Estrogen-Related Receptor γ Maintains Pancreatic Acinar Cell Function and Identity by Regulating Cellular Metabolism.

BACKGROUND & AIMS: Mitochondrial dysfunction disrupts the synthesis and secretion of digestive enzymes in pancreatic acinar cells and plays a primary role in the etiology of exocrine pancreas disorders. However, the transcriptional mechanisms that regulate mitochondrial function to support acinar cell physiology are poorly understood. Here, we aim to elucidate the function of estrogen-related receptor γ (ERRγ) in pancreatic acinar cell mitochondrial homeostasis and energy production. METHODS: Two models of ERRγ inhibition, GSK5182-treated wild-type mice and ERRγ conditional knock-out (cKO) mice, were established to investigate ERRγ function in the exocrine pancreas. To identify the functional role of ERRγ in pancreatic acinar cells, we performed histologic and transcriptome analysis with the pancreas isolated from ERRγ cKO mice. To determine the relevance of these findings for human disease, we analyzed transcriptome data from multiple independent human cohorts and conducted genetic association studies for ESRRG variants in 2 distinct human pancreatitis cohorts. RESULTS: Blocking ERRγ function in mice by genetic deletion or inverse agonist treatment results in striking pancreatitis-like phenotypes accompanied by inflammation, fibrosis, and cell death. Mechanistically, loss of ERRγ in primary acini abrogates messenger RNA expression and protein levels of mitochondrial oxidative phosphorylation complex genes, resulting in defective acinar cell energetics. Mitochondrial dysfunction due to ERRγ deletion further triggers autophagy dysfunction, endoplasmic reticulum stress, and production of reactive oxygen species, ultimately leading to cell death. Interestingly, ERRγ-deficient acinar cells that escape cell death acquire ductal cell characteristics, indicating a role for ERRγ in acinar-to-ductal metaplasia. Consistent with our findings in ERRγ cKO mice, ERRγ expression was significantly reduced in patients with chronic pancreatitis compared with normal subjects. Furthermore, candidate locus region genetic association studies revealed multiple single nucleotide variants for ERRγ that are associated with chronic pancreatitis. CONCLUSIONS: Collectively, our findings highlight an essential role for ERRγ in maintaining the transcriptional program that supports acinar cell mitochondrial function and organellar homeostasis and provide a novel molecular link between ERRγ and exocrine pancreas disorders.

Enantioseparation and molecular modeling study of chiral amines as three naphthaldimine derivatives using amylose or cellulose trisphenylcarbamate chiral stationary phases.

This study describes the enantioseparation of three chiral amines as naphthaldimine derivatives, using normal phase HPLC with amylose and cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phases (CSPs). Three chiral amines were derivatized using three structurally similar naphthaldehyde derivatizing agents, and the enantioselectivity of the CSPs toward the derivatives was examined. The degree of enantioseparation and resolution was affected by the amylose or cellulose-derived CSPs and aromatic moieties as well as a kind of chiral amine. Especially, efficient enantiomer separation was observed for 2-hydroxynapthaldimine derivatives on cellulose-derived CSPs. Molecular docking studies of three naphthaldimine derivatives of leucinol on cellulose tris(3,5-dimethylphenylcarbamate) were performed to estimate the binding energies and conformations of the CSP-analyte complexes. The obtained binding energies were in good agreement with the experimentally determined enantioseparation and elution order.

Chiral LC-MS/MS method for the discrimination of triiodothyronine enantiomers on a crown ether-based chiral stationary phase.

Considering the greater pharmaceutical and clinical interest of triiodothyronine (T3 ) thyroid hormone, an effective D/L-T3 enantiomer separation was performed on a crown ether-based chiral stationary phase by LC-MS/MS. In optimal analytical condition and selected reaction monitoring mode, the two enantiomers of T3 were baseline separated within 10 min. The limit of detection and limit of quantitation were found to be 0.05 and 0.10 ng/µl; 0.20 and 0.50 ng/µl for D- and L-T3 , respectively. During validation, this method proved to be feasible, accurate as well as enantioselective and sensitive for the resolution of T3 enantiomers. For commercial D- and L-T3 chemicals, the enantiomeric impurities as the other enantiomer were 0.11% and 4.61%. On the other hand, the impurity as D-T3 for commercial pharmaceutical products (liothyronine sodium tablets, two suppliers) was 0.68% and 6.57%.

