Mining Xanthine Oxidase Inhibitors from an Edible Seaweed Pterocladiella capillacea by Using In Vitro Bioