study. In general, our results provide an insight into the behaviors of bacterial responding to KAN stress, and demonstrate the intrinsic antibiotics adaptive mechanism of A. hydrophila. In this study, the differentially expressed proteins (DEPs) of A. hydrophila strain between with and without kanamycin (KAN) were compared by using a data-independent acquisition (DIA) - based quantitative proteomics method. Bioinformatics analysis showed that stress - related proteins are hub proteins that significantly increased under KAN stress. Moreover, several metallic pathways, such as oxidative phosphorylation and citrate cycle (TCA cycle) pathways, can affect KAN resistance. Finally, our antibiotics susceptibility assay showed that the protein A0KI26 of the OmpA II family, and the AtoC of the two-component system may involve in KAN resistance in this study. These results provide insights into the antibiotics adaptation mechanism of A. hydrophila when responding to KAN stress. [Display omitted] • A total of 374 DEPs were detected under the stress of KAN. • Stress-related proteins increased significantly in the presence of kanamycin. • A0KI26 and AtoC may involve in the KAN resistance. [ABSTRACT FROM AUTHOR]

The efficiency of two extraction techniques-ultrasound-assisted extraction and pressurized liquid extraction-are compared and evaluated in the determination of parabens in compost samples. The extraction parameters for each technique were accurately optimized. The selected compounds were detected and quantified using ultra-performance LC MS/ MS, operating in negative ESI and in SRM mode. The analytes were separated in less than 5 min. Ethylparaben (ring-13C6 labeled) was used as an internal standard. Two selective, sensitive, and accurate analytical methods were developed and validated. The LODs of the methods ranged from 3 to 7 ng/g and the LOQs from 10 to 23 ng/g, while inter- and intraday variability was under 6% in all cases. The methods were validated separately by using matrix-matched calibration and recovery assays with spiked samples. Recovery rates ranged from 94.0 to 105.0%. Compost samples were taken from different composting plants. Although the statistical comparison demonstrated no statistically significant differences between the two extraction techniques, the method based on pressurized liquid extraction was more sensitive than the ultrasound extraction based method. [ABSTRACT FROM AUTHOR]

ABSTRACT A convenient and precise chiral method was developed and validated for measuring indoxacarb enantiomers in cucumber and tomato using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a reversed-phase Chiralpak AD-RH column. The target analytes were extracted by acetonitrile and then purified by solid phase extraction (SPE) using NH2/Carb combined-cartridge. Parameters including the matrix effect, linearity, precision, accuracy, and stability were used. Then the proposed method was successfully applied to investigate the possible enantioselective degradation of rac-indoxacarb in cucumber and tomato under open conditions. The results indicated that the degradation of indoxacarb enantiomers followed first-order kinetics in cucumber and tomato. The half-lives of (+)- S-indoxacarb in cucumber and tomato were 3.0 and 5.9 days, respectively; while the (-)- R-indoxacarb were 7.3 and 12.2 days, respectively. The data of the half-lives showed that (+)- S-indoxacarb was preferentially degraded in cucumber and tomato. Moreover, indoxacarb degraded faster in cucumber than in tomato. Chirality 25:350-354:, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Objectives The objective of the current investigation was to develop a simple, rapid method for determining in-vivo 5-hydroxytryptamine type 4 receptor ( 5-HT4R) occupancy in rat brain using non-radiolabelled SB207145 as a tracer for accelerating the drug discovery process. Methods In-vivo tracer optimization studies for tracer dose, survival intervals and brain distribution profile were carried out in rats. The tracer was pharmacologically validated using potent well-characterized 5-HT4R ligands. The brain regional concentrations of tracer ( SB207145); plasma and brain concentrations of 5-HT4R ligands were quantified using high-performance liquid chromatography coupled with a tandem mass spectrometric detector ( LC-MS/ MS). Key findings SB207145 showed a higher specific binding in striatum (1.96 ng/g) and lower binding in cerebellum (0.66 ng/g), which is consistent with findings of other published 5-HT4R expression studies. Pretreatment with potent 5-HT4 ligands dose-dependently reduced striatal SB207145 concentration and the effective dose to achieve 50% receptor occupancy ( ED50) values were 4.8, 2.0, 7.4, 9.9, 3.8 and 0.02 mg/kg for GR113808, piboserod, prucalopride, RS67333, TD8954 and PF04995274, respectively. Conclusions Results from the mass spectrometry approach to determine 5-HT4R occupancy in rat brain are comparable with those reported using radiolabelled scintillation spectroscopy methods. In conclusion, the LC-MS/ MS characterization permits use of tracer at a preclinical stage in high-throughput fashion as well as characterization of target expression. [ABSTRACT FROM AUTHOR]