Metabolomic analysis of amino acids and organic acids in aging mouse eyes using gas chromatography-tandem mass spectrometry.

This is a metabolomics study for monitoring altered amino acid (AA) and organic acid (OA) metabolism of in eyes from aging an mouse model at 8 and 18 weeks and 18 months. Simultaneous metabolic profiling analysis of OAs and AAs was performed as ethoxycarbonyl/methoxime/tert-butyldimethylsilyl derivatives by gas chromatography-tandem mass spectrometry. A total of 42 metabolites-24 AAs and 18 OAs-were determined and their composition values were normalized to the corresponding mean values of 8-week-old mice as the control group. Then their normalized values were plotted as star graphs, which were distorted and readily distinguishable for each age-related group. Among the 42 metabolites, 18 AAs and 11 OAs were age dependent and significantly different (p < 0.05). Principal component analysis and partial least squares discriminant analysis showed unclear separation between 8- and 18-week-old mice but clear separation between these and 18-month-old mice. In particular, the variable importance in projection scores of 4-hydroxyproline, cis-aconitic acid, glycine, isocitric acid, leucine, pipecolic acid and lysine from partial least-squares-discriminant analysis were higher than 1.3. A heatmap for the classification and visualization of 42 metabolites showed differences in metabolite changes with aging. Altered AA and OA profiles were monitored, which may explain the metabolic disturbance of AA and OA. These findings are related to mitochondrial dysfunctions related to energy metabolism and the impaired antioxidant system in the aging eye. Therefore, the present metabolomics results of the association between physiological states and altered metabolism of AA and OA will be useful for understanding the aging eye and related diseases.

Comprehensive Targeted Metabolomic Study in the Lung, Plasma, and Urine of PPE/LPS-Induced COPD Mice Model.

(1) Background: Progression of chronic obstructive pulmonary disease (COPD) leads to irreversible lung damage and inflammatory responses; however, biomarker discovery for monitoring of COPD progression remains challenging. (2) Methods: This study evaluated the metabolic mechanisms and potential biomarkers of COPD through the integrated analysis and receiver operating characteristic (ROC) analysis of metabolic changes in lung, plasma, and urine, and changes in morphological characteristics and pulmonary function in a model of PPE/LPS-induced COPD exacerbation. (3) Results: Metabolic changes in the lungs were evaluated as metabolic reprogramming to counteract the changes caused by the onset of COPD. In plasma, several combinations of phenylalanine, 3-methylhistidine, and polyunsaturated fatty acids have been proposed as potential biomarkers; the α-aminobutyric acid/histidine ratio has also been reported, which is a novel candidate biomarker for COPD. In urine, a combination of succinic acid, isocitric acid, and pyruvic acid has been proposed as a potential biomarker. (4) Conclusions: This study proposed potential biomarkers in plasma and urine that reflect altered lung metabolism in COPD, concurrently with the evaluation of the COPD exacerbation model induced by PPE plus LPS administration. Therefore, understanding these integrative mechanisms provides new insights into the diagnosis, treatment, and severity assessment of COPD.

Acremonamide, a Cyclic Pentadepsipeptide with Wound-Healing Properties Isolated from a Marine-Derived Fungus of the Genus Acremonium.

