Pseudotargeted metabolomics revealed the adaptive mechanism of Draba oreades Schrenk at high altitude

Strong ultraviolet radiation and low temperature environment on Gangshika Mountain, located in the eastern part of the Qilian Mountains in Qinghai Province, can force plants to produce some special secondary metabolites for resisting severe environmental stress. However, the adaptive mechanism of Draba oreades Schrenk at high altitude are still unclear. In the current study, Draba oreades Schrenk from the Gangshika Mountain at altitudes of 3800 m, 4000 m and 4200 m were collected for comprehensive metabolic evaluation using pseudotargeted metabolomics method. Through KEGG pathway enrichment analysis, we found that phenylpropanoid biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis and phenylalanine metabolism related to the biosynthesis of flavonoids were up-regulated in the high-altitude group, which may enhance the environmental adaptability to strong ultraviolet intensity and low temperature stress in high altitude areas. By TopFc20 distribution diagram, the content of flavonoids gradually increased with the elevation of altitude, mainly including apigenin, luteolin, quercetin, hesperidin, kaempferol and their derivatives. Based on the random forest model, 10 important metabolites were identified as potential biomarkers. L-phenylalanine, L-histidine, naringenin-7-O-Rutinoside-4’-O-glucoside and apigenin related to the flavonoids biosynthesis and plant disease resistance were increased with the elevation of altitude. This study provided important insights for the adaptive mechanism of Draba oreades Schrenk at high altitude by pseudotargeted metabolomics

Human Ribonuclease A Superfamily Members, Eosinophil-Derived Neurotoxin and Pancreatic Ribonuclease, Induce Dendritic Cell Maturation and Activation

A number of mammalian antimicrobial proteins produced by neutrophils and cells of epithelial origin have chemotactic and activating effects on host cells, including cells of the immune system. Eosinophil granules contain an antimicrobial protein known as eosinophil-derived neurotoxin (EDN), which belongs to the RNase A superfamily. EDN has antiviral and chemotactic activities in vitro. In this study, we show that EDN, and to a lesser extent human pancreatic RNase (hPR), another RNase A superfamily member, activates human dendritic cells (DCs), leading to the production of a variety of inflammatory cytokines, chemokines, growth factors, and soluble receptors. Human angiogenin, a RNase evolutionarily more distant to EDN and hPR, did not display such activating effects. Additionally, EDN and hPR also induced phenotypic and functional maturation DCs. These RNases were as efficacious as TNF-α, but induced a different set of cytokine mediators. Furthermore, EDN production by human macrophages could be induced by proinflammatory stimuli. The results reveal the DC-activating activity of EDN and hPR and suggest that they are likely participants of inflammatory and immune responses. A number of endogenous mediators in addition to EDN have been reported to have both chemotactic and activating effects on APCs, and can thus amplify innate and Ag-specific immune responses to danger signals. We therefore propose these mediators be considered as endogenous multifunctional immune alarmins

Characterization of the xiamenmycin biosynthesis gene cluster in Streptomyces xiamenensis 318.

Xiamenmycin (1) is a prenylated benzopyran derivative with anti-fibrotic activity. To investigate the genetic basis of xiamenmycin biosynthesis, we performed genome mining in the xiamenmycin-producing Streptomyces xiamenensis wild-type strain 318 to identify a candidate gene cluster. The complete gene cluster, consisting of five genes, was confirmed by a series of gene inactivations and heterologous expression. Based on bioinformatics analyses of each gene and feeding experiments, we found that the structure of an intermediate xiamenmycin B (3) accumulated in a ximA inactivation mutant, allowing us to propose a biosynthetic pathway. All five of the genes in the pathway were genetically and biochemically characterized. XimA was biochemically characterized as an ATP-dependent amide synthetase, catalyzing an amide bond formation in the presence of ATP as the final step in Xiamenmycin biosynthesis. The Km value of XimA was determined to be 474.38 µM for the substrate xiamenmycin B. These studies provide opportunities to use genetic and chemo-enzymatic methods to create new benzopyran derivatives as potential therapeutic agents

Deduced ORFs and their predicted functions in the <i>xim</i> gene cluster.

a<p>aa, amino acids.</p>b<p>genome annotation based on <i>Streptomyces himastatinicus</i> ATCC 53653 whole genome shotgun sequence cont1.771.</p>c<p>DNA sequence identity of 86% was observed in the un-annotated ORF in <i>Streptomyces himastatinicus</i> ATCC 53653 cont1.771, whole genome shotgun sequence.</p

Heterologus expression of the xiamenmycin biosynthetic pathway in <i>S. lividans</i>.

<p>UPLC- total ion chromatography MS profiles of the metabolites produced by (A) <i>S. xiamenensis</i> wild type; (B) <i>S. lividans</i> harboring the empty vector pSET152; (C) <i>S. lividans</i> containing pLMO09404.</p

Mutational analysis of the xiamenmycin gene cluster.

<p>(A) Schematic representation of the construction of the gene inactivation mutant (<i>ximA</i>), where <i>ximA</i> was replaced by the <i>apr</i> cassette. The <i>ximA</i> mutant gives a 1041 bp PCR product, while the wild type strain is 1663 bp. Marker (1 kb DNA Ladder, Fermantas). (B) UPLC profiles of 1, 3 and products of wild type and xim mutants. Disruption of ximA to ximE individually abolished production of 1, but the intermediate 3 only accumulated in ΔximA.</p

Proposed biosynthetic pathway for xiamenmycin.

<p>Proposed biosynthetic pathway for xiamenmycin.</p

<i>In vitro</i> assay of XimA.

<p>UPLC-extracted ion chromatography MS (EIC-MS) in the negative ionization mode of XimA <i>in vitro</i> assay. (A) <b>xiamenmycin</b> (<b>1</b>); (B) XimA incubated with <b>xiamenmycin B</b> (<b>3</b>) and L-threonine; (C) boiled XimA incubated with <b>xiamenmycin B</b> (<b>3</b>) and L-threonine.</p

<i>In vitro</i> assay of XimB.

<p>UPLC-total ion chromatography MS in the negative ionization mode of the XimB <i>in </i><i>vitro</i> assay. (A) HR-MS/MS of compound <b>2</b>; the lines display the proposed permutations and combination pattern; (B) UPLC-total ion chromatography MS of the membrane fraction without XimB incubated with 4HB and GPP; (C) UPLC-total ion chromatography MS of XimB incubated with 4HB and GPP.</p

Confirmation of 4HB in the core structure of xiamenmycin.

<p>HR-MS of <b>1</b> after [ring-¹³C₆] 4HB was fed to <i>S. xiamenensis</i> wild type strain, (A) in the positive ionization mode; (B) in the negative ionization mode.</p