A metabolomics perspective on 2 years of high-intensity training in horses.

The plasma metabolomic profile of elite harness horses subjected to different training programmes was explored. All horses had the same training programme from 1.5 until 2 years of age and then high-intensity training was introduced, with horses divided into high and low training groups. Morning blood samples were collected at 1.5, 2, 2.5 and 3.5 years of age. The plasma was analysed using targeted absolute quantitative analysis and a combination of tandem mass spectrometry, flow-injection analysis and liquid chromatography. Differences between the two training groups were observed at 2 years of age, when 161 metabolites and sums and ratios were lower (e.g. ceramide and several triglycerides) and 51 were higher (e.g. aconitic acid, anserine, sum of PUFA cholesteryl esters and solely ketogenic AAs) in High compared with low horses. The metabolites aconitic acid, anserine, leucine, HArg synthesis and sum of solely ketogenic AAs increased over time, while beta alanine synthesis, ceramides and indole decreased. Therefore high-intensity training promoted adaptations linked to aerobic energy production and amino acid metabolism, and potentially also affected pH-buffering and vascular and insulin responses.

Regulation of tricarboxylate transport and metabolism in Acinetobacter baylyi ADP1.

Despite the significant presence of plant-derived tricarboxylic acids in some environments, few studies detail the bacterial metabolism of trans-aconitic acid (Taa) and tricarballylic acid (Tcb). In a soil bacterium, Acinetobacter baylyi ADP1, we discovered interrelated pathways for the consumption of Taa and Tcb. An intricate regulatory scheme tightly controls the transport and catabolism of both compounds and may reflect that they can be toxic inhibitors of the tricarboxylic acid cycle. The genes encoding two similar LysR-type transcriptional regulators, TcuR and TclR, were clustered on the chromosome with tcuA and tcuB, genes required for Tcb consumption. The genetic organization differed from that in Salmonella enterica serovar Typhimurium, in which tcuA and tcuB form an operon with a transporter gene, tcuC. In A. baylyi, tcuC was not cotranscribed with tcuAB. Rather, tcuC was cotranscribed with a gene, designated pacI, encoding an isomerase needed for Taa consumption. TcuC appears to transport Tcb and cis-aconitic acid (Caa), the presumed product of PacI-mediated periplasmic isomerization of Taa. Two operons, tcuC-pacI and tcuAB, were transcriptionally controlled by both TcuR and TclR, which have overlapping functions. We investigated the roles of the two regulators in activating transcription of both operons in response to multiple effector compounds, including Taa, Tcb, and Caa.IMPORTANCEIngestion of Taa and Tcb by grazing livestock can cause a serious metabolic disorder called grass tetany. The disorder, which results from Tcb absorption by ruminants, focuses attention on the metabolism of tricarboxylic acids. Additional interest stems from efforts to produce tricarboxylic acids as commodity chemicals. Improved understanding of bacterial enzymes and pathways for tricarboxylic acid metabolism may contribute to new biomanufacturing strategies.

Metabolomics reveals the impact of nitrogen combined with the zinc supply on zinc availability in calcareous soil via root exudates of winter wheat (Triticum aestivum).

A possible mechanism for the improved availability of zinc (Zn) in soil by combining nitrogen (N) with Zn supply was investigated based on the root exudates of winter wheat. N, Zn supply as well as their combination significantly regulated nine root exudates in winter wheat; in which, the secretion of cis-aconitic acid involving in the TCA cycle, C5-branched dibasic acid metabolism, glyoxylate and dicarboxylate metabolism and 2-oxocarboxylic acid metabolism was upregulated by N, Zn supply as well as their combination. N-Zn combination induced the activities of citrate synthase and cis-aconitase in roots and shoots of winter wheat thus to increase the concentrations of citric and aconitic acid; the decrease of isocitric acid concentrations in shoots indicated the inhibited conversion of aconitic acid to isocitric acid by N-Zn combination. It revealed a possible reason for the enhanced secretion of cis-aconitic acid by N-Zn combination. Exogenous addition of 10 µ plant-1 cis-aconitate significantly increased available Zn concentrations in soil and Zn concentrations in winter wheat under N-Zn combination. Thus, the N-Zn combination regulated the metabolism of cis-aconitic acid in winter wheat, thus enhancing the secretion of cis-aconitic acid to increase the bioavailability of Zn in soil.

Identification of a specific exporter that enables high production of aconitic acid in Aspergillus pseudoterreus.