ABSTRACT Metoprolol is available for clinical use as a racemic mixture. The S-(−)-metoprolol enantiomer is the one expressing higher activity in the blockade of the β1-adrenergic receptor. The α-hydroxymetoprolol metabolite also has activity in the blockade of the β1-adrenergic receptor. The present study describes the development and validation of a stereoselective method for sequential analysis of metoprolol and of α-hydroxymetoprolol in plasma using high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). 1-ml aliquots of plasma were extracted with dichloromethane : diisopropyl ether (1:1, v/v). Metoprolol enantiomers and α-hydroxymetoprolol isomers were separated on a Chiralpak AD column (Daicel Chemical Industries, New York, NY, USA) and quantitated by LC-MS/MS. The limit of quantitation obtained was 0.2 ng of each metoprolol enantiomer/ml plasma and 0.1 ng/ml of each α-hydroxymetoprolol isomer/ml plasma. The method was applied to the study of kinetic disposition of metoprolol in plasma samples collected up to 24 h after the administration of a single oral dose of 100-mg metoprolol tartrate to a hypertensive parturient with a gestational age of 42 weeks. The clinical study showed that the metoprolol pharmakokinetics is enantioselective, with the observation of higher area under the curve (AUC)0−∞ values for S-(−)-metoprolol (AUC S-(−)/AUC R-(+) = 1.81) and the favoring of the formation of the new chiral center 1′ R of α-hydroxymetoprolol (AUC0−∞1′ R/1′ S = 2.78). Chirality, 25:1-7, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Molecular approaches in ecotoxicology have greatly enhanced mechanistic understanding of the impact of aquatic pollutants in organisms. These methods have included high throughput Omics technologies, including quantitative proteomics methods such as 2D differential in-gel electrophoresis (DIGE) and isobaric tagging for relative and absolute quantitation (iTRAQ). These methods are becoming more widely used in ecotoxicology studies to identify and characterize protein bioindicators of adverse effect. In teleost fish, iTRAQ has been used successfully in different fish species (e.g. fathead minnow, goldfish, largemouth bass) and tissues (e.g. hypothalamus and liver) to quantify relative protein abundance. Of interest for ecotoxicology is that many proteins commonly utilized as bioindicators of toxicity or stress are quantifiable using iTRAQ on a larger scale, providing a global baseline of biological effect from which to assess changes in the proteome. This review highlights the successes to date for high throughput quantitative proteomics using DIGE and iTRAQ in aquatic toxicology. Current challenges for the iTRAQ method for biomarker discovery in fish are the high cost and the lack of complete annotated genomes for teleosts. However, the use of protein homology from teleost fishes in protein databases and the introduction of hybrid LTQ–FT (Linear ion trap–Fourier transform) mass spectrometers with high resolution, increased sensitivity, and high mass accuracy are able to improve significantly the protein identification rates. Despite these challenges, initial studies utilizing iTRAQ for ecotoxicoproteomics have exceeded expectations and it is anticipated that the use of non-gel based quantitative proteomics will increase for protein biomarker discovery and for characterization of chemical mode of action. [Copyright &y& Elsevier]

In this study, three methods of protein extraction from the seeds of the Chinese fir were compared by examining the quality (including the number of protein spots observed) in the two-dimensional gel electrophoresis (2-DE), obtained by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis of the total released protein. Three protein extraction methods were: TCA-acetone precipitation, SDS extraction/acetone precipitation, and phenol extraction methanol/ammonium acetate precipitation. The results showed that TCA-acetone precipitation was the most effective method for protein extraction; it gave the highest yield of total protein (8.9 mg protein per g seed weight) and the greatest number of proteins spots (1,034 spots) on the 2-DE gel. Further, several proteins were identified by liquid chromatography mass spectrometry (LC MS/MS), which are legumin-like storage protein, similar to AMP binding/acetate-CoA ligase, similar to 40S ribosomal protein S20, actin, ascorbate peroxidase, Similar to cysteine synthase, and unknown protein. These data demonstrates that TCA-acetone precipitation followed by 2-DE and LC MS/MS is a suitable method for proteomic analysis of coniferous species, such as Chinese fir and provides a valuable starting point for similar proteomic analysis of other coniferous tree species. [ABSTRACT FROM AUTHOR]

