Pore-Forming Cardiotoxin VVA2 (Volvatoxin A2) Variant I82E/L86K Is an Atypical Duplex-Specific Nuclease.

VVA2 (volvatoxin A chain 2) is a cardiotoxic protein purified from Volvariella volvacea. Its biological activities include hemolysis, writhing reaction, neurotoxicity, and ventricular systolic arresting activity. The cytotoxicity of VVA2 was mainly considered due to its pore-forming activity. Here we report a novel biological activity of its variants VVA2 I82E/K86K as a duplex-specific nuclease. Recombinant VVA2 variant I82E/L86K (Re-VVA2 I82E/L86K), deprived of the oligomerization property, shows increased nuclease activity compared to VVA2. Re-VVA2 I82E/L86K converts supercoiled DNA (Replicative form I, RF I) into nicked form (RF II) and linear form (RF III) in the presence of Mg2+ or Mn2+. Besides plasmid DNA, it also exhibits nuclease activity on E. coli genomic DNA rather than ssDNA or RNA. Re-VVA2 I82E/L86K preferentially cleaves dG-dC-rich dsDNA regions and shows the best performance at pH 6-9 and 55 °C. Our structure-function study has revealed amino acid E111 may take an active part in nuclease activity through interacting with metal ions. Based on the sequences of its cleavage sites, a "double-hit" mechanism was thereby proposed. Given that Re-VVA2 I82E/L86K did not exhibit the conserved nuclease structure and sequence, it is considered an atypical duplex-specific nuclease.

Metabolomics Analysis Reveals Specific Novel Tetrapeptide and Potential Anti-Inflammatory Metabolites in Pathogenic Aspergillus species.

Infections related to Aspergillus species have emerged to become an important focus in infectious diseases, as a result of the increasing use of immunosuppressive agents and high fatality associated with invasive aspergillosis. However, laboratory diagnosis of Aspergillus infections remains difficult. In this study, by comparing the metabolomic profiles of the culture supernatants of 30 strains of six pathogenic Aspergillus species (A. fumigatus, A. flavus, A. niger, A. terreus, A. nomius and A. tamarii) and 31 strains of 10 non-Aspergillus fungi, eight compounds present in all strains of the six Aspergillus species but not in any strain of the non-Aspergillus fungi were observed. One of the eight compounds, Leu-Glu-Leu-Glu, is a novel tetrapeptide and represents the first linear tetrapeptide observed in Aspergillus species, which we propose to be named aspergitide. Two other closely related Aspergillus-specific compounds, hydroxy-(sulfooxy)benzoic acid and (sulfooxy)benzoic acid, may possess anti-inflammatory properties, as 2-(sulfooxy)benzoic acid possesses a structure similar to those of aspirin [2-(acetoxy)benzoic acid] and salicylic acid (2-hydroxybenzoic acid). Further studies to examine the potentials of these Aspergillus-specific compounds for laboratory diagnosis of aspergillosis are warranted and further experiments will reveal whether Leu-Glu-Leu-Glu, hydroxy-(sulfooxy)benzoic acid and (sulfooxy)benzoic acid are virulent factors of the pathogenic Aspergillus species.

Lipid mediators of inflammation as novel plasma biomarkers to identify patients with bacteremia.

OBJECTIVES: Rapid diagnostic tests for bacteremia are important for early treatment to improve clinical outcome. We sought to identify plasma biomarkers that can identify patients with bacteremia using an untargeted global metabolomic analysis. METHODS: Plasma metabolomic profiles were analyzed for 145 adult patients with (cases) and without (controls) bacteremia using ultra-high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). All metabolites were compared between cases and controls using a 2-tier filtering approach, and each metabolite underwent receiver operating characteristic (ROC) curve analysis. Individual metabolites that distinguish between cases and controls were characterized. Subgroup analysis was performed to identify metabolites with prognostic significance. RESULTS: After 2-tier filtering, 128 molecular features were identified to be potential biomarkers that could distinguish cases from controls. Five metabolites had an area under the ROC curve (AUC) of >0.8 in ROC curve analysis, including a sphingolipid, an acylcarnitine, a fatty acid ester, and 2 glycerophosphocholines. These metabolites could distinguish cases from controls in the unsupervised hierarchical clustering analysis. Subgroup analysis of bacteremic patients showed that the level of trans-2,3,4-trimethoxycinnamate was lower in fatal than non-fatal cases. CONCLUSIONS: Plasma lipid mediators of inflammation can distinguish bacteremia cases from non-bacteremia controls. These biomarkers may be used as targets for rapid test in clinical practice.