Acremonamide (1) was isolated from a marine-derived fungus belonging to the genus Acremonium. The chemical structure of 1 was established using MS, UV, and NMR spectroscopic data analyses. Acremonamide (1) was found to contain N-Me-Phe, N-Me-Ala, Val, Phe, and 2-hydroxyisovaleric acid. The absolute configurations of the four aforementioned amino acids were determined through acid hydrolysis followed by the advanced Marfey's method, whereas the absolute configuration of 2-hydroxyisovaleric acid was determined through GC-MS analysis after formation of the O-pentafluoropropionylated derivative of the (-)-menthyl ester of 2-hydroxyisovaleric acid. As an intrinsic biological activity, acremonamide (1) did not exert cytotoxicity to cancer and noncancer cells and increased the migration and invasion. Based on these activities, the wound healing properties of acremonamide (1) were confirmed in vitro and in vivo.

Silica-coated magnetic nanoparticles activate microglia and induce neurotoxic D-serine secretion.

BACKGROUND: Nanoparticles have been studied for brain imaging, diagnosis, and drug delivery owing to their versatile properties due to their small sizes. However, there are growing concerns that nanoparticles may exert toxic effects in the brain. In this study, we assessed direct nanotoxicity on microglia, the resident macrophages of the central nervous system, and indirect toxicity on neuronal cells exerted by silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)]. METHODS: We investigated MNPs@SiO2(RITC)-induced biological changes in BV2 murine microglial cells via RNA-sequencing-based transcriptome analysis and gas chromatography-mass spectrometry-based intracellular and extracellular amino acid profiling. Morphological changes were analyzed by transmission electron microscopy. Indirect effects of MNPs@SiO2(RITC) on neuronal cells were assessed by Transwell-based coculture with MNPs@SiO2(RITC)-treated microglia. MNPs@SiO2(RITC)-induced biological changes in the mouse brain in vivo were examined by immunohistochemical analysis. RESULTS: BV2 murine microglial cells were morphologically activated and the expression of Iba1, an activation marker protein, was increased after MNPs@SiO2(RITC) treatment. Transmission electron microscopy analysis revealed lysosomal accumulation of MNPs@SiO2(RITC) and the formation of vesicle-like structures in MNPs@SiO2(RITC)-treated BV2 cells. The expression of several genes related to metabolism and inflammation were altered in 100 µg/ml MNPs@SiO2(RITC)-treated microglia when compared with that in non-treated (control) and 10 µg/ml MNPs@SiO2(RITC)-treated microglia. Combined transcriptome and amino acid profiling analyses revealed that the transport of serine family amino acids, including glycine, cysteine, and serine, was enhanced. However, only serine was increased in the growth medium of activated microglia; especially, excitotoxic D-serine secretion from primary rat microglia was the most strongly enhanced. Activated primary microglia reduced intracellular ATP levels and proteasome activity in cocultured neuronal cells, especially in primary cortical neurons, via D-serine secretion. Moreover, ubiquitinated proteins accumulated and inclusion bodies were increased in primary dopaminergic and cortical neurons cocultured with activated primary microglia. In vivo, MNPs@SiO2(RITC), D-serine, and ubiquitin aggresomes were distributed in the MNPs@SiO2(RITC)-treated mouse brain. CONCLUSIONS: MNPs@SiO2(RITC)-induced activation of microglia triggers excitotoxicity in neurons via D-serine secretion, highlighting the importance of neurotoxicity mechanisms incurred by nanoparticle-induced microglial activation.

Silica-coated magnetic-nanoparticle-induced cytotoxicity is reduced in microglia by glutathione and citrate identified using integrated omics.