Aconitic acid is an unsaturated tricarboxylic acid that is attractive for its potential use in manufacturing biodegradable and biocompatible polymers, plasticizers, and surfactants. Previously Aspergillus pseudoterreus was engineered as a platform to produce aconitic acid by deleting the cadA (cis-aconitic acid decarboxylase) gene in the itaconic acid biosynthetic pathway. In this study, the aconitic acid transporter gene (aexA) was identified using comparative global discovery proteomics analysis between the wild-type and cadA deletion strains. The protein AexA belongs to the Major Facilitator Superfamily (MFS). Deletion of aexA almost abolished aconitic acid secretion, while its overexpression led to a significant increase in aconitic acid production. Transportation of aconitic acid across the plasma membrane is a key limiting step in its production. In vitro, proteoliposome transport assay further validated AexA's function and substrate specificity. This research provides new approaches to efficiently pinpoint and characterize exporters of fungal organic acids and accelerate metabolic engineering to improve secretion capability and lower the cost of bioproduction.

Antagonistic Activity of Oroxylin A against Fusarium graminearum and Its Inhibitory Effect on Zearalenone Production.

Fusarium graminearum produces zearalenone (ZEA), a mycotoxin that is widely found in food and feed products and is toxic to humans and livestock. Piper sarmentosum extract (PSE) inhibits F. graminearum, and Oroxylin A appears to be a major antifungal compound in PSE. The aim of this study is to quantify the Oroxylin A content in PSE using UPLC-QTOF-MS/MS, and to investigate the antagonistic activity of Oroxylin A against F. graminearum and its inhibitory effect on ZEA production. The results indicate that Oroxylin A inhibits both fungal growth and ZEA production in a dose-dependent manner. Oroxylin A treatment downregulated the mRNA expression of zearalenone biosynthesis protein 1 (ZEB1) and zearalenone biosynthesis protein 2 (ZEB2). The metabolomics analysis of F. graminearum mycelia indicated that the level of ribose 5-phosphate (R5P) deceased (p < 0.05) after Oroxylin A treatment (64-128 ng/mL). Moreover, as the Oroxylin A treatment content increased from 64 to 128 ng/mL, the levels of cis-aconitate (p < 0.05) and fumarate (p < 0.01) were upregulated successively. A correlation analysis further showed that the decreased R5P level was positively correlated with ZEB1 and ZEB2 expression, while the increased cis-aconitate and fumarate levels were negatively correlated with ZEB1 and ZEB2 expression. These findings demonstrate the potential of Oroxylin A as a natural agent to control toxigenic fungi and their mycotoxin.

Microbial production of trans-aconitic acid.

Trans-aconitic acid (TAA) is a promising bio-based chemical with the structure of unsaturated tricarboxylic acid, and also has the potential to be a non-toxic nematicide as a potent inhibitor of aconitase. However, TAA has not been commercialized because the traditional production processes of plant extraction and chemical synthesis cannot achieve large-scale production at a low cost. The availability of TAA is a serious obstacle to its widespread application. In this study, we developed an efficient microbial synthesis and fermentation production process for TAA. An engineered Aspergillus terreus strain producing cis-aconitic acid and TAA was constructed by blocking itaconic acid biosynthesis in the industrial itaconic acid-producing strain. Through heterologous expression of exogenous aconitate isomerase, we further designed a more efficient cell factory to specifically produce TAA. Subsequently, the fermentation process was developed and scaled up step-by-step, achieving a TAA titer of 60 g L-1 at the demonstration scale of a 20 m3 fermenter. Finally, the field evaluation of the produced TAA for control of the root-knot nematodes was performed in a field trial, effectively reducing the damage of the root-knot nematode. Our work provides a commercially viable solution for the green manufacturing of TAA, which will significantly facilitate biopesticide development and promote its widespread application as a bio-based chemical.

Dietary flaxseed oil and vitamin E improve semen quality via propionic acid metabolism.

