Macrophage-specific FGFR1 deletion alleviates high-fat-diet-induced liver inflammation by inhibiting the MAPKs/TNF pathways.

Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disease that is substantially associated with obesity-induced chronic inflammation. Macrophage activation and macrophage-medicated inflammation play crucial roles in the development and progression of NAFLD. Furthermore, fibroblast growth factor receptor 1 (FGFR1) has been shown to be essentially involved in macrophage activation. This study investigated the role of FGFR1 in the NAFLD pathogenesis and indicated that a high-fat diet (HFD) increased p-FGFR1 levels in the mouse liver, which is associated with increased macrophage infiltration. In addition, macrophage-specific FGFR1 knockout or administration of FGFR1 inhibitor markedly protected the liver from HFD-induced lipid accumulation, fibrosis, and inflammatory responses. The mechanistic study showed that macrophage-specific FGFR1 knockout alleviated HFD-induced liver inflammation by suppressing the activation of MAPKs and TNF signaling pathways and reduced fat deposition in hepatocytes, thereby inhibiting the activation of hepatic stellate cells. In conclusion, the results of this research revealed that FGFR1 could protect the liver of HFD-fed mice by inhibiting MAPKs/TNF-mediated inflammatory responses in macrophages. Therefore, FGFR1 can be employed as a target to prevent the development and progression of NAFLD.

Long duration of atrial high-rate episode is more favorable in predicting ischemic stroke than high CHA2 DS2 -VASc score.

OBJECTIVE: This study aimed to explore the roles of duration and burden of atrial high-rate episode (AHRE) on ischemic stroke in patients with pacemaker implantation. METHODS: Patients with pacemaker implantation for bradycardia from 2013 to 2017 were consecutively enrolled. Data such as gender, age, combined diseases, type of AF, left atrial size, left ventricular size, left ventricular ejection fraction, CHA2 DS2 -VASc score, and anticoagulants were collected. The burden and duration of AHRE based on different interval partition were also recorded in detail to evaluate the impacts on ischemic stroke. Cox regression analysis with time-dependent covariates was conducted. RESULTS: A total of 220 patients with AHRE were enrolled. The average follow-up time was 48.42 ± 17.20 months. Univariate regression analysis showed that diabetes (p = .024), high CHA2 DS2 -VASc score (≥ 2) (p = .021), long mean AHRE burden (p = .011), long maximal AHRE burden (p = .015), long AHRE duration lasting≥48 h (p = .001) or 24 h (p = .001) or 12 h (p = .005) were prone to ischemic stroke. Further multivariate regression analysis showed that long duration of AHRE (≥48 h) (HR 10.77; 95% CI 3.22-55.12; p = .030) were significantly correlated with stroke in patients with paroxysmal AF. There was no significant correlation between the type of AF and stroke (p = .927). CONCLUSION: The longer duration of AHRE (≥48 h) was more favorable in predicting ischemic stroke than high CHA2 DS2 -VASc score (≥2).

Predictors of pacing-induced cardiomyopathy detection and outcomes demonstration after conduction system pacing upgrade on patients with long-term persistent atrial fibrillation.