BACKGROUND: Nanoparticles have been utilized in brain research and therapeutics, including imaging, diagnosis, and drug delivery, owing to their versatile properties compared to bulk materials. However, exposure to nanoparticles leads to their accumulation in the brain, but drug development to counteract this nanotoxicity remains challenging. To date, concerns have risen about the potential toxicity to the brain associated with nanoparticles exposure via penetration of the brain blood barrier to address this issue. METHODS: Here the effect of silica-coated-magnetic nanoparticles containing the rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)] were assessed on microglia through toxicological investigation, including biological analysis and integration of transcriptomics, proteomics, and metabolomics. MNPs@SiO2(RITC)-induced biological changes, such as morphology, generation of reactive oxygen species, intracellular accumulation of MNPs@SiO2(RITC) using transmission electron microscopy, and glucose uptake efficiency, were analyzed in BV2 murine microglial cells. Each omics data was collected via RNA-sequencing-based transcriptome analysis, liquid chromatography-tandem mass spectrometry-based proteome analysis, and gas chromatography- tandem mass spectrometry-based metabolome analysis. The three omics datasets were integrated and generated as a single network using a machine learning algorithm. Nineteen compounds were screened and predicted their effects on nanotoxicity within the triple-omics network. RESULTS: Intracellular reactive oxygen species production, an inflammatory response, and morphological activation of cells were greater, but glucose uptake was lower in MNPs@SiO2(RITC)-treated BV2 microglia and primary rat microglia in a dose-dependent manner. Expression of 121 genes (from 41,214 identified genes), and levels of 45 proteins (from 5918 identified proteins) and 17 metabolites (from 47 identified metabolites) related to the above phenomena changed in MNPs@SiO2(RITC)-treated microglia. A combination of glutathione and citrate attenuated nanotoxicity induced by MNPs@SiO2(RITC) and ten other nanoparticles in vitro and in the murine brain, protecting mostly the hippocampus and thalamus. CONCLUSIONS: Combination of glutathione and citrate can be one of the candidates for nanotoxicity alleviating drug against MNPs@SiO2(RITC) induced detrimental effect, including elevation of intracellular reactive oxygen species level, activation of microglia, and reduction in glucose uptake efficiency. In addition, our findings indicate that an integrated triple omics approach provides useful and sensitive toxicological assessment for nanoparticles and screening of drug for nanotoxicity.

Metabolomic analysis of organic acids, amino acids, and fatty acids in plasma of Hanwoo beef on a high-protein diet.

INTRODUCTION: Ketoacidosis of metabolic disease showed in beef cattle although body weight was increased by high-grain diets (HGDs). However, few studies have examined for health status related with metabolic disease of ketoacidosis following high-protein diet (HPD). OBJECTIVES: Metabolomic analysis was performed for the monitoring of health status associated with metabolic disease of ketoacidosis in the plasma of Hanwoo heifers following a HPD. METHODS: Hanwoo heifers of 24 months with 459 ± 42 kg weight were used under a 2 × 2 crossover design. The plasma was collected from control (n = 5) and HPD group (n = 5) on day 21 following diet adaptation for 20 days. Metabolic profiling analysis of organic acids (OAs), amino acids (AAs) and fatty acids (FAs) by gas chromatography-tandem mass spectrometry combined with star pattern analysis was performed in plasma. Levels of OAs, AAs and FAs were evaluated by Mann-Whitney test, PCA and PLS-DA. RESULTS: In HPD group, ketoacidosis as metabolic disease was monitored by elevated acetoacetic acid and 3-hydroxybutyric acid. In addition, the elevation of ketogenic AAs, reduction of medium chain FAs and OAs with energy metabolism in TCA cycle were monitored in HPD group. Star graphic pattern was characteristic and readily distinguished between control and HPD groups. In PLS-DA, two groups were separated with VIP score of top-ranked 10 FAs as important metabolites for discrimination. CONCLUSION: Elevation of ketone body including ketogenic AAs and reduced energy metabolism of FAs and OAs may useful for evaluation of health states associated with ketoacidosis from metabolic event by HPD in beef cattle.

Antidepressant-Like Activities of Hispidol and Decursin in Mice and Analysis of Neurotransmitter Monoamines.

