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.

Development of ion pairing LC-MS/MS method for itaconate and cis-aconitate in cell extract and cell media.

Accumulation of Immune Responsive Gene 1(IRG1) in macrophage induced by lipopolysaccharide (LPS) and interferon gamma (IFN-γ) leads to production of itaconate by decarboxylation of cis-aconitate. The biology associated with IRG1 and itaconate is not fully understood. A rapid and sensitive method for measurement of itaconate will benefit the study of IRG1 biology. Multiple HPLC and derivatization methods were tested. An ion pairing LC-MS/MS method using tributylamine/formic acid as ion pairing agents and a HypercarbTM guard column we proposed demonstrated better peak shape and better sensitivity for itaconate. The current protocol allows baseline separation of itaconate, citraconate, and cis-aconitate without derivatization and direct analysis of analytes in 80% methanol/water solution to avoid the dry-down step. It provides the limit of quantitation (LOQ) of 30 pg itaconate on column with a 4.5-minute run time. This method is validated for measurement of itaconate and cis-aconitate in RAW264.7 cell extract and cell media in a 96-well plate format. We applied this method to successfully measure the increase of itaconate and the decrease of cis-aconitate in RAW cell extract and cell media after LPS/IFN-γ treatment.

Contribution of trans-aconitic acid to DPPH scavenging ability in different media.

The antioxidant properties of trans-aconitic acid (TAA) alone or in the presence of usual antioxidants were assessed by DPPH assay. The IC50 value equal to 70mM was very high compared to usual antioxidants (vitamin C and trolox). A joint experimental/theoretical study suggested that hydrogen atom abstraction in TAA by DPPH was located on -CH2- methylene bridge because the corresponding radical was more stabilized than COO(·) and CC(·) radicals. In combination with antioxidants (vitamin C, gallic acid, caffeic acid, trolox), synergy or additivity effects were noticed. The magnitude of the synergistic effect varied between 1.06 and 1.24 depending on the type and concentration of antioxidant for a concentration of TAA equal to 22.3mM. Especially, the addition of TAA at a concentration below 32mM to a solution containing 20µM of vitamin C had a synergy effect. Beyond this concentration, TAA showed an additive effect.

Antiedematogenic activity and phytochemical composition of preparations from Echinodorus grandiflorus leaves.

The leaves of Echinodorus grandiflorus (Alismataceae) are traditionally used in Brazil to treat inflammatory conditions. The aim of the present study was to evaluate the antidematogenic activity of crude aqueous, dichloromethane and hydroethanolic extracts from E. grandiflorus leaves using the carrageenan-induced paw edema model in mice, along with of fractions enriched in diterpenes, flavonoids and hydroxycinnamoyltartaric acids (HCTA). Significant inhibitions of paw edema were elicited by the 50% and 70% EtOH extracts (1000 mg/kg, p.o.), as well as by the fractions enriched in diterpenes (70-420 mg/kg, p.o.) and flavonoids (7.2-36 mg/kg, p.o.). Isovitexin, isoorientin, trans-aconitic and chicoric acids were identified in all extracts by HPLC analysis. Trans-aconitic acid itself exhibited significant antiedematogenic effect (270 mg/kg, p.o.). The biological activity correlated positively with the contents of flavonoids and diterpenes, but negatively with HCTA concentrations, demonstrating the participation of the two classes of compounds in the antiedematogenic activity of E. grandiflorus.

High-speed counter-current chromatography for the study of defense metabolites in wheat. Characterization of 5,6-O-methyl trans-aconitic acid.

High-speed counter-current chromatography (HSCCC) methods were developed for the study of induced defense metabolites in wheat (Triticum aestivum) against powdery mildew (Blumeria graminis f. sp. tritici). A single HSCCC purification step afforded extraction of mg-quantities of an induced compound with antifungal activity. Subsequent LC-MS and NMR analyses have led to the characterization of 5,6-O-methyl trans-aconitic acid, the first such report of this compound in a plant species. The inducible nature of aconitic acid was evidenced by comparing the metabolite profiles of leaf extracts from plants treated or not with soluble silicon and infected or not with powdery mildew. In a second step, dual-mode HSCCC was used to enhance the separation of other forms of aconitic acid in wheat. Based on these results, it was concluded that 5,6-O-methyl trans-aconitic acid plays an important role as a defense molecule in wheat plants and that HSCCC is a powerful separation method for purifying such compounds from complex plant-pathogen interactions.

Chemical interactions of Brachiaria plantaginea with Commelina bengalensis and Acanthospermum hispidum in soybean cropping systems.

Previous results obtained in soybean-wheat rotations under no-tillage conditions showed reductions in the seedbank of the weed species Commelina benghalensis, but no alteration in the seedbank of Acanthospermum hispidum in areas infested with Brachiaria plantaginea. Analyses of the soluble fraction of B. plantaginea indicated the predominance of aconitic acid (AA) among the aliphatic acids and ferulic acid (FA) among the phenolic acids. Laboratory bioassays using C. benghalensis and A. hispidum were carried out to evaluate phytotoxic effects of pure organic acid solutions and dilute extracts of B. plantaginea on seed germination, root development, and fungal germination. Solutions of AA and FA were prepared at 0.25, 0.50, and 1.0 mM. Extracts of B. plantaginea were diluted to obtain concentrations of AA similar to those in the prepared solutions. Seeds were sown on 0.5% agar (containing AA, FA, or diluted extract) in plastic-covered receptacles and maintained in a germination chamber for 10 days. AA and FA solutions and the B. plantaginea extract reduced germination and root length, mainly of C. benghalensis. AA also stimulated the development of endophytic fungi (Fusarium solani), which had complementary adverse effects on C. benghalensis germination. FA and AA may play important roles in reducing the seedbank of some weed species, acting directly on germination and development and, indirectly, by stimulating endophytic fungi that alter germination.

Automated docking in crystallography: analysis of the substrates of aconitase.

Automated docking of substrates to proteins of known structure aids the process of crystallographic analysis in two ways. First, automated docking can be used to generate a small number of starting models for substrates using only protein coordinates from an early stage of refinement. Second, automated docking provides a method for exploring aspects of catalysis that are inaccessible to crystallography by postulating binding modes of catalytic intermediates. This paper describes the use of automated docking to explore the binding of substrates to aconitase. The technique starts with a substrate molecule in an arbitrary configuration and position and finds favorable docked configurations in a (static) protein active site based on a molecular mechanics type force field. Using protein coordinates from an early stage of refinement of an aconitase-isocitrate complex, we successfully predicted the binding configuration of isocitrate. Four configurations were found, the energetically most favorable of which fit the observed electron density well and was used as a starting model for further refinement. Two configurations were found in citrate docking experiments, the second of which approximates the mode of substrate binding in an aconitase-nitrocitrate complex. We were also able to propose two binding modes of the catalytic intermediate cis-aconitate. These correspond closely to the isocitrate and the citrate binding modes. The relation of these new results to the proposed reaction mechanism is discussed.

Kinetic studies of cytoplasmic and mitochondrial aconitate hydratases from rat liver.

The kinetic properties of the cytoplasmic and mitochondrial aconitate hydratases of rat liver have been studied by measuring the formation of the two products from each of the three tricarboxylic acids used as substrate. The kinetic properties of the two enzymes are very similar; the similarity of the Km values for each of the three substrates is particularly remarkable. The results are discussed with particular reference to a possible role of the cytoplasmic aconitate hydratase in the process of gluconeogenesis. With both aconitate hydratases, substrate activation by citrate and D-isocitrate has been observed.