Genetic variation in glia-neuron signalling modulates ageing rate.

The rate of behavioural decline in the ageing population is remarkably variable among individuals. Despite the considerable interest in studying natural variation in ageing rate to identify factors that control healthy ageing, no such factor has yet been found. Here we report a genetic basis for variation in ageing rates in Caenorhabditis elegans. We find that C. elegans isolates show diverse lifespan and age-related declines in virility, pharyngeal pumping, and locomotion. DNA polymorphisms in a novel peptide-coding gene, named regulatory-gene-for-behavioural-ageing-1 (rgba-1), and the neuropeptide receptor gene npr-28 influence the rate of age-related decline of worm mating behaviour; these two genes might have been subjected to recent selective sweeps. Glia-derived RGBA-1 activates NPR-28 signalling, which acts in serotonergic and dopaminergic neurons to accelerate behavioural deterioration. This signalling involves the SIR-2.1-dependent activation of the mitochondrial unfolded protein response, a pathway that modulates ageing. Thus, natural variation in neuropeptide-mediated glia-neuron signalling modulates the rate of ageing in C. elegans.

Stable Isotope Labeling-Based Nontargeted Strategy for Characterization of the In Vitro Metabolic Profile of a Novel Doping BPC-157 in Doping Control by UHPLC-HRMS.

Traditional strategies for the metabolic profiling of doping are limited by the unpredictable metabolic pathways and the numerous proportions of background and chemical noise that lead to inadequate metabolism knowledge, thereby affecting the selection of optimal detection targets. Thus, a stable isotope labeling-based nontargeted strategy combined with ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) was first proposed for the effective and rapid metabolism analysis of small-molecule doping agents and demonstrated via its application to a novel doping BPC-157. Using 13C/15N-labeled BPC-157, a complete workflow including automatic 13C0,15N0-13C6,15N2m/z pair picking based on the characteristic behaviors of isotope pairs was constructed, and one metabolite produced by a novel metabolic pathway plus eight metabolites produced by the conventional amide-bond breaking metabolic pathway were successfully discovered from two incubation models. Furthermore, a specific method for the detection of BPC-157 and the five main metabolites in human urine was developed and validated with satisfactory detection limits (0.01~0.11 ng/mL) and excellent quantitative ability (linearity: 0.02~50 ng/mL with R2 > 0.999; relative error (RE)% < 10% and relative standard deviation (RSD)% < 5%; recovery > 90%). The novel metabolic pathway and the in vitro metabolic profile could provide new insights into the biotransformation of BPC-157 and improved targets for doping control.

Qualitative and quantitative analysis of ephedrine stimulants in urine by ultra-performance liquid chromatography-tandem mass spectrometry.

RATIONALE: Ephedrine analogues are stimulants that are explicitly required to be quantified and characterized in the Anti-Doping Prohibited List of the World Anti-Doping Agency. Given the difficulty of distinguishing diastereoisomers, the qualitative and quantitative analyses of ephedrine diastereoisomers are difficult. METHODS: An ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS) method was developed to detect five ephedrine analogues, and two pairs of diastereoisomers were identified using this method. The samples were analyzed qualitatively and quantitatively using a tandem mass spectrometer with an electrospray ionization source in multiple reaction detection mode after one-step dilution. RESULTS: The effective detection limits of this method were below 0.5 ng/mL. A matrix effect (range: 83.4% to 102%) was observed in quality control samples. The intra- and inter-day precision was lower than 9.16% and 8.60%, respectively, and the accuracy was within ±8.0%. CONCLUSIONS: The method is efficient, accurate, stable and sensitive, and fully meets the requirements for the detection of ephedrine substances in stimulants.

Integrated metabolomics and transcriptomics study of traditional herb Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao reveals global metabolic profile and novel phytochemical ingredients.