With the genomes of three Frankia strains available, high-throughput proteomics methods can be used to reveal the set of proteins expressed by these bacteria in symbiosis with plants. A question we address is the degree to which the known genomes can be used to study proteomes of uncharacterized frankiae growing in field-collected root nodules. To this end, we have characterized the symbiotic proteomes of Frankia from three plant species, Alnus incana subsp. rugosa, Ceanothus americanus, and Elaeagnus angustifolia. Root nodule proteins were identified using two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC MS/MS) of trypsin-digested protein samples. We identified 1300 Frankia proteins in A. incana nodules using the Frankia alni ACN14a genome and 1100 proteins from E. angustifolia nodules using the EAN1pec genome . In addition, over 100 proteins were identified from C. americanus nodules using a more limited one dimensional LC MS/MS analysis . Many of the most abundant proteins identified are involved in energy and nitrogen metabolism. The enzyme nitrogenase and the nitrogenase iron protein were among the most abundant proteins, reflecting the major process occurring in symbiosis. Several hundred plant proteins were also identified. We highlight the power of proteomics to uncover the physiology of symbiotic Frankia in the environment using heterologous genome information. [ABSTRACT FROM AUTHOR]

Abstract: Resveratrol, a phenolic compound found in plant tissues, berries, and wine exists in trans and cis stereo isomeric forms. Naturally, due to differences in energetic state the trans form is the more common, but in wines the cis form is also present, resulting from photoisomerisation reactions. It is thought that from this reaction the only product arising is the cis-resveratrol. After UV irradiation of the trans-resveratrol solution for 10min a third component appears with a growing concentration till 1h of irradiation. From the MS/MS results we can assert that this compound is a product derived from oxidation, where the double bond at the centre of the resveratrol molecule, changes into a triple bond, forming a diphenylacetylene derivative (3,4′,5-trihydroxy-diphenylacetylene). [Copyright &y& Elsevier]

Abstract: Dietary supplements on soy based foods and beverages are increasingly gaining prominence all over the world. In this study, liquid chromatography coupled with positive electrospray ionisation tandem mass spectrometry (LC-ESI-MS) and diode array detection was used for the quantitation and characterisation of isoflavones in fermented and unfermented soymilk made from soy protein isolate SUPRO 590. Bifidobacterium animalis ssp. lactis Bb12 was used for the fermentation of soymilk. The isoflavones were found to produce characteristic radical ions as well as molecules of H2O, CO2, a sugar unit, and an alcohol through collision-induced fragmentation. Product ion fragments revealed unique fragmentation pathways for each isoflavone compound. Characteristic fragmentation of different isoflavones were unequivocally identified and differentiated. The occurrence of aldehydes such as pentanal, ethanal and methanal was shown to be specifically linked with isoflavone aglycones, daidzein, genistein and glycitein, respectively. Main glycosides such as genistin, daidzin and glycitin as well as the acetyl-, and malonyl forms also showed respective aglycone ions in their spectra fragmentation. Thus positive ion fragmentation was important in the absolute confirmation of isoflavones and to reveal the occurrence of other related compounds such as aldehydes in soymilk. [Copyright &y& Elsevier]

Abstract: Soil salinization has become a severe global problem and salinity is one of the most severe abiotic stresses inhibiting growth and survival of mycorrhizal fungi and their host plants. Salinity tolerance of ectomycorrhizal fungi and survival of ectomycorrhizal inocula is essential to reforestation and ecosystem restoration in saline areas. Proteomic changes of an ectomycorrhizal fungus, Boletus edulis, when exposed to salt stress conditions (4% NaCl, w/v) were determined using two-dimensional electrophoresis (2DE) and mass spectrometry (MS) techniques. Twenty-two protein spots, 14 upregulated and 8 downregulated, were found changed under salt stress conditions. Sixteen changed protein spots were identified by nanospray ESI Q-TOF MS/MS and liquid chromatography MS/MS. These proteins were involved in biosynthesis of methionine and S-adenosylmethionine, glycolysis, DNA repair, cell cycle control, and general stress tolerance, and their possible functions in salinity adaptation of Boletus edulis were discussed. [Copyright &y& Elsevier]

Cationic peanut peroxidase (CP) was isolated from peanut (Arachis hypogaea) cell suspension culture medium. CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185. ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans. Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS. The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS. The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site. Good agreement was found with the one glycan previously analyzed by 1H NMR. This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry. [Copyright &y& Elsevier]