Introduction: Flaxseed oil (FO) and vitamin E (VE) both have antioxidant effects on sperm. The present study investigated the effects of dietary supplementation with FO and/or VE on semen quality. Methods: 16 fertile Simmental bulls were selected and randomly divided into 4 groups (n = 4): the control group (control diet), FO group (control diet containing 24 g/kg FO), VE group (control diet containing 150 mg/kg VE) and FOVE group (control diet containing 150 mg/kg VE and 24 g/kg FO), and the trial lasted 10 weeks. Results: The results showed that the addition of FO independently can increase sperm motion parameters, the levels of catalase (CAT), glutathione peroxidase (GSH-Px), testosterone (T) and estradiol (E2), while reduce oxidative stress in seminal plasma (P < 0.05). Supplement of VE independently can increased the motility, motility parameters, CAT and superoxide dismutase (SOD) levels, and reduce oxidative stress in seminal plasma (P < 0.05). There was an interaction effect of FO × VE on motility and reactive oxygen species (ROS), while GSH-Px and ROS were affected by week × VE 2-way interaction, levels of T and E2 were also affected by the dietary FO × week interaction (P < 0.05). The triple interaction effects of FO, VE and week were significant for malondialdehyde (MDA) (P < 0.05). Compared with the control group, sperm from the FOVE group had a significantly higher in vitro fertilization (IVF) rate, and subsequent embryos had increased developmental ability with reduced ROS levels at the eight-cell stage, then increased adenosine triphosphate (ATP) content and gene expression levels of CAT, CDX2, Nanog, and SOD at the blastocyst stage (P < 0.05). Metabolomic and transcriptomic results indicated that dietary supplementation of FO and VE increased the expression of the metabolite aconitic acid, as well as the expression of ABAT and AHDHA genes. Conclusion: With in-silico analysis, it can be concluded that the effects of dietary FO and VE on improving semen quality and embryo development may be related to increased aconitic acid via the ABAT and AHDHA genes involved in the propionic acid metabolism pathway.

Singlet oxygen-induced signalling depends on the metabolic status of the Chlamydomonas reinhardtii cell.

Using a mutant screen, we identified trehalose 6-phosphate phosphatase 1 (TSPP1) as a functional enzyme dephosphorylating trehalose 6-phosphate (Tre6P) to trehalose in Chlamydomonas reinhardtii. The tspp1 knock-out results in reprogramming of the cell metabolism via altered transcriptome. As a secondary effect, tspp1 also shows impairment in 1O2-induced chloroplast retrograde signalling. From transcriptomic analysis and metabolite profiling, we conclude that accumulation or deficiency of certain metabolites directly affect 1O2-signalling. 1O2-inducible GLUTATHIONE PEROXIDASE 5 (GPX5) gene expression is suppressed by increased content of fumarate and 2-oxoglutarate, intermediates in the tricarboxylic acid cycle (TCA cycle) in mitochondria and dicarboxylate metabolism in the cytosol, but also myo-inositol, involved in inositol phosphate metabolism and phosphatidylinositol signalling system. Application of another TCA cycle intermediate, aconitate, recovers 1O2-signalling and GPX5 expression in otherwise aconitate-deficient tspp1. Genes encoding known essential components of chloroplast-to-nucleus 1O2-signalling, PSBP2, MBS, and SAK1, show decreased transcript levels in tspp1, which also can be rescued by exogenous application of aconitate. We demonstrate that chloroplast retrograde signalling involving 1O2 depends on mitochondrial and cytosolic processes and that the metabolic status of the cell determines the response to 1O2.

Metabolic profiles of Sri Lankan cassava mosaic virus-infected and healthy cassava (Manihot esculenta Crantz) cultivars with tolerance and susceptibility phenotypes.

BACKGROUND: Cassava mosaic disease (CMD) of cassava (Manihot esculenta Crantz) has expanded across many continents. Sri Lankan cassava mosaic virus (SLCMV; family Geminiviridae), which is the predominant cause of CMD in Thailand, has caused agricultural and economic damage in many Southeast Asia countries such as Vietnam, Loas, and Cambodia. The recent SLCMV epidemic in Thailand was commonly found in cassava plantations. Current understanding of plant-virus interactions for SLCMV and cassava is limited. Accordingly, this study explored the metabolic profiles of SLCMV-infected and healthy groups of tolerant (TME3 and KU50) and susceptible (R11) cultivars of cassava. Findings from the study may help to improve cassava breeding, particularly when combined with future transcriptomic and proteomic research. RESULTS: SLCMV-infected and healthy leaves were subjected to metabolite extraction followed by ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS/MS). The resulting data were analyzed using Compound Discoverer software, the mzCloud, mzVault, and ChemSpider databases, and published literature. Of the 85 differential compounds (SLCMV-infected vs healthy groups), 54 were differential compounds in all three cultivars. These compounds were analyzed using principal component analysis (PCA), hierarchical clustering dendrogram analysis, heatmap analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. Chlorogenic acid, DL-carnitine, neochlorogenic acid, (E)-aconitic acid, and ascorbyl glucoside were differentially expressed only in TME3 and KU50, with chlorogenic acid, (E)-aconitic acid, and neochlorogenic acid being downregulated in both SLCMV-infected TME3 and KU50, DL-carnitine being upregulated in both SLCMV-infected TME3 and KU50, and ascorbyl glucoside being downregulated in SLCMV-infected TME3 but upregulated in SLCMV-infected KU50. Furthermore, 7-hydroxycoumarine was differentially expressed only in TME3 and R11, while quercitrin, guanine, N-acetylornithine, uridine, vorinostat, sucrose, and lotaustralin were differentially expressed only in KU50 and R11. CONCLUSIONS: Metabolic profiling of three cassava landrace cultivars (TME3, KU50, and R11) was performed after SLCMV infection and the profiles were compared with those of healthy samples. Certain differential compounds (SLCMV-infected vs healthy groups) in different cultivars of cassava may be involved in plant-virus interactions and could underlie the tolerance and susceptible responses in this important crop.