BACKGROUND: Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao is one of the most common herbs widely used in South and East Asia, to enhance people's health and reinforce vital energy. Despite its prevalence, however, the knowledge about phytochemical compositions and metabolite biosynthesis in Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao is very limited. RESULTS: An integrated metabolomics and transcriptomics analysis using state-of-the-art UPLC-Q-Orbitrap mass spectrometer and advanced bioinformatics pipeline were conducted to study global metabolic profiles and phytochemical ingredients/biosynthesis in Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao. A total of 5435 metabolites were detected, from which 2190 were annotated, representing an order of magnitude increase over previously known. Metabolic profiling of Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao tissues found contents and synthetic enzymes for phytochemicals were significantly higher in leaf and stem in general, whereas the contents of the main bioactive ingredients were significantly enriched in root, underlying the value of root in herbal remedies. Using integrated metabolomics and transcriptomics data, we illustrated the complete pathways of phenylpropanoid biosynthesis, flavonoid biosynthesis, and isoflavonoid biosynthesis, in which some were first reported in the herb. More importantly, we discovered novel flavonoid derivatives using informatics method for neutral loss scan, in addition to inferring their likely synthesis pathways in Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao. CONCLUSIONS: The current study represents the most comprehensive metabolomics and transcriptomics analysis on traditional herb Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao. We demonstrated our integrated metabolomics and transcriptomics approach offers great potentials in discovering novel metabolite structure and associated synthesis pathways. This study provides novel insights into the phytochemical ingredients, metabolite biosynthesis, and complex metabolic network in herbs, highlighting the rich natural resource and nutritional value of traditional herbal plants.

Novel affinity monolithic column modified with cuprous sulfide nanoparticles for the selective enrichment of low-molecular-weight electron-rich analytes.

A novel monolithic column modified with cuprous sulfide nanoparticles was developed and its affinity characteristics towards low-molecular-weight electron-rich analytes were investigated. In the synthesis process, home-made cuprous oxide nanocubes were immobilized on the surface of monolithic skeleton with the moderate thickness based on the strong interaction between imidazole groups and cuprous oxide, then the cuprous oxide layer was transformed into the more stable cuprous sulfide layer through the treatment by sodium sulfide. The resulting cuprous sulfide modified monolithic column presented good permeability and stability in a wide pH range from 2 to 10. Two kinds of typical electron-rich analytes, kanamycin A and purine, were chosen to assess its affinity characteristics. Compared with the commercial Cu(2+) - and Ni(2+) -based affinity sorbents, a larger binding capacity of cuprous sulfide modified column toward kanamycin A was obtained under basic condition and the recovery of kanamycin A in a milk sample was over 70%. Moreover, the binding capacity of cuprous sulfide modified column for purine was up to 5.57 mg/mL in frontal elution mode. These results suggested that the Cu2 S column has a promising application for the enrichment of electron-rich analytes.

Facile synthesis of boronate-decorated polyethyleneimine-grafted hybrid magnetic nanoparticles for the highly selective enrichment of modified nucleosides and ribosylated metabolites.

Ribosylated metabolites, especially modified nucleosides, have been extensively evaluated as cancer-related biomarkers. Boronate adsorbents are considered to be promising materials for extracting them from complex matrices. However, the enrichment of ribosylated metabolites in low abundance is still a challenge due to the limited capacity and selectivity of the existing boronate adsorbents. In this study, a novel type of magnetic nanoparticles named Fe3O4@SiO2@PEI-FPBA was synthesized by grafting polyethyleneimine (PEI) onto the surface of Fe3O4@SiO2 before modification by boronate groups. The high density of the amino groups on the PEI chains supplied a large number of binding sites for boronate groups. Thus, the adsorption capacity (1.34 ± 0.024 mg/g) of the nanoparticles, which is 6- to 7-fold higher than that of analogous materials, was greatly improved. The unreacted secondary amines and tertiary amines of the PEI enhanced the aqueous solubility of the nanoparticles, which could efficiently reduce nonspecific adsorption. The nanoparticles were able to capture 1,2 cis-diol nucleosides from 1000-fold interferences. Moreover, the flexible chains of PEI were favorable for effective enrichment and quick equilibration (<2 min). Finally, 60 ribose conjugates were enriched from human urine using the nanoparticles. Among them, 43 were identified to be nucleosides and other ribosylated metabolites. Nine low abundance modified nucleosides were detected for the first time. In conclusion, Fe3O4@SiO2@PEI-FPBA is an attractive candidate material for the highly selective enrichment of 1,2-cis-diol compounds.