• Alterations of epileptogenesis-associated calcium signaling proteins were analyzed. • Calcium signaling protein expression showed stage-specific changes in expression. • CD38 levels increase in hippocampus with kindling and relate to NAD+ decline. • MGluR1 level was altered more than other CD38 upstream receptors. • CD38/cADPR signaling pathways emerges as interesting new targets in epilepsy. In spite of long-term intensive scientific research efforts, there are still many issues concerning the mechanisms of epileptogenesis and epilepsy to be resolved. Temporal lobe, in particular hippocampus, is vulnerable to epileptogenic process. Herein, electrical kindling model of temporal lobe were analyzed using proteomic approach. A dramatic decrease in nicotinamide adenine dinucleotide (NAD+) level was exhibited during the kindling procedure in hippocampus. After stage 3, high CD38 expression was detected by qPCR, nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) and western blot analysis. An increase in expression of CD38/NADase activity was observed during the kindling procedure in hippocampus that suggest it as one of the most important NAD+ degrading enzymes during epileptogenesis. Subsequently, gene expression of CD38 metabolite related proteins (Ryr2, FKBP-12.6, Chrm1, mGluR1 and Cnx43) were examined. Among them, changes in the expression level of mGluR1 was more than other genes, which was also confirmed by LC MS/MS and western blotting analysis. These findings provided valuable information about changes in the expression of CD38/cADPR signaling pathway and suggest its crucial role during epileptogenesis. [ABSTRACT FROM AUTHOR]

Many plants that are commonly used in folk medicine have multidirectional biological properties confirmed by scientific research. One of them is Aerva lanata (L.) Juss. (F. Amaranthaceae). It is widely used, but there are very few scientific data about its chemical composition and pharmacological activity. The aim of the present study was to investigate the chemical composition of phenolic acid (PA)-rich fractions isolated from methanolic extracts of A. lanata (L.) Juss. herb using the liquid/liquid extraction method and their potential antioxidant, anti-inflammatory, and anti-diabetic properties. The free PA fraction (FA), the PA fraction (FB) released after acid hydrolysis, and the PA fraction (FC) obtained after alkaline hydrolysis were analysed using liquid chromatography/electrospray ionization triple quadrupole mass spectrometry (LC-ESI-MS/MS). The phenolic profile of each sample showed a high concentration of PAs and their presence in A. lanata (L.) Juss. herb mainly in bound states. Thirteen compounds were detected and quantified in all samples, including some PAs that had not been previously detected in this plant species. Bioactivity assays of all fractions revealed high 2,2-diphenyl-1-picrylhydrazyl (DPPH•) (2.85 mM Trolox equivalents (TE)/g) and 2,2-azino-bis-3(ethylbenzthiazoline-6-sulphonic acid) (ABTS•+) (2.88 mM TE/g) scavenging activity. Fraction FB definitely exhibited not only the highest antiradical activity but also the strongest xanthine oxidase (XO) (EC50 = 1.77 mg/mL) and lipoxygenase (LOX)(EC50 = 1.88 mg/mL) inhibitory potential. The fraction had the best anti-diabetic properties, i.e., mild inhibition of α-amylase (EC50 = 7.46 mg/mL) and strong inhibition of α-glucosidase (EC50 = 0.30 mg/mL). The activities of all analysed samples were strongly related to the presence of PA compounds and the total PA content. [ABSTRACT FROM AUTHOR]

Organophosphorus (OP) nerve agents represent a class of highly toxic chemical warfare agents banned by the Chemical Weapons Convention. Nevertheless, in the past few years they have been used repeatedly for warfare, assassination and attempted murder. In addition, the chemically related OP pesticides were frequently used for suicide and may be deployed for terroristic attacks. Therefore, sensitive and selective bioanalytical methods are indispensable to investigate biological specimens as pieces of evidence to prove poisoning. OP agents form long-lived covalent reaction products (adducts) with endogenous proteins like human serum albumin (HSA) and butyrylcholinesterase (BChE). The adducted nonapeptide (NP) obtained by proteolysis of the BChE-adduct is one of the most sensitive and important biomarkers. We herein present a novel class of NP-adducts propionylated at its N-terminal phenylalanine residue (F195). The biomarker derivative is produced by addition of propionic anhydride to the NP-adduct inducing its quantitative conversion in aqueous buffer within 5 min at room temperature. Afterwards the mixture is directly analyzed by micro-liquid chromatography-electrospray ionization tandem-mass spectrometry (µLC-ESI MS/MS). The sensitivity of the method is comparable to that of the non-derivatized NP-adduct. These characteristics make the method highly beneficial for forensic analysis especially in cases in which the OP agent does not form adducts with HSA that are typically targeted as a second biomarker of exposure. This novel procedure was successfully applied to nerve agent-spiked samples sent by the Organisation for the Prohibition of Chemical Weapons (OPCW) as well as to plasma samples of real cases of pesticide poisoning. [ABSTRACT FROM AUTHOR]