Metabolomics profiling of seminal plasma in obesity-induced asthenozoospermia.

BACKGROUND: Asthenozoospermia is one of the essential causes of male infertility, and its incidence is significantly higher in obese men. Due to its complex etiology and unknown pathomechanism, the diagnosis and treatment of obesity-induced asthenozoospermia is a prevalent problem in reproductive medicine. OBJECTIVE: This study aims to explore major differential metabolites and metabolic pathways in seminal plasma and pathological mechanisms for obesity-induced asthenozoospermia. MATERIALS AND METHODS: We performed non-target metabolomic studies on the seminal plasma of healthy men with normal semen parameters (HN group, n = 20), obese men with normal semen parameters (ON group, n = 20), and men with obesity-induced asthenozoospermia (OA group, n = 20) based on gas chromatography-mass spectrometry. Metabolic profilings and related pathway analyses were conducted to discriminate differential metabolites and metabolic pathways. RESULTS: A total of 20 differential metabolites including fructose, succinic acid, aconitic acid, methylmaleic acid, glucopyranose, serine, valine, leucine, phenylalanine, glycine, glutamic acid, alanine, proline and threonine were identified in HN group and ON group; 24 differential metabolites including glucose, fructose, pyruvic acid, citric acid, succinic acid, aconitic acid, glucopyranose, glutamic acid, valine, leucine, glycine, phenylalanine, lysine, citrulline, proline and alanine were produced in OA group and ON group; and 28 differential metabolites including glucose, fructose, citric acid, succinic acid, glucopyranose, valine, glycine, serine, leucine, phenylalanine, alanine, threonine, proline, glutamic acid, citrulline, lysine and tyrosine were produced in OA group and HN group. In addition, abnormal energy metabolism including carbohydrate metabolism (TCA cycle, glycolysis/gluconeogenesis, and pyruvate metabolism) and amino acid metabolism (phenylalanine, tyrosine, and tryptophan biosynthesis, D-glutamine and D-glutamate metabolism; phenylalanine metabolism, etc.) were found in ON group and OA group. CONCLUSION: Obesity could affect the metabolite composition in seminal plasma and abnormal energy metabolism in seminal plasma mainly including carbohydrate metabolism and amino acid metabolism were closely related to obesity-induced asthenozoospermia.

Insecticidal potential of extracts, fractions, and molecules of Aconitum heterophyllum Wall ex. Royle against aphid Aphis craccivora Koch (Hemiptera: Aphididae).