Multi-analyte screening of small peptides by alkaline pre-activated solid phase extraction coupled with liquid chromatography-high resolution mass spectrometry in doping controls.

Peptidic drugs with wide spectrum of physiological activity are of interest for cheating athletes and can be misused as doping in sports. A growing number of small peptide drugs capable of enhancing performance are included in the prohibited list issued by World Anti-Doping Agency (WADA), therefore the improvement of the detection methods is constantly needed. In the present study, a screening assay was developed comprising 54 prohibited small peptides and the related substances in urine by means of the alkaline pre-activated weak cation exchange-solid phase extraction (WCX-SPE) with liquid chromatography-high resolution mass spectrometry (LCHRMS). This method performed good enrichment and purification effect of traditional WCX for basic peptides, and also improve the purification power of acidic peptides, which significantly expanded the coverage of detection substances. The method was validated in accordance with WADA relevant criteria and validated with a main focus on qualitative parameters including selectivity, limits of detection (0.02-0.2 ng/mL), linearity (0.1-20 ng/mL for 46 analytes and 0.2-20 ng/mL for 9 analytes), accuracy and precision (RE% and RSD% < 20% at 1, 5 and 10 ng/mL), recovery (39.2%-100.1% except for the TB500(1-2) free acid: 9.2%), matrix effects (ion suppression effect: 0 to 49.4% and ion enhancement effect: 100% and 264.6%), carryover, reliability and sample extract stability. As a proof-of-principle, urine samples from two patients received a single injection of leuprorelin acetate microspheres (3.75 mg) 30 days before were analyzed and the results proved the applicability of the method.

Automated online dried blood spot sample preparation and detection of anabolic steroid esters for sports drug testing.

Dried blood spots (DBSs) provide a valuable complementary sample matrix for routine doping analysis, and the full automation of analysis for DBS samples is achievable to avoid extensive and repetitive manual laboratory work. In the current study, a fully automated online DBS preparation and detection method for the screening and quantification analysis of 13 anabolic steroid esters by means of the DBSA-TLX-HRMS system was developed and validated, based on the purpose for the determination of the abuse of anabolic steroid esters in athletes. Validation of the method yielded linear (R2 > 0.99), precise, and accurate (RSD% and Re% < 20% at low, medium, and high concentration levels) results. The LOD of testosterone laurate was established at 0.5 ng/ml and at 0.2 ng/ml for other steroid esters. The extraction recovery of the target compounds from DBS ranged from 10.5% to 88.9%, and matrix effects were moderate. Furthermore, the developed and validated method was applied in the analysis of DBS samples collected after the oral administration of a single dose of 80 mg testosterone undecanoate demonstrating its applicability. Evaluation of analyte stability showed that testosterone undecanoate are more stable (8 weeks) in DBS samples of administration study when stored in frozen (-20°C) condition compared with cold storage (4°C). Collectively, these findings demonstrate the applicability of automated DBS analysis in doping control for detection of anabolic steroid esters.

Expanding the Coverage of Metabolic Landscape in Cultivated Rice with Integrated Computational Approaches.