The antidepressant activities of hispidol and decursin (both potent monoamine oxidase A (MAO-A) inhibitors) were evaluated using the forced swimming test (FST) and the tail suspension test (TST) in mice, and thereafter, levels of neurotransmitter monoamines and metabolites in brain tissues were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hispidol (15 mg/kg) caused less or comparable immobility than fluoxetine (15 mg/kg; the positive control) in immobility time, as determined by FST (9.6 vs 32.0 s) and TST (53.1 vs 48.7 s), respectively, and its effects were dose-dependent and significant. Decursin (15 mg/kg) also produced immobility comparable to that of fluoxetine as determined by FST (47.0 vs 43.4 s) and TST (55.6 vs 63.4 s), and its effects were also dose-dependent and significant. LC-MS/MS analysis after FST showed that hispidol (15 mg/kg) greatly increased dopamine (DA) and serotonin levels dose-dependently in brain tissues as compared with the positive control. Decursin (15 mg/kg) dose-dependently increased DA level after TST. Slight changes in norepinephrine and 3,4-dihydroxyphenylacetic acid levels were observed after FST and TST in hispidol- or decursin-treated animals. It was observed that hispidol and decursin were effective and comparable to fluoxetine in immobility tests. These immobility and monoamine level results suggest that hispidol and decursin are potential antidepressant agents for the treatment of depression, and that they act mainly through serotonergic and/or dopaminergic systems.

Discrimination of Lycium chinense and L. barbarum Based on Metabolite Analysis and Hepatoprotective Activity.

Lycii Fructus is a traditional medicine used to prevent liver and kidney diseases, which commonly derives from Lycium chinense and Lycium barbarum. Here, the extracts and ethyl acetate-soluble fractions of L. chinense fruits exhibited better hepatoprotective effects than those of L. barbarum, which was likely due to differences in their composition. Therefore, GC-MS and HPLC analyses were conducted to characterize the metabolite differences between L. chinense and L. barbarum. Based on amino acid (AA) and phenolic acid (PA) profiling, 24 AAs and 9 PAs were identified in the two species. Moreover, each species exhibited unique and readily distinguishable AA and PA star graphic patterns. HPLC analysis elucidated composition differences between the ethyl acetate-soluble layers of the two compounds. Further, NMR analysis identified their chemical structures as 4-(2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl)butanoic acid and p-coumaric acid. The higher content of 4-(2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl)butanoic acid was detected in L. chinense, whereas the content of p-coumaric acid was higher in L. barbarum. Therefore, the differences in the relative contents of these two secondary metabolites in the ethyl acetate-soluble layer of Lycii Fructus could be a good marker to discriminate between L. chinense and L. barbarum.

Metabolomic study of polyamines in rat urine following intraperitoneal injection of γ-hydroxybutyric acid.

INTRODUCTION: Recently, illegal abuse of γ-hydroxybutyric acid (GHB) has increased in drug-facilitated crimes, but the determination of GHB exposure and intoxication is difficult due to rapid metabolism of GHB. Its biochemical mechanism has not been completely investigated. And a metabolomic study by polyamine profile and pattern analyses was not performed in rat urine following intraperitoneal injection with GHB. OBJECTIVES: Urinary polyamine (PA) profiling by gas chromatography-tandem mass spectrometry was performed to monitor an altered PA according to GHB administration. METHODS: Polyamine profiling analysis by gas chromatography-mass spectrometry combined with star pattern recognition analysis was performed in this study. The multivariate statistical analysis was used to evaluate discrimination among control and GHB administration groups. RESULTS: Six polyamines were determined in control, single and multiple GHB administration groups. Star pattern showed distorted hexagonal shapes with characteristic and readily distinguishable patterns for each group. N1-Acetylspermine (p < 0.001), putrescine (p < 0.006), N1-acetylspermidine (p < 0.009), and spermine (p < 0.027) were significantly increased in single administration group but were significantly lower in the multiple administration group than in the control group. N1-Acetylspermine was the main polyamine for discrimination among control, single and multiple administration groups. Spermine showed similar levels in single and multiple administration groups. CONCLUSIONS: The polyamine metabolic pattern was monitored in GHB administration groups. N1-Acetylspermine and spermine were evaluated as potential biomarkers of GHB exposure and addiction.