OBJECTIVE: To identify the predictors of pacing-induced cardiomyopathy (PICM) and illustrate the safety and feasibility of conduction system pacing (CSP) upgrade on patients with long-term persistent atrial fibrillation (AF). METHODS: All patients with long-term persistent AF and normal left ventricular ejection fraction (LVEF) ≥50% were consecutively enrolled from January 2008 to December 2017, and all the patients with atrioventricular block (AVB) and high right ventricular pacing (RVP) percentage of at least 40%. The predictors of PICM were identified, and patients with PICM were followed up for at least 1 year regardless of CSP upgrade. Cardiac performances and lead outcomes were investigated in all patients before and after CSP upgrade. RESULTS: The present study included 139 patients, out of which 37 (26.62%) developed PICM, resulting in a significant decrease in the left ventricular ejection fraction (LVEF) from 56.11 ± 2.56% to 38.10 ± 5.81% (p< .01). The median duration for the development of PICM was 5.43 years. Lower LVEF (≤52.50%), longer paced QRS duration (≥175 ms), and higher RVP percentage (≥96.80%) were identified as independent predictors of PICM. Furthermore, the morbidity of PICM progressively increased with an increased number of predictors. The paced QRS duration (183.90 ± 22.34 ms vs. 136.57 ± 20.71 ms, p < .01), LVEF (39.35 ± 2.71% vs. 47.50 ± 7.43%, p < .01), and left ventricular end-diastolic diameter (LVEDD) (55.53 ± 5.67 mm vs. 53.20 ± 5.78 mm, p = .03) improved significantly on patients accepting CSP upgrade. CSP responses and complete reverse remodeling (LVEF ≥50% and LVEDD < 50 mm) were detected in 80.95% (17/21) and 42.9% (9/21) of patients. The pacing threshold (1.52 ± 0.78 V/0.4 ms vs. 1.27 ± 0.59 V/0.4 ms, p = .16) was stable after follow-up. CONCLUSION: PICM is very common in patients with long-term persistent AF, and CSP upgrade was favorable for better cardiac performance in this patient population.

Comprehensive insights into the metabolism characteristics of small RNA Qrr4 in Vibrio alginolyticus.

Vibrio alginolyticus is an important foodborne pathogen that can infect both humans and marine animals and cause massive economic losses in aquaculture. Small noncoding RNAs (sRNAs) are emerging posttranscriptional regulators that affect bacterial physiology and pathological processes. In the present work, a new cell density-dependent sRNA, Qrr4, was characterized in V. alginolyticus based on a previously reported RNA-seq analysis and bioinformatics approach. The effects of Qrr4 actions on the physiology, virulence, and metabolism of V. alginolyticus were comprehensively investigated based on molecular biology and metabolomics approaches. The results showed that qrr4 deletion markedly inhibited growth, motility and extracellular protease activities. Additionally, nontargeted metabolism and lipidomics analyses revealed that qrr4 deletion induced significant disturbance of multiple metabolic pathways. The key metabolic remodelling that occurred in response to qrr4 deletion was found to involve phospholipid, nucleotide, carbohydrate and amino acid metabolic pathways, providing novel clues about a potential mechanism via which mutation of qrr4 could interfere with cellular energy homeostasis, modulate membrane phospholipid composition and inhibit nucleic acid and protein syntheses to regulate the motility, growth and virulence characteristics of V. alginolyticus. Overall, this study provides a comprehensive understanding of the regulatory roles of the new cell density-dependent sRNA Qrr4 in V. alginolyticus. KEY POINTS: • A novel cell density-dependent sRNA, Qrr4, was cloned in V. alginolyticus. •Qrr4 regulated growth and virulence factors of V. alginolyticus. • Phospholipid, nucleotide and energy metabolisms were modulated obviously by Qrr4.

Effect of ethanolamine utilization on the pathogenicity and metabolic profile of enterotoxigenic Escherichia coli.

Bacterial pathogenicity is greatly affected by nutrient recognition and utilization in the host microenvironment. The characterization of enteral nutrients that promote intestinal pathogen virulence is helpful for developing new adjuvant therapies and inhibiting host damage. Ethanolamine (EA), as a major component of intestinal epithelial cells and bacterial membranes, is abundant in the intestine. Here, we provide the first demonstration that the critical human and porcine pathogen enterotoxigenic Escherichia coli (ETEC) can utilize EA as a nitrogen source, which affects its virulence phenotype. We found that compared with that in M9 medium (containing NH4Cl), EA inhibited ETEC growth to a certain extent; however, the relative expression levels of virulence-related genes, such as ltA (3.0-fold), fimH (2.9-fold), CfaD (2.6-fold), gspD (3.6-fold), and qesE (1.3-fold), increased significantly with 15 mM EA as a nitrogen source (P < 0.05), and the adhesion efficiency of ETEC to Caco-2 cells increased approximately 4.2-fold. In Caco-2 cells, the relative cell viability decreased from 74.8 to 63.4%, and the transepithelial electrical resistance (TEER) cells decreased to 74.8% with intestinal EA (4 mM). In addition, the relative expression levels of proinflammatory factors, such as TNF-α (3.2-fold), INF-γ (2.9-fold), and IL-1ß (1.98-fold), in ETEC-infected Caco-2 cells were significantly upregulated (P < 0.05) under EA exposure; however, the above virulence changes were not found in ΔeutR and ΔeutB ETEC. A gas chromatography-mass spectrometry (GC-MS)-based untargeted metabolomics approach was then employed to reveal EA-induced metabolic reprogramming related to ETEC virulence. The data showed that most metabolites related to carbohydrate, aspartate and glutamate metabolism, shikimic acid metabolism, and serine metabolism in ETEC exhibited a decreasing trend with increases in the EA concentration from 0 to 15 mM, but the branched-chain amino acid (BCAA) levels in ETEC increased in a dose-dependent manner under EA exposure. Our data suggest that the intestinal EA concentration can significantly affect the virulence phenotype, metabolic profile, and pathogenicity of ETEC. KEY POINTS: • ETEC growth and virulence gene expression could be regulated by ethanolamine. • The intestinal concentration of EA promoted the damaging effect of ETEC on the host epithelial barrier. • The promoting effect of EA on ETEC toxicity may be related to BCAA metabolism.