Genome-scale metabolomics analysis is increasingly used for pathway and function discovery in the post-genomics era. The great potential offered by developed mass spectrometry (MS)-based technologies has been hindered, since only a small portion of detected metabolites were identifiable so far. To address the critical issue of low identification coverage in metabolomics, we adopted a deep metabolomics analysis strategy by integrating advanced algorithms and expanded reference databases. The experimental reference spectra and in silico reference spectra were adopted to facilitate the structural annotation. To further characterize the structure of metabolites, two approaches were incorporated into our strategy, i.e., structural motif search combined with neutral loss scanning and metabolite association network. Untargeted metabolomics analysis was performed on 150 rice cultivars using ultra-performance liquid chromatography coupled with quadrupole-Orbitrap MS. Consequently, a total of 1939 out of 4491 metabolite features in the MS/MS spectral tag (MS2T) library were annotated, representing an extension of annotation coverage by an order of magnitude in rice. The differential accumulation patterns of flavonoids between indica and japonica cultivars were revealed, especially O-sulfated flavonoids. A series of closely-related flavonolignans were characterized, adding further evidence for the crucial role of tricin-oligolignols in lignification. Our study provides an important protocol for exploring phytochemical diversity in other plant species.

Chemical proteomics reveal CD147 as a functional target of pseudolaric acid B in human cancer cells.

CD147 is a glycosylated transmembrane protein highly expressed on the surface of various tumor cells which plays vital roles in tumor invasion, progression and metastasis. We report the discovery of the natural product pseudolaric acid B (PAB) directly targeting CD147 by chemical proteomics utilizing a PAB-derived photoaffinity probe which could serve as a novel type of anticancer reagent.

Preparation and evaluation of a novel hybrid monolithic column based on pentafluorobenzyl imidazolium bromide ionic liquid.

To develop a novel hybrid monolithic column based on pentafluorobenzyl imidazolium bromide ionic liquid, a new ionic liquid monomer was synthesized from 1-vinylimidazole and pentafluorobenzyl bromide. By employing a facile one-step copolymerization of polyhedral-oligomeric-silsesquioxane-type (POSS) cross-linking agent and the home-made ionic liquid monomer, the hybrid monolithic columns were in situ fabricated in fused-silica capillary. The morphology of monolithic column was characterized by scanning electron microscope (SEM) and the chemical composition was confirmed by Fourier-transform infrared spectroscopy (FT-IR) and elemental analysis. Excellent mechanical stability and slight swelling propensity were exhibited which was ascribed to the rigid hybrid monolithic skeleton. Reproducibility results of run-to-run, column-to-column, batch-to-batch and day-to-day were investigated and the RSDs were less than 0.46%, 1.84%, 3.96% and 3.17%, respectively. The mixed-mode retention mechanism with hydrophobic interaction, π-π stacking, ion-exchange, electrostatic interaction and dipole-dipole interaction was explored systematically using analytes with different structure types. Satisfied separation capability and column efficiency were achieved for the analysis of small molecular compounds such as alkylbenzenes, polycyclic aromatic hydrocarbons, nucleosides and halogenated compounds.

[Preparation and application of mixed-mode capillary polymer monolithic column].

Based on the diversity of the retention mechanism, mixed-mode capillary monolithic columns have broad application prospect. In this work, a novel capillary polymer monolithic column was prepared with [2-(methacryloyloxy) ethyl] dimethyl-(3-sulfopropyl) ammonium hydroxide (SPE) as monomer, ethylene dimethacrylate (EDMA) as crosslinking agent, azobisisobutyronitrile (AIBN) as initiator and butylalcohol/1, 4-butanediol/water as ternary porogens. Under the optimized reaction conditions including the proportion of monomer and porogens, amount of initiator, reaction temperature and polymerization time, the monolithic column showed good mechanical strength up to 10 MPa, high permeability of 2. 17 x 10(-14) m2 and good repeatability. The peak area RSDs of column-to-column and batch-to-batch reproducibility were 1.0% and 4.6%, respectively. Finally, the capillary monolithic column was evaluated with polar and non-polar test mixtures. It showed hydrophilic interaction under high organic phase while hydrophobic interaction under low organic phase, indicating it a mixed-mode monolithic column.

Study of surface-bonded dicationic ionic liquids as stationary phases for hydrophilic interaction chromatography.