• An improved QuEChERS for multiclass EOCs analysis in marine biota was developed. • The method was validated for various validation parameters. • High accuracy, precision and sensitivity was achieved for the optimized method. • The method was verified by determining EOCs in biota from field sampling. Marine biota is one of the important components of an ecosystem, and it has been widely used as a pollution indicator for emerging organic contaminants (EOCs) in the coastal zone. Previous studies divulged the concentration of EOCs in various types of biota matrices at trace level detection. EOCs have been associated with several adverse human health effects. Bioaccumulation of EOCs in the food web has been the main concern, and thus a sensitive and selective method for trace quantification of EOCs in biota matrices need to be developed and optimized. Considering various matrix interferences for EOC analysis in marine biota, a combination technique of QuEChERS and solid phase extraction cleanup with detection based on liquid chromatography with tandem mass spectrometry (LC MS/MS) is presented in this study. A method for 16 compounds grouped into four different classes, namely, pharmaceutically active chemicals, phenolic endocrine disrupter compounds, estrogenic hormones, and pesticides, was developed and validated for biota extraction. Satisfactory extraction was obtained for the optimized method with percentage of recovery from 64% to 114% and excellent sensitivity with detection limit in the range of 0.02–3.50 ng/g. Linearity of the standards (in the solvent) in the LC MS-MS analysis ranged from 0.991 to 0.999. The relative standard deviation for intra-day and inter-day repeatability was less than 20%, indicating good-precision analysis. Assessment on the matrix effects showed ionization suppression for all the developed compounds. The developed method was verified by analyzing biota matrices collected from the Klang River estuary. Trace concentrations of EOCs, ranging from 0.05 to 10.76 ng/g, were found in those matrices. Of the 16 targeted compounds, 10 were detected, namely, diclofenac, bisphenol A, sulfamethoxazole, amoxicillin, E2, E1, progesterone, testosterone, primidone, and 4-octylphenol. The other compounds were below the method detection limit. [ABSTRACT FROM AUTHOR]

Our previous studies have profiled lysine acetylation and succinylation modifications in Aeromonas hydrophila protein and have found that CobB may be involved in lysine deacylation; however, its effects on bacterial biological function are still unknown. In this study, a data-independent acquisition (DIA)-based proteomics method was used to compare the protein abundance between cob -deleted mutants and wild-type strains. Of the total 2385 identified proteins, 385 were found to have increased abundance, while only 46 showed decreased abundance. Data analysis revealed that many proteins in six metabolic pathways, ribosome, the bacterial secretion system, protein export, RNA degradation, beta-Lactam resistance and oxidative phosphorylation, were affected by the deletion of cobB. Some proteins, such as outer membrane proteins, the two-component regulatory systems and transcriptional factor, were also regulated by cobB. The following phenotype assays confirmed that the ΔcobB mutant produced more biofilm, migrated farther in soft agar, and was more sensitive to oxidative stress than its WT parent. Taken together, the results presented herein provide insights into the behaviors of sirtuin protein CobB in bacteria and demonstrate its important biological functions in A. hydrophila. The sirtuin protein CobB play crucial roles on lysine deacylation, such as desuccinylation and deacetylation in many bacterial species, while the intrinsic behavior of CobB on bacteria remains elusive. The current DIA-based quantitative proteomics analysis showed that the deletion of A. hydrophila cobB significantly affect the intracellular biological processes. Further phenotype assays validated proteomics results. Overall, our data further confirmed the important roles of CobB on the complex protein-protein interaction network regulation in A. hydrophila. Unlabelled Image • The protein profiles of ΔahcobB and WT strain were compared. • 385 proteins were increased abundance while 46 were decreased. • Six biological pathways were affected by the deletion of ahcobB. • The ahcobB phenotypes were further validated. [ABSTRACT FROM AUTHOR]