Process optimization of vanillin production by conversion of ferulic acid by Bacillus megaterium.

BACKGROUND: Vanillin is an important flavoring and aromatic ingredient found mainly in the pods of the tropical plant vanilla and is widely used in the food industry. Attempts have been made to produce vanillin from ferulic acid esters in agricultural residues of wheat bran. RESULTS: The results showed that a strain with high tolerance to the substrate ferulic acid was isolated and screened from soil and identified as belonging to the genus Bacillus (Bacillus megaterium). The concentration of vanillin produced by this strain was 0.048 g L-1 , and the molar conversion of vanillin was 12.25%. The production of vanillin was optimized by orthogonal experiments. Beef pastes 6.0 g L-1 , soybean meal 5.0 g L-1 , magnesium sulfate heptahydrate 1.0 g L-1 , iron(II) sulfate heptahydrate 1.0 g L-1 , calcium chloride 1.0 g L-1 , dipotassium hydrogen phosphate trihydrate 1.0 g L-1 ; fermentation culture conditions were pH 7.0, inoculum level 5%, loading volume 20%, ferulic acid 1.0 g L-1 , fermentation culture temperature 35 °C. The concentration of vanillin obtained was 0.218 g L-1 . Finally, transcriptomic analysis of the strain samples before and after the optimization of the fermentation conditions was carried out to study the effect of the optimization of the fermentation conditions on the concentration of vanillin produced by the strain. CONCLUSION: This study provides a theoretical basis for further improving the yield of vanillin and gradually realizing efficient industrial production. © 2022 Society of Chemical Industry.

Vanillic acid combats Vibrio alginolyticus by cell membrane damage and biofilm reduction.

Antibiotics are the most powerful weapon against bacterial infectious diseases in aquaculture. However, the indiscriminate usage of antibiotics often culminates in the emerging development of antibiotic-resistant bacteria, making it imperative to search for novel types of antimicrobial agents. This study investigated the antibacterial and antivirulence effects of vanillic acid (VA) against the fish pathogen, Vibrio alginolyticus. We showed that VA had a good anti-Vibrio activity with minimal inhibitory concentration (MIC) of 1.0 mg/ml. In addition, VA wielded its antibacterial action in a dose-/time-dependent manner by causing cell membrane damage and increasing membrane permeability, which is evidenced by increasing the conductivity and malondialdehyde content in the treated cell cultures and the scanning electron microscopy images. Furthermore, VA significantly reduced the biofilm-forming capability, mobility and exotoxin production (protease and exopolysaccharide) and downregulation of the expression of biofilm- and virulence-associated genes (sypG, fliS, fliK, lafA, lafK, asp and luxR) was seen in the V. alginolyticus that exposed to VA at subinhibitory concentrations. Overall, our findings suggested that VA may be of interest for treating V. alginolyticus-associated infections in aquaculture.

Antidepressant effects of total iridoids of Valeriana jatamansi via the intestinal flora-blood-brain barrier pathway.