Metabolomic study on bleomycin and polyhexamethylene guanidine phosphate-induced pulmonary fibrosis mice models.

INTRODUCTION: Polyhexamethylene guanidine phosphate (PHMG) has been used as a disinfectant and biocide, and was known to be harmless and non-toxic. However, in 2011, PHMG used as a humidifier disinfectant was reported to be associated with lung diseases, such as, fibrosis in the toxicant studies on pulmonary fibrosis by PHMG. However, no metabolomics study has been performed in PHMG-induced mouse models of pulmonary fibrosis. OBJECTIVES: We performed a metabolomic study to understand the biochemical events that occur in bleomycin (BLM)- and PHMG-induced mouse models of pulmonary fibrosis using gas chromatography-mass spectrometry (GC-MS), LC-tandem MS, and GC-tandem MS. RESULTS: The levels of 61 metabolites of 30 amino acids, 13 organic acids, 12 fatty acids, 5 polyamines, and oxidized glutathione were determined in the pulmonary tissues of mice with BLM- and PHMG-induced pulmonary fibrosis and in normal controls. Principal component analysis and partial least squares discriminant analysis used to compare level of these 61 metabolites in pulmonary tissues. Levels of metabolites were significantly different in the BLM and PHMG groups as compared with the control group. In particular, the BLM- and PHMG-induced pulmonary fibrosis models showed elevated collagen synthesis and oxidative stress and metabolic disturbance of TCA related organic acids including fumaric acid by NADPH oxidase. In addition, polyamine metabolism showed severe alteration in the PHMG group than that of the BLM group. CONCLUSION: This result suggests PHMG will be able to induce pulmonary fibrosis by arginine metabolism and NADPH oxidase signaling.

Metabolomic approaches to polyamines including acetylated derivatives in lung tissue of mice with asthma.

INTRODUCTION: Recently, the relationship between polyamine (PA) metabolism and asthma has been studied in severe asthmatic therapy, but systematic PA metabolism including their acetylated derivatives was not fully understood. OBJECTIVES: Profiling analysis of polyamines (PAs) was performed to understand the biochemical events and monitor altered PA metabolism in lung tissue of mice with asthma. METHODS: Polyamine profiling of lung tissue of mice with asthma was performed without derivatization by liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with star pattern recognition analysis. The PA levels between control and asthma groups were evaluated by multivariate analysis. RESULTS: In mouse lung tissue, seven PAs were determined by LC-MS/MS in multiple reaction monitoring (MRM) mode. Their levels were normalized to the corresponding mean levels of the control group for star pattern analysis, which showed distorted heptagonal shapes with characteristic and readily distinguishable patterns for each group. Levels of putrescine (p < 0.0034), N1-acetylputrescine (p < 0.0652), and N8-acetylspermidine (p < 0.0827) were significantly increased in asthmatic lung tissue. The separation of the two groups was evaluated using multivariate analysis. In unsupervised learning, acetylated PAs including N1-acetylspermine were the main metabolites for discrimination. In supervised learning, putrescine and N1-acetylputrescine were evaluated as important metabolites. CONCLUSIONS: The present results provide basic data for understanding polyamine metabolism in asthma and may help to improve the therapy for severe asthma patients.

Silica-coated magnetic nanoparticles induce glucose metabolic dysfunction in vitro via the generation of reactive oxygen species.