BACKGROUND: Aphis craccivora is the major sap-sucking pest of leguminous crops and vector of plant viruses that cause damage to plants and reduce yield. Indiscriminate and nonjudicious use of synthetic insecticides led to resistance development and harmful to environment. Therefore, it is important to discover plant-based lead(s) which can replace synthetic insecticides. In the current study the residual toxicity of extracts, fractions, and isolated compounds of Aconitum heterophyllum were evaluated against A. craccivora to identify lead(s) for further development of botanical formulation. RESULTS: In residual contact assay, ethanolic (LC50  = 2837.17 mg L-1 ) and aqueous methanolic extracts (LC50  = 2971.59 mg L-1 ) were effective against A. craccivora. Among fractions, the n-butanol fraction of the aqueous methanolic extract (LC50  = 986.96 mg L-1 ) was found to be most effective, followed by the ethyl acetate fraction of the ethanolic extract (LC50  = 1037.52 mg L-1 ) and the n-hexane fraction of both extracts (LC50  = 1113.85 to 1233.11 mg L-1 ). Among pure molecules, aconitic acid was found to be the most effective (68% mortality; LC50  = 1313.19 mg L-1 ) and was on a par with azadirachtin 0.15% EC (66% mortality; LC50  = 1921.10 mg L-1 ). Furthermore, from the effect of ethanoic extract on detoxification enzyme inhibition in A. craccivora we concluded that the target site of action of this extract in A. craccivora might be glutathione S-transferase. CONCLUSIONS: The parent extract/fractions of A. heterophylum showed promising activity against A. craccivora. Among phytoconstituents of the active extract and fractions, aconitic acid was found to be on a par with azadirachtin 0.15% EC. © 2022 Society of Chemical Industry.

Flavonifractor plautii Protects Against Elevated Arterial Stiffness.

BACKGROUND: Dysbiosis of gut microbiota plays a pivotal role in vascular dysfunction and microbial diversity was reported to be inversely correlated with arterial stiffness. However, the causal role of gut microbiota in the progression of arterial stiffness and the specific species along with the molecular mechanisms underlying this change remain largely unknown. METHODS: Participants with elevated arterial stiffness and normal controls free of medication were matched for age and sex. The microbial composition and metabolic capacities between the 2 groups were compared with the integration of metagenomics and metabolomics. Subsequently, Ang II (angiotensin II)-induced and humanized mouse model were employed to evaluate the protective effect of Flavonifractor plautii (F plautii) and its main effector cis-aconitic acid. RESULTS: Human fecal metagenomic sequencing revealed a significantly high abundance and centrality of F plautii in normal controls, which was absent in the microbial community of subjects with elevated arterial stiffness. Moreover, blood pressure only mediated part of the effect of F plautii on lower arterial stiffness. The microbiome of normal controls exhibited an enhanced capacity for glycolysis and polysaccharide degradation, whereas, those of subjects with increased arterial stiffness were characterized by increased biosynthesis of fatty acids and aromatic amino acids. Integrative analysis with metabolomics profiling further suggested that increased cis-aconitic acid served as the main effector for the protective effect of F plautii against arterial stiffness. Replenishment with F plautii and cis-aconitic acid improved elastic fiber network and reversed increased pulse wave velocity through the suppression of MMP-2 (matrix metalloproteinase-2) and inhibition of MCP-1 (monocyte chemoattractant protein-1) and NF-κB (nuclear factor kappa-B) activation in both Ang II-induced and humanized model of arterial stiffness. CONCLUSIONS: Our translational study identifies a novel link between F plautii and arterial function and raises the possibility of sustaining vascular health by targeting gut microbiota.

An integrated strategy to study the combination mechanisms of Bupleurum chinense DC and Paeonia lactiflora Pall for treating depression based on correlation analysis between serum chemical components profiles and endogenous metabolites profiles.