CONTEXT: Valeriana jatamansi Jones [syn. V. wallichii DC, (Valerianaceae)] (VJJ) is used to treat depression. OBJECTIVE: To explore the effects of total iridoids of VJJ extract (TIV) on chronic unpredictable mild stress (CUMS) in mice. MATERIALS AND METHODS: VJJ roots and rhizomes were extracted with 70% ethanol. CUMS rats were treated daily with fluoxetine (2.6 mg/kg, i.g.) or TIV (5.7, 11.4, and 22.8 mg/kg, i.g.) for 14 days. Male Kun Ming mice on normal chow and 0.5% CMC-Na solution were used as a control. Behavioural tests included the tail suspension (TST) and sucrose preference tests (SPT). Evans blue staining was used to evaluate blood-brain barrier (BBB) permeability. Western blotting was used to measure zonula occludens-1 (ZO-1) and occludin expression. 16S rRNA sequencing was used to analyse intestinal flora abundance. Tax4Fun was used to predict KEGG metabolic pathways. RESULTS: TIV treatment reduced TST time (117.35 ± 8.23 or 108.95 ± 6.76 vs. 144.45 ± 10.30 s), increased SPT (55.83 ± 7.24 or 53.12 ± 13.85 vs. 38.98 ± 5.43%), increased the abundance of phylum Firmicutes (86.99 ± 0.03 vs. 60.88 ± 0.19%) and genus Lactobacillus (75.20 ± 0.19 vs. 62.10 ± 0.13%), reduced the abundance of phylum Bacteroidetes (6.69 ± 0.06 or 11.50 ± 0.09 vs. 25.07 ± 0.20%). TIV increased carbohydrate metabolism (14.50 ± 3.00 × 10-3 or 14.60 ± 2.00 × 10-3 or 14.90 ± 2.00 × 10-3 vs.13.80 ± 4.00 × 10-3%), replication and repair functions (5.60 ± 1.00 × 10-3 or 5.60 ± 1.00 × 10-3 vs. 5.10 ± 4.00 × 10-3%), reduced the frequency of infectious disease (1.60 ± 2.00 × 10-4 or 1.90 ± 5.00 × 10-4 or 1.80 ± 3.00 × 10-4 vs. 2.20 ± 7.00 × 10-3%), BBB permeability (0.77 ± 0.30 vs. 1.81 ± 0.33 µg/g), and up-regulated the expression of ZO-1 (1.42-fold, 1.60-fold, 1.71-fold) and occludin (1.79-fold, 2.20-fold). CONCLUSIONS: TIV may modulate the intestinal flora, thereby inducing the expression of ZO-1 and occludin, protecting the BBB and exerting an antidepressant effect.

Changes in transcriptomic and metabolomic profiles of morphotypes of Ophiocordyceps sinensis within the hemocoel of its host larvae, Thitarodes xiaojinensis.

BACKGROUND: Ophiocordyceps sinensis (Berk.) is a well-known entomopathogenic and medicinal fungus. It parasitizes and mummifies the underground ghost moth larvae to produce a fruiting body named Chinese cordyceps. Specific for the fungus, O. sinensis experiences a biotrophic vegetative growth period spanning over 5 months. During this vegetative growth, it appears successively in the host hemocoel in three/four morphotypes, namely, the yeast-like blastospores (subdivided into proliferative (BP) and stationary phase (BS)), prehyphae (PreHy) and the hyphae (Hy). This peculiar morphogenesis has been elucidated through morphological and ultrastructural observations, but its molecular basis remains cryptic. In this study, transcriptome and metabolome profiling of BP, BS, PreHy and Hy stages were performed to characterize the key genes, metabolites, and signaling pathways that regulated the vegetative development of O. sinensis in Thitarodes xiaojinensis larva. RESULTS: The molecular events and metabolic pathways that regulated different intracellular processes at various stages were examined. Cluster analyses of differentially expressed genes across the four stages revealed the stage specifically enriched pathways. Analysis of metabolome profiles showed that carbon metabolism and several amino acids biosynthesis were significantly perturbed during the tested development stages of O. sinensis in the host hemocoel. Genes homologous to Saccharomyces cerevisiae MAPK cascade were significantly up-regulated during the transition from blastospore to hypha. The up-regulation of Sho1, a regulator protein, suggested nutrient starvation act a role in activation of MAPK pathway and filamentous growth. In addition, up-regulation of several fatty acid synthesis genes and their corresponding products accumulation in the samples of BS might explain more lipid droplets were observed in BS than in BP. Coupled with the up-regulation of fatty acid degradation during PreHy and Hy stages, it is presumed that lipid accumulation and mobilization play important roles in filamentous development. CONCLUSIONS: This is the first report comprehensively describing developmental transcriptomics and metabolomics of O. sinensis in vivo. Our findings provide new perspectives into the key pathways and hub genes involved in morphological changes of fungus developed in the hemocoel of its host, and are expected to guide future studies on morphogenesis and morphotype changes of entomopathogenic fungi in vivo.