Nanoparticles are a useful material in biomedicine given their unique properties and biocompatibility; however, there is increasing concern regarding the potential toxicity of nanoparticles with respect to cell metabolism. Some evidence suggests that nanoparticles can disrupt glucose and energy homeostasis. In this study, we investigated the metabolomic, transcriptomic, and integrated effects of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)] on glucose metabolism in human embryonic kidney 293 (HEK293) cells. Using gas chromatography-tandem mass spectrometry, we analysed the metabolite profiles of 14 organic acids (OAs), 20 amino acids (AAs), and 13 fatty acids (FAs) after treatment with 0.1 or 1.0 µg/µl MNPs@SiO2(RITC) for 12 h. The metabolic changes were highly related to reactive oxygen species (ROS) generation and glucose metabolism. Additionally, effects on the combined metabolome and transcriptome or "metabotranscriptomic network" indicated a relationship between ROS generation and glucose metabolic dysfunction. In the experimental validation, MNPs@SiO2(RITC) treatment significantly decreased the amount of glucose in cells and was associated with a reduction in glucose uptake efficiency. Decreased glucose uptake efficiency was also related to ROS generation and impaired glucose metabolism in the metabotranscriptomic network. Our results suggest that exposure to high concentrations of MNPs@SiO2(RITC) produces maladaptive alterations in glucose metabolism and specifically glucose uptake as well as related metabolomic and transcriptomic disturbances via increased ROS generation. These findings further indicate that an integrated metabotranscriptomics approach provides useful and sensitive toxicological assessment for nanoparticles.

Role of Th2 cytokines on the onset of asthma induced by meta-xylene in mice.

meta-Xylene (m-xylene) is one of three isomers of xylene, which is widely used as a solvent and detergent in various industries and medical technology. Exposure to volatile organic compounds, such as m-xylene, causes pulmonary inflammation and airway inflammation, thereby contributing to the onset of asthma. Exposure to m-xylene increases acute wheezing and intensity of asthma symptom. However, the mechanism of the onset of asthma by m-xylene has not been studied yet. C57BL/6 mice were sensitized and challenged by m-xylene at 100 or 300 mg/kg. The mice were then sacrificed after the last challenge. Exposure to m-xylene increased the total number of inflammatory cells and the production of interleukin (IL)-4, IL-5, IL-13, and immunoglobulin E related to the Th2 immune response. In contrast, the production of interferon-γ related to the Th1 immune response was decreased. In addition, the airway resistance increased according to the airway hyper-responsiveness measurements. Finally, a histological analysis revealed infiltration of inflammatory cells, mucus production, and lung fibrosis. These results suggest that m-xylene is a potential risk factor for asthma and the onset of asthma is caused by TH2 cytokines.

Effective Soil Extraction Method for Cultivating Previously Uncultured Soil Bacteria.

Here, a new medium, named intensive soil extract medium (ISEM), based on new soil extract (NSE) using 80% methanol, was used to efficiently isolate previously uncultured bacteria and new taxonomic candidates, which accounted for 49% and 55% of the total isolates examined (n = 258), respectively. The new isolates were affiliated with seven phyla (Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria, Verrucomicrobia, Planctomycetes, and Bacteroidetes). The result of chemical analysis showed that NSE included more diverse components of low-molecular-weight organic substances than two conventional soil extracts made using distilled water. Cultivation of previously uncultured bacteria is expected to extend knowledge through the discovery of new phenotypic, physiological, and functional properties and even roles of unknown genes.IMPORTANCE Both metagenomics and single-cell sequencing can detect unknown genes from uncultured microbial strains in environments, and either method may find the significant potential metabolites and roles of these strains. However, such gene/genome-based techniques do not allow detailed investigations that are possible with cultures. To solve this problem, various approaches for cultivation of uncultured bacteria have been developed, but there are still difficulties in maintaining pure cultures by subculture.

Monitoring of altered amino acid metabolic pattern in rat urine following intraperitoneal injection with γ-hydroxybutyric acid.