ETHNOPHARMACOLOGICAL RELEVANCE: Bupleurum chinense DC-Paeonia lactiflora Pall (BCD-PLP) is a common clinical herb pair in traditional Chinese medicine (TCM) prescriptions commonly used to treat depression. However, its combination mechanisms with its anti-depressive effects remain highly unclear. AIM OF THE STUDY: Here, an effective strategy has been developed to study the combination mechanisms of Bupleurum chinense DC (BCD) and Paeonia lactiflora Pall (PLP) by integrating serum pharmacochemistry analysis, metabolomics technology, and molecular docking technology. MATERIALS AND METHODS: First, the depression model rats were replicated by the chronic unpredictable mild stress (CUMS) procedure, and the difference in the chemical composition in vivo before and after the combination of BCD and PLP was analyzed by integrating background subtraction and multivariate statistical analysis techniques. Then, UPLC/HRMS-based serum metabolomics was performed to analyze the synergistic effect on metabolite regulation before and after the combination of BCD and PLP. Further, the correlation analysis between the differential exogenous chemical components and the differential endogenous metabolites before and after the combination was employed to dissect the combination mechanisms from a global perspective of combining metabolomics and serum pharmacochemistry. Finally, the molecular docking between the differential chemical components and the key metabolic enzymes was applied to verify the regulatory effect of the differential exogenous chemical components on the differential endogenous metabolites. RESULTS: The serum pharmacochemistry analysis results demonstrated that the combination of BCD and PLP could significantly affect the content of 10 components in BCD (including 5 prototype components were significantly decreased and 5 metabolites were significantly increased) and 8 components in PLP (including 4 prototype components and 3 metabolites were significantly increased, 1 metabolite was significantly decreased), which indicated that the combination could enhance BCD prototype components' metabolism and the absorption of the PLP prototype components. Besides, metabolomics results indicated that the BCD-PLP herb pair group significantly reversed more metabolites (8) than BCD and PLP single herb group (5 & 4) and has a stronger regulatory effect on metabolite disorders caused by CUMS. Furthermore, the correlation analysis results suggested that saikogenin F and saikogenin G were significantly positively correlated with the endogenous metabolite itaconate, an endogenous anti-inflammatory metabolite; and benzoic acid was significantly positively correlated with D-serine, an endogenous metabolite with an antidepressant effect. Finally, the molecular docking results further confirmed that the combination of BCD and PLP could affect the activities of cis-aconitic acid decarboxylase and D-amino acid oxidase by increasing the in vivo concentration of saikogenin F and benzoic acid, which further enhances its anti-inflammatory activity and anti-depressive effect. CONCLUSIONS: In this study, an effective strategy has been developed to study the combination mechanisms of BCD and PLP by integrating serum pharmacochemistry analysis, multivariate statistical analysis, metabolomics technology, and molecular docking technology. Based on this strategy, the present study indicated that the combination of BCD and PLP could affect the activities of cis-aconitic acid decarboxylase and D-amino acid oxidase by increasing the concentration of saikogenin F and benzoic acid in vivo, which further enhances its anti-depressive effect. In short, this strategy will provide a reliable method for elucidating the herb-herb compatibility mechanism of TCM.

Redundancy in Citrate and cis-Aconitate Transport in Pseudomonas aeruginosa.

Tricarboxylates such as citrate are the preferred carbon sources for Pseudomonas aeruginosa, an opportunistic pathogen that causes chronic human infections. However, the membrane transport process for the tricarboxylic acid cycle intermediates citrate and cis-aconitate is poorly characterized. Transport is thought to be controlled by the TctDE two-component system, which mediates transcription of the putative major transporter OpdH. Here, we search for previously unidentified transporters of citrate and cis-aconitate using both protein homology and RNA sequencing approaches. We uncover new transporters and show that OpdH is not the major citrate importer; instead, citrate transport primarily relies on the tripartite TctCBA system, which is encoded in the opdH operon. Deletion of tctA causes a growth lag on citrate and loss of growth on cis-aconitate. Combinatorial deletion of newly discovered transporters can fully block citrate utilization. We then characterize transcriptional control of the opdH operon in tctDE mutants and show that loss of tctD blocks citrate utilization due to an inability to express opdH-tctCBA. However, tctE and tctDE mutants evolve heritable adaptations that restore growth on citrate as the sole carbon source. IMPORTANCE Pseudomonas aeruginosa is a bacterium that infects hospitalized patients and is often highly resistant to antibiotic treatment. It preferentially uses small organic acids called tricarboxylates rather than sugars as a source of carbon for growth. The transport of many of these molecules from outside the cell to the interior occurs through unknown channels. Here, we examined how the tricarboxylates citrate and cis-aconitate are transported in P. aeruginosa. We then sought to understand how production of proteins that permit citrate and cis-aconitate transport is regulated by a signaling system called TctDE. We identified new transporters for these molecules, clarified the function of a known transport system, and directly tied transporter expression to the presence of an intact TctDE system.

Metabolic alterations of short-chain fatty acids and TCA cycle intermediates in human plasma from patients with gastric cancer.