Characterization of a cell density-dependent sRNA, Qrr, and its roles in the regulation of the quorum sensing and metabolism in Vibrio alginolyticus.

Vibrio alginolyticus is an important fish pathogen causing pandemic diseases in marine animals. Small noncoding RNAs (sRNAs) are important posttranscriptional modulators of gene expression and involved in the pathogenesis of bacterial pathogens. Thus far, no cell density-dependent sRNA has been reported in V. alginolyticus. In this study, a cell density-dependent sRNA, Qrr, predicted based on the previous RNA-Seq analysis of V. alginolyticus cultured at low cell density (LCD) and high cell density (HCD), was characterized. The Qrr mutant showed significantly impaired growth and decreased swimming and swarming ability, and increased biofilm formation, extracellular polysaccharide content, serine protease production, and LD50 values during zebrafish infection in contrast to the wild-type strain. Qrr modulates the master regulators LuxR and AphA in quorum sensing (QS) pathways possibly at the posttranscriptional level by base pairing with the 5'-untranslated regions (5'-UTRs). Meanwhile, both LuxR and AphA could directly bind to the promoter of qrr to activate or repress its transcription, respectively. Moreover, our unbiased metabolic approaches revealed that Qrr modulates a large quantity of metabolic and lipidomic pathways, including amino acids, purine and pyrimidine derivatives, tricarboxylic acid cycle (TCA cycle) intermediates, and lipids. Collectively, this work contributes to a systematic understanding of regulatory roles of the cell density-dependent sRNA, Qrr, in V. alginolyticus.

Vegetative development and host immune interaction of Ophiocordyceps sinensis within the hemocoel of the ghost moth larva, Thitarodes xiaojinensis.

Ophiocordyceps sinensis is an entomopathogenic fungus that infects ghost moth larva, forming the most valuable and rare traditional Chinese medicine, Chinese cordyceps. Our knowledge of the basic morphology and developmental biology of Chinese cordyceps is limited. In this study, morphological and ultrastructural observations of O. sinensis development in the hemocoel of Thitarodes xiaojinensis were obtained by multiple light and electron microscopy techniques, and the host immune reaction activities were determined. Our results indicated that fungal cells in the host hemocoel underwent morphotype transformations from blastospores to prehyphae to hyphae in sequence. The fusiform yeast-like blastospores were the initial cell type present in the host hemocoel and remained for 5 months or more; the encapsulation reaction and phenoloxidase activity of T. xiaojinensis hemolymph were inhibited during this period. When larvae entered the last instar, the blastospores switched to prehyphae and expanded throughout the host tissues, and then hyphae germinated from the prehyphae and mycelia formed, which finally led to host death. Considering the distinct differences between blastospores and hyphae, we identified prehyphae, which play important roles in fungal expansion, hyphae germination, and fusion formation among filaments. Notably, the elongation of prehyphae was strongly presumed to occur through fission but without separation of the two sister cells, in contrast to blastospore budding. During the morphotype transformation, the amount and composition of lipid droplets changed greatly, suggesting their important roles in these events. Overall, we provide a morphological and ultrastructural characterization of O. sinensis vegetative development within the hemocoel of T. xiaojinensis, identify and name the prehypha fungal cell type in entomopathogenic fungi for the first time, and conclude that O. sinensis infection causes sustained immunosuppression in T. xiaojinensis.