In the present study, several geminal dicationic ionic liquids based on 1,4-bis(3-allylimidazolium)butane and 1,8-bis(3-allylimidazolium)octane in combination with different anions bromide and bis(trifluoromethanesulphonyl)imide were prepared and then bonded to the surface of 3-mercaptopropyl modified silica materials through the "thiol-ene" click chemistry as stationary phases for hydrophilic interaction chromatography (HILIC). Compared with their monocationic analogues, the dicationic ionic liquids stationary phases presented effective retention and good selectivity for typical hydrophilic compounds under HILIC mode with the column efficiency as high as 130,000 plates/m. Moreover, the influence of different alkyl chain spacer between dications and combined anions on the retention behavior and selectivity of the dicationic ionic liquids stationary phases under HILIC mode was displayed. The results indicated that the longer linkage chain would decrease the hydrophilicity and retention on the dicationic ionic liquid stationary phase, and while differently combined anions had no difference due to the exchangeability under the common HILIC mobile phase with buffer salt. Finally, the retention mechanism was investigated by evaluating the effect of chromatographic factors on retention, including the water content in the mobile phase, the mobile phase pH and buffer salt concentration. The results showed that the dicationic ionic liquids stationary phases presented a mixed-mode retention behavior with HILIC mechanism and anion exchange.

A novel surface-confined glucaminium-based ionic liquid stationary phase for hydrophilic interaction/anion-exchange mixed-mode chromatography.

Glucaminium-based ionic liquids are a new class of recently developed ionic liquids and prepared by functionalizing the amine group of N-methyl-d-glucamine, which renders them good hydrophilicity due to the presence of the glucose structure and charged quaternary ammonium group. In the present study, a glucaminium-based ionic liquid N,N-diallyl-N-methyl-d-glucaminium bromide was synthesized and subsequently bonded to the surface of 3-mercaptopropyl modified silica materials through "thiol-ene" click chemistry. The obtained stationary phase was characterized by elemental analysis and infrared spectroscopy, and then packed as a HPLC column. A mixture of five nucleosides was used to characterize the separation performance of the obtained column under HILIC mode and the column efficiency was determined with cytidine as the test solute, reaching 80,000plates/m. Then, the retention behavior was evaluated by investigating the effect of various chromatographic factors on retention of different types of solutes, and the results revealed that the developed surface-confined glucaminium-based ionic liquid stationary phase exhibited a hydrophilic interaction/anion-exchange mixed-mode retention mechanism. Finally, two mixtures of nucleotides and flavonoids were separated on the glucaminium-based ionic liquid column, respectively under hydrophilic interaction and hydrophilic interaction/anion-exchange mixed-mode chromatography. In conclusion, the multimodal retention capabilities of the glucaminium-based ionic liquid column could offer a wider range of retention behavior and flexible selectivity toward polar and hydrophilic compounds.

Development and evaluation of new imidazolium-based zwitterionic stationary phases for hydrophilic interaction chromatography.

Hydrophilic interaction liquid chromatography (HILIC) has been widely used for separating polar compounds as a complement mode to reversed-phase liquid chromatography. The development of new stationary phases for HILIC is significant to improve the coverage of various polar and hydrophilic compounds. The present study described the preparation and application of novel imidazolium-based zwitterionic stationary phases. 1-Vinyl-3-(butyl-4-sulfonate) imidazolium was synthesized from 1-vinylimidazole and 1,4-butane sultone, then bonded to the surface of 3-mercaptopropyl modified silica particles (core-shell silica and totally porous silica) by "thiol-ene" click chemistry to obtain the resulting zwitterionic stationary phase with a positively charged imidazole ring and a negatively charged sulfonate group. The zwitterionic stationary phases exhibited good selectivity and favorable retention for a wide range of polar solutes (nucleosides, nucleic acid bases, benzoic organic acids, uric acid and its methyl derivatives, water-soluble vitamins) as compared to a bare silica column. The column efficiency could reach up to 100,000 theoretical plates/m with cytosine as the test solute. The retention changes of various types of test solutes were investigated under different chromatographic conditions including water content, pH, buffer salt concentration in mobile phase and column temperature. The results indicated that the retention of solutes on the stationary phase was the outcome of a mixed-mode retention mechanism (i.e. a combination of adsorptive and partitioning interactions). In conclusion, the new imidazolium-based zwitterionic stationary phases have shown excellent chromatographic behavior for a variety of polar solutes under HILIC mode, and have a great potential as a new type of stationary phases for HILIC.