INTRODUCTION: γ-Hydroxybutyric acid is a well-known prescription medicine that is used for the clinical treatment of alcohol dependence and narcolepsy. However, the biochemical mechanism underlying γ-hydroxybutyric acid intoxication remains unclear, and metabolomic amino acid profiling and pattern analyses have not been attempted following treatment with γ-hydroxybutyric acid. OBJECTIVES: We carried out urinary amino acid profiling and pattern analyses in rats to determine the biochemical events associated with altered amino acid metabolism and biomarker detection of intoxication following treatment with γ-hydroxybutyric acid. METHODS: Metabolic profiling analysis of amino acids in rat urine samples was performed as ethoxycarbonyl/tert-butyldimethylsilyl derivatives by gas chromatography-mass spectrometry following intraperitoneal administration of γ-hydroxybutyric acid once per day for 1 and 10 consecutive days. RESULTS: A total of 28 amino acids were positively identified in urine samples from the control, single and multiple groups treated with γ-hydroxybutyric acid. Their levels from the single and multiple treated groups were normalized to the corresponding mean control values. The star graphic pattern of the amino acids was characteristic and readily distinguishable for each group owing to its distorted nonacosagonal shape. In the principle component analysis, we monitored phenylalanine, glutamic acid, aspartic acid, asparagine, and methionine as contributing factors that discriminated the three groups. CONCLUSION: The present metabolomic study may explain the altered metabolism of amino acids following administration, and intoxication with γ-hydroxybutyric acid.

Analysis of the free fatty acid metabolome in the plasma of patients with systemic lupus erythematosus and fever.

INTRODUCTION: Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease with heterogeneous clinical manifestations mediated by immune dysregulation. OBJECTIVES: We aimed to analyze the metabolomic differences in free fatty acids (FFAs) between patients with SLE and healthy controls (HCs). METHODS: In this study, the levels of 24 FFAs, as their tert-butyldimethylsilyl derivatives, in the plasma of 41 patients with SLE and 41 HCs, were investigated using gas chromatography with mass spectrometry in selected-ion monitoring mode. RESULTS: The results showed that patients with SLE and HCs had significantly different levels of 13 of the 24 FFAs. The levels of myristic, palmitoleic, oleic, and eicosenoic acids were significantly higher, whereas the levels of caproic, caprylic, linoleic, stearic, arachidonic, eicosanoic, behenic, lignoceric, and hexacosanoic acids were significantly lower in patients with SLE, than in the HCs. In the partial-correlation analysis of the FFA profiles and markers of disease activity of SLE, several metabolic markers correlated with SLE disease activity. CONCLUSIONS: Our results provide a comprehensive understanding of the relationship between FFAs and markers of SLE disease activity. Thus, this approach has promising potential for the discovery of metabolic biomarkers of SLE.

Effects of diisononyl phthalate on osteopenia in intact mice.

Osteopenia is characterized by bone loss and deterioration of trabecular bone, which leads to osteoporotic fractures. This disease is highly prevalent in industrialized areas and is associated with exposure to endocrine disrupting chemicals (EDCs). Diisononyl phthalate (DINP) is one of these EDCs and is mainly used as a plasticizer in flexible polyvinyl chloride (PVC) products. Although it is well known that exposure to DINP is harmful to humans, no studies have been reported concerning its contribution to osteopenia. Therefore, in this study, we injected DINP (2, 20, and 200mg/kg) into C3H/HeN mice for 6weeks and found that the uterus weight, bone (femur and tibia) weight, and bone length of the DINP-exposed mice were reduced compared to those of the SHAM group. On the other hand, body weight, the serum alkaline phosphatase (ALP) and inorganic phosphorus (IP) levels in the DINP treated mice were increased compared with those of the SHAM group. The tartrate-resistant acid phosphatase (TRAP) activity (bone resorption marker) was increased and the bone alkaline phosphatase (BALP) activity was lowered by the treatment with DINP as compared with the SHAM group. Furthermore, the microarchitecture of the femur and tibia in the intact mice was destroyed by the DINP injection. The tissue volume (TV), bone volume (BV), BV/TV, bone surface (BS), BS/TV, trabecular thickness (Tb.Th), and trabecular number (Tb.N) were reduced and the trabecular pattern factor (Tb.Pf), structure model index (SMI), and trabecular separation (Tb.Sp) were increased by the DINP injection. The bone mineral density (BMD) of the femur and tibia was lower in the DINP group than in the SHAM group. These results indicate that DINP contributes to an increased risk of osteopenia via destruction of the microarchitecture and enhancement of osteoclast activity.