Structurally Defined Ru(II) Metallointercalators for Real-Time Monitoring of DNA Amplification Reactions.

The low cost and convenience of fluorescent DNA binding dyes make them widely used for real-time DNA analysis. Even though several dyes for real-time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assays are commercially available, most of them rely on organic parent molecules with an indefinite chemical structure, making it difficult to predict their behavior in nucleic acid amplification and to get the best performance. Herein, we demonstrate for the first time the use of the structurally defined dipyridophenazine complexes of ruthenium(II) for real-time monitoring of PCR and LAMP. These inorganic metallointercalators exhibit less inhibition on DNA amplification. They can quantify a range of initial template concentrations and provide the product melting curve to differentiate the unspecific amplification and even perform multiplex assays. These ruthenium(II) complexes have the advantages of the defined structure, nontoxicity, good stability, and facile synthesis, and they may act as excellent alternative DNA binding dyes with wide applications in the field of nucleic acid research.

Metabolic changes in primary, secondary, and lipid metabolism in tobacco leaf in response to topping.

As an important cultivation practice used for flue-cured tobacco, topping affects diverse biological processes in the later stages of development and growth. Some studies have focused on using tobacco genes to reflect the physiological changes caused by topping. However, the complex metabolic shifts in the leaf resulting from topping have not yet been investigated in detail. In this study, a comprehensive metabolic profile of primary, secondary, and lipid metabolism in flue-cured tobacco leaf was generated with use of a multiple platform consisting of gas chromatography-mass spectrometry, capillary electrophoresis-mass spectrometry, and liquid chromatography-mass spectrometry/ultraviolet spectroscopy. A total of 367 metabolites were identified and determined. Both principal component analysis and the number of significantly different metabolites indicated that topping had the greatest influence on the upper leaves. During the early stage of topping, great lipid level variations in the upper leaves were observed, and antioxidant defense metabolites were accumulated. This indicated that the topping activated lipid turnover and the antioxidant defense system. At the mature stage, lower levels of senescence-related metabolites and higher levels of secondary metabolites were found in the topped mature leaves. This implied that topping delayed leaf senescence and promoted secondary metabolite accumulation. This study provides a global view of the metabolic perturbation in response to topping. Graphical abstract Metabolic alterations in tobacco leaf in response to topping using a multiplatform metabolomics.

Metabolic Profiling with Gas Chromatography-Mass Spectrometry and Capillary Electrophoresis-Mass Spectrometry Reveals the Carbon-Nitrogen Status of Tobacco Leaves Across Different Planting Areas.

The interaction between carbon (C) and nitrogen (N) metabolism can reflect plant growth status and environmental factors. Little is known regarding the connections between C-N metabolism and growing regions under field conditions. To comprehensively investigate the relationship in mature tobacco leaves, we established metabolomics approaches based on gas chromatography-mass spectrometry (GC-MS) and capillary electrophoresis-time-of-flight-mass spectrometry (CE-TOF-MS). Approximately 240 polar metabolites were determined. Multivariate statistical analysis revealed that the growing region greatly influenced the metabolic profiles of tobacco leaves. A metabolic correlation network and related pathway maps were used to reveal the global overview of the alteration of C-N metabolism across three typical regions. In Yunnan, sugars and tricarboxylic acid (TCA) cycle intermediates were closely correlated with amino acid pools. Henan tobacco leaves showed positive correlation between the pentose phosphate pathway (PPP) intermediates and C-rich secondary metabolism. In Guizhou, the proline and asparagine had significant links with TCA cycle intermediates and urea cycle, and antioxidant accumulation was observed in response to drought. These results demonstrate that combined analytical approaches have great potential to detect polar metabolites and provide information on C-N metabolism related to planting regional characteristics.

A Novel Strategy for Large-Scale Metabolomics Study by Calibrating Gross and Systematic Errors in Gas Chromatography-Mass Spectrometry.