[Triacyliglyeride profiling in serum by silver ion high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry].

A simple and effective method was developed to investigate triacylglyceride (TAG) compounds in biological matrices using silver ion high performance liquid chromatography-mass spectrometry (Ag+ -HPLC-MS). The TAG compounds from mouse serum were extracted by a modified Folch method using the classical CHC13-MeOH solvent system. The extract was separated on a Varian ChromSpher 5 Lipids column with isocratic elution using acetonitrile-hexane (1: 99, v/v) as mobile phase at a flow rate of 0.75 mL/min, and detected by a mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source in positive ion mode to acquire sufficient MS information of TAG compounds by enhanced mass spectrometry (EMS), enhanced product ion (EPI) and neutral loss (NL) scans. The identification of TAG compounds was based on their chromatographic behaviors and MS data. Besides, the NL scans of thirty fatty acids were performed to further validate the results. Finally, 66 TAG compounds as well as 5 cholesteryl ether (CE) compounds were obtained from the mouse serum extract. This method is simple, reproducible and also suitable for the analysis of TAG compounds in other samples.

On-line two dimensional liquid chromatography/mass spectrometry for the analysis of triacylglycerides in peanut oil and mouse tissue.

Triacylglycerides (TAGs) are a large class of complex neutral lipids that naturally occur in both plants and animals. In the present work, an on-line comprehensive silver-ion liquid chromatography (silver-ion LC) × reversed-phase liquid chromatography (RPLC) system was constructed to analyze these compounds. A micro bore silver-ion modified column was employed in the first dimension with the commonly used hexane-based mobile phase. After a series of C18 columns were assessed, a wide bore column packed with 1.5 µm particles was selected as the second dimension column to reduce the negative effect caused by the large volume and strong solvent injection in the second dimension. The system coupled with mass spectrometry was applied to the analysis of an edible peanut oil and a mouse liver extract. Twenty-eight TAGs from the peanut oil and forty-four from the mouse liver were identified based on the TAGs' retention behaviors on the comprehensive two-dimensional LC system and their APCI MS fragments.

A fully automated system with on-line micro solid-phase extraction combined with capillary liquid chromatography-tandem mass spectrometry for high throughput analysis of microcystins and nodularin-R in tap water and lake water.

Microcystins and nodularins are cyclic peptide hepatotoxins and tumour promoters from cyanobacteria. The present study describes the development, validation and practical application of a fully automated analytical method based on on-line micro solid-phase extraction-capillary liquid chromatography-tandem mass spectrometry for the simultaneous determination of seven microcystins and nodularin-R in tap water and lake water. Aliquots of just 100 µL of water samples are sufficient for the detection and quantification of all eight toxins. Selected reaction monitoring was used to obtain the highest sensitivity. Good linear calibrations were obtained for microcystins (50-2000ng/L) and nodularin-R (25-1000 ng/L) in spiked tap water and lake water samples. Excellent interday and intraday repeatability were achieved for eight toxins with relative standard deviation less than 15.7% in three different concentrations. Acceptable recoveries were achieved in the three concentrations with both tap water matrix and lake water matrix and no significant matrix effect was found in tap water and lake water except for microcystin-RR. The limits of detection (signal to noise ratio=3) of toxins were lower than 56.6 ng/L which is far below the 1 µg/L defined by the World Health Organization provisional guideline for microcystin-LR. Finally, this method was successfully applied to lake water samples from Tai lake and proved to be useful for water quality monitoring.