AIMS: Short-chain fatty acids (SCFAs) are produced by gut microbiota from dietary fiber. Since absorbed SCFAs could be introduced into the tricarboxylic acid (TCA) cycle in host cells, the relationships between SCFAs and TCA cycle intermediates might influence to energy metabolism in the human body. For this reason, information on profile changes between SCFAs and TCA cycle intermediates could help unveil pathological mechanisms of gastric cancer. MAIN METHODS: A gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed to simultaneously determine SCFAs and TCA cycle intermediates in human plasma from patients with chronic superficial gastritis (CSG), intestinal metaplasia (IM), and gastric cancer. We applied a tetra-alkyl ammonium pairing method to prevent loss of volatile SCFAs and base decarboxylation of TCA cycle intermediates during sample preparation. To assess gastric diseases, metabolic alterations of SCFAs and TCA cycle intermediates in human plasma with gastric disorders were analyzed by their plasma levels. KEY FINDINGS: Significantly different metabolic alterations based on the plasma levels of SCFAs and TCA cycle intermediates were investigated in cancer metabolic pathways. Not only propionate and butyrate, mainly produced by gut microbiota, were significantly decreased, but also cis-aconitate, α-ketoglutarate, and fumarate were significantly increased in plasma with IM or gastric cancer, compared to CSG. Further, based on ratios of product to precursor, three metabolic pathways (succinate/propionate, succinate/α-ketoglutarate, and cis-aconitate/citrate) were supposed to be distorted between gastric diseases. SIGNIFICANCE: In conclusion, propionate, cis-aconitate, α-ketoglutarate, and fumarate could be used to assess the progression of gastric cancer.

Metabolic engineering of Schizosaccharomyces pombe for itaconic acid production.

The economical production of value-added chemicals from renewable biomass is a promising aspect of producing a sustainable economy. Itaconic acid (IA) is a high value-added compound that is expected to be an alternative to petroleum-based chemicals. In this study, we developed a metabolic engineering strategy for the large-scale production of IA from glucose using the fission yeast Schizosaccharomyces pombe. Heterologous expression of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus, which encodes cis-aconitate decarboxylase in the cytosol, led to the production of 0.132 g/L of IA. We demonstrated that mitochondrial localization of CAD enhanced the production of IA. To prevent the leakage of carbon flux from the TCA cycle, we generated a strain in which the endogenous malate exporter, citrate lyase, and citrate transporter genes were disrupted. A titer of 1.110 g/L of IA was obtained from a culture of this strain started with 50 g/L of glucose. By culturing the multiple mutant strain at increased cell density, we succeeded in enhancing the IA production to 1.555 g/L. The metabolic engineering strategies presented in this study have the potential to improve the titer of the biosynthesis of derivatives of intermediates of the TCA cycle.

A Kinetic Model for Predicting Trace Gas Uptake and Reaction.

A model is developed to describe trace gas uptake and reaction with applications to aerosols and microdroplets. Gas uptake by the liquid is formulated as a coupled equilibria that links gas, surface, and bulk regions of the droplet or solution. Previously, this framework was used in explicit stochastic reaction-diffusion simulations to predict the reactive uptake kinetics of ozone with droplets containing aqueous aconitic acid, maleic acid, and sodium nitrite. With the use of prior data and simulation results, a new equation for the uptake coefficient is derived, which accounts for both surface and bulk reactions. Lambert W functions are used to obtain closed form solutions to the integrated rate laws for the multiphase kinetics; similar to previous expressions that describe Michaelis-Menten enzyme kinetics. Together these equations couple interface and bulk processes over a wide range of conditions and do not require many of the limiting assumptions needed to apply resistor model formulations to explain trace gas uptake and reaction.

Dioxin-elicited decrease in cobalamin redirects propionyl-CoA metabolism to the ß-oxidation-like pathway resulting in acrylyl-CoA conjugate buildup.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that induces diverse biological and toxic effects, including reprogramming intermediate metabolism, mediated by the aryl hydrocarbon receptor. However, the specific reprogramming effects of TCDD are unclear. Here, we performed targeted LC-MS analysis of hepatic extracts from mice gavaged with TCDD. We detected an increase in S-(2-carboxyethyl)-L-cysteine, a conjugate from the spontaneous reaction between the cysteine sulfhydryl group and highly reactive acrylyl-CoA, an intermediate in the cobalamin (Cbl)-independent ß-oxidation-like metabolism of propionyl-CoA. TCDD repressed genes in both the canonical Cbl-dependent carboxylase and the alternate Cbl-independent ß-oxidation-like pathways as well as inhibited methylmalonyl-CoA mutase (MUT) at lower doses. Moreover, TCDD decreased serum Cbl levels and hepatic cobalt levels while eliciting negligible effects on gene expression associated with Cbl absorption, transport, trafficking, or derivatization to 5'-deoxy-adenosylcobalamin (AdoCbl), the required MUT cofactor. Additionally, TCDD induced the gene encoding aconitate decarboxylase 1 (Acod1), the enzyme responsible for decarboxylation of cis-aconitate to itaconate, and dose-dependently increased itaconate levels in hepatic extracts. Our results indicate MUT inhibition is consistent with itaconate activation to itaconyl-CoA, a MUT suicide inactivator that forms an adduct with adenosylcobalamin. This adduct in turn inhibits MUT activity and reduces Cbl levels. Collectively, these results suggest the decrease in MUT activity is due to Cbl depletion following TCDD treatment, which redirects propionyl-CoA metabolism to the alternate Cbl-independent ß-oxidation-like pathway. The resulting hepatic accumulation of acrylyl-CoA likely contributes to TCDD-elicited hepatotoxicity and the multihit progression of steatosis to steatohepatitis with fibrosis.

The Difference of Gut Microbiota and Their Correlations With Urinary Organic Acids Between Autistic Children With and Without Atopic Dermatitis.

Autism is a kind of biologically based neurodevelopmental condition, and the coexistence of atopic dermatitis (AD) is not uncommon. Given that the gut microbiota plays an important role in the development of both diseases, we aimed to explore the differences of gut microbiota and their correlations with urinary organic acids between autistic children with and without AD. We enrolled 61 autistic children including 36 with AD and 25 without AD. The gut microbiota was sequenced by metagenomic shotgun sequencing, and the diversity, compositions, and functional pathways were analyzed further. Urinary organic acids were assayed by gas chromatography-mass spectrometry, and univariate/multivariate analyses were applied. Spearman correlation analysis was conducted to explore their relationships. In our study, AD individuals had more prominent gastrointestinal disorders. The alpha diversity of the gut microbiota was lower in the AD group. LEfSe analysis showed a higher abundance of Anaerostipes caccae, Eubacterium hallii, and Bifidobacterium bifidum in AD individuals, with Akkermansia muciniphila, Roseburia intestinalis, Haemophilus parainfluenzae, and Rothia mucilaginosa in controls. Meanwhile, functional profiles showed that the pathway of lipid metabolism had a higher proportion in the AD group, and the pathway of xenobiotics biodegradation was abundant in controls. Among urinary organic acids, adipic acid, 3-hydroxyglutaric acid, tartaric acid, homovanillic acid, 2-hydroxyphenylacetic acid, aconitic acid, and 2-hydroxyhippuric acid were richer in the AD group. However, only adipic acid remained significant in the multivariate analysis (OR = 1.513, 95% CI [1.042, 2.198], P = 0.030). In the correlation analysis, Roseburia intestinalis had a negative correlation with aconitic acid (r = -0.14, P = 0.02), and the latter was positively correlated with adipic acid (r = 0.41, P = 0.006). Besides, the pathway of xenobiotics biodegradation seems to inversely correlate with adipic acid (r = -0.42, P = 0.18). The gut microbiota plays an important role in the development of AD in autistic children, and more well-designed studies are warranted to explore the underlying mechanism.

cis-Aconitic Acid, a Constituent of Echinodorus grandiflorus Leaves, Inhibits Antigen-Induced Arthritis and Gout in Mice.

cis-Aconitic acid is a constituent from the leaves of Echinodorus grandiflorus, a medicinal plant traditionally used in Brazil to treat inflammatory conditions, including arthritic diseases. The present study aimed to investigate the anti-arthritic effect of cis-aconitic acid in murine models of antigen-induced arthritis and monosodium urate-induced gout. The possible underlying mechanisms of action was evaluated in THP-1 macrophages. Oral treatment with cis-aconitic acid (10, 30, and 90 mg/kg) reduced leukocyte accumulation in the joint cavity and C-X-C motif chemokine ligand 1 and IL-1ß levels in periarticular tissue. cis-Aconitic acid treatment reduced joint inflammation in tissue sections of antigen-induced arthritis mice and these effects were associated with decreased mechanical hypernociception. Administration of cis-aconitic acid (30 mg/kg p. o.) also reduced leukocyte accumulation in the joint cavity after the injection of monosodium urate crystals. cis-Aconitic acid reduced in vitro the release of TNF-α and phosphorylation of IκBα in lipopolysaccharide-stimulated THP-1 macrophages, suggesting that inhibition of nuclear factor kappa B activation was an underlying mechanism of cis-aconitic acid-induced anti-inflammatory effects. In conclusion, cis-aconitic acid has significant anti-inflammatory effects in antigen-induced arthritis and monosodium urate-induced arthritis in mice, suggesting its potential for the treatment of inflammatory diseases of the joint in humans. Additionally, our findings suggest that this compound may contribute to the anti-inflammatory effect previously reported for E. grandiflorus extracts.