Metabolomics is increasingly applied to discover and validate metabolite biomarkers and illuminate biological variations. Combination of multiple analytical batches in large-scale and long-term metabolomics is commonly utilized to generate robust metabolomics data, but gross and systematic errors are often observed. The appropriate calibration methods are required before statistical analyses. Here, we develop a novel correction strategy for large-scale and long-term metabolomics study, which could integrate metabolomics data from multiple batches and different instruments by calibrating gross and systematic errors. The gross error calibration method applied various statistical and fitting models of the feature ratios between two adjacent quality control (QC) samples to screen and calibrate outlier variables. Virtual QC of each sample was produced by a linear fitting model of the feature intensities between two neighboring QCs to obtain a correction factor and remove the systematic bias. The suggested method was applied to handle metabolic profiling data of 1197 plant samples in nine batches analyzed by two gas chromatography-mass spectrometry instruments. The method was evaluated by the relative standard deviations of all the detected peaks, the average Pearson correlation coefficients, and Euclidean distance of QCs and non-QC replicates. The results showed the established approach outperforms the commonly used internal standard correction and total intensity signal correction methods, it could be used to integrate the metabolomics data from multiple analytical batches and instruments, and it allows the frequency of QC to one injection of every 20 real samples. The suggested method makes a large amount of metabolomics analysis practicable.

Sample-directed pseudotargeted method for the metabolic profiling analysis of rice seeds based on liquid chromatography with mass spectrometry.

Rice is one of the most important food crops in the world. Metabolite composition in rice seeds varies significantly depending on genetic variety, climatic alternation and agricultural practice. Metabolomics is a powerful tool to reveal the metabolic response of rice to various conditions. In this work, a rice seed sample-directed pseudotargeted metabolomics method was first established and validated based on ultra high performance liquid chromatography with triple quadrupole mass spectrometry in the multiple reaction monitoring mode. A total of 749 and 617 ion pairs in positive and negative modes were achieved, respectively. Among them, about 200 metabolites were identified or tentatively identified. The developed method showed better linearity and repeatability than those of non-targeted metabolomics method. Good intra-day and inter-day precisions, recoveries and wide linear range were also obtained. Furthermore, the method was applied for the investigation of metabolic variation of rice seeds with two wild cultivars and their transgenic lines that were grown in two locations. Principal component analysis indicated that the effects of cultivar and location on metabolic variations were far more than those of gene modification. The nonparametric Mann-Whitney U test revealed that most metabolites were influenced by cultivar, location and gene modifications together.

Lipidome and metabolome analysis of fresh tobacco leaves in different geographical regions using liquid chromatography-mass spectrometry.

The combination of the lipidome and the metabolome can provide much more information in plant metabolomics studies. A method for the simultaneous extraction of the lipidome and the metabolome of fresh tobacco leaves was developed. Method validation was performed on the basis of the optimal ratio of methanol to methyl tert-butyl ether to water (37:45:68) from the design of experiments. Good repeatability was obtained. We found that 92.2% and 91.6% of the peaks for the lipidome and the metabolome were within a relative standard deviation of 20%, accounting for 94.6% and 94.6% of the total abundance, respectively. The intraday and interday precisions were also satisfactory. A total of 230 metabolites, including 129 lipids, were identified. Significant differences were found in lipidomic and metabolomic profiles of fresh tobacco leaves in different geographical regions. Highly unsaturated galactolipids, phosphatidylethanolamines, predominant phosphatidylcholines, most of the polyphenols, amino acids, and polyamines had a higher content in Yunnan province, and low-unsaturation-degree galactolipids, triacylglycerols, glucosylceramides with trihydroxy long-chain bases, acylated sterol glucosides, and some organic acids were more abundant in Henan province. Correlation analysis between differential metabolites and climatic factors indicated the vital importance of temperature. The fatty acid unsaturation degree of galactolipids could be influenced by temperature. Accumulation of polyphenols and decreases in the ratios of stigmasterols to sitosterols and glucosylstigmasterols to glucosylsitosterols were also correlated with lower temperature in Yunnan province. Furthermore, lipids were more sensitive to climatic variations than other metabolites.

[Analysis of factors associated with decline of FEV1 among community population in urban area of Beijing].

OBJECTIVE: To investigate risk factors correlated to the decline of FEV1among community population in the urban area of Beijing. METHOD: Subjects no younger than 40 years old were recruited from three communities in the urban area of Beijing. All of them were asked to fill in a questionnaire in regard to general health conditions, past medical history, medication usage, smoking history, etc. FEV1and FEV6 were measured by Vitalograph COPD-6 spirometer using the standard protocol. Two years after the first visit, questionnaire survey and spirometry were repeated. RESULT: Four hundred and fifty two subjects fulfilled the inclusion criteria and finished the 2nd visit, with an average age of (58.8 ± 8.6) years, 29% male and 71% female. The mean decline rate of FEV1in the cohort was (43 ± 114) ml per year. There was no significant difference of mean FEV1decline between different gender and age groups. A mean decline of FEV1by (64 ± 125) ml per year was observed in smokers (including former smokers and current smokers) whereas the decline rate in non-smokers was (36 ± 109) ml per year (P = 0.030). There was no significant statistical difference among current smokers, former smokers, passive smokers and subjects who never smoke. A higher decline rate of FEV1was observed in subjects with a history of COPD or airway hyperreactivity, chronic cough, diabetes, hypertension and coronary heart disease. The difference, however, was not statistically significant. Binary logistic regression was used to screen risk factors affected the FEV1decline rate between rapid decline (ΔFEV1 ≥ 30 ml/y) and non-rapid decline (ΔFEV1 < 30 ml/y), and found smoking was an independent risk factor of FEV1decline rate. CONCLUSION: The mean rate of FEV1 decline in 2.6 years in the surveyed community population in the urban area of Beijing was (43 ± 114) ml per year; Smoking is an independent risk factor of FEV1decline.

Metabolomics study of cured tobacco using liquid chromatography with mass spectrometry: method development and its application in investigating the chemical differences of tobacco from three growing regions.

Cured tobacco is an important plant material. Component studies are a big challenge for its significantly diverse chemical properties and vastly different concentrations. In this work, liquid chromatography with quadrupole time-of-flight mass spectrometry was used to perform a metabolomics study of cured tobacco owing to its efficient separation and detection of semipolar metabolites. A solvent of methanol/water (8:2, v/v) and 30 min of ultrasound time were found to be optimal to perform extraction. 95, 92, and 93% of metabolite features had within 20% of coefficient of variation for repeatability, intraday and interday precision analysis, respectively, indicating a good stability of the method developed. 113 metabolites were identified in cured tobacco based on accurate mass, retention time, and MS/MS fragments. The developed method was applied to a metabolomics study of cured tobacco from three growing regions. Forty three metabolites were found to be contributed to the classification. It is shown that the developed method can be applied to metabolomics analysis of plant materials.

Study of metabolite differences of flue-cured tobacco from different regions using a pseudotargeted gas chromatography with mass spectrometry selected-ion monitoring method.

A pseudotargeted method based on gas chromatography and mass spectrometry with selected-ion monitoring was established to investigate the metabolite differences of flue-cured tobacco from three different growing regions. The mixed solvent of acetonitrile/isopropanol/water (3:3:2, v/v/v) was chosen as the optimal extraction system based on the good repeatability and extraction efficiency. A self-developed software coupled with commercial software was used to establish the pseudotargeted method including 289 peaks and 47 groups. Multivariable statistical analysis indicated that tobacco samples can be obviously separated based on the geographical origins. On the basis of a Mann-Whitney U test, organic acids, phenols, and alkaloids had higher levels in Hunan province. In contrast, a large proportion of amino acids (including L-tyrosine, L-proline, and serine), sucrose, and linoleic acid were the highest in Yunnan province. Meanwhile, multiple metabolic pathways (including carbohydrate metabolism, tricarboxylic acid cycle, and nitrogen metabolism) were influenced by growing regions. Twenty-eight differential metabolites, which had great contributions to the classification of tobacco samples of three growing regions, were further defined. The results demonstrated that the developed pseudotargeted method was a powerful tool to investigate the metabolic profiling of tobacco leaves and discriminate tobacco leaves of different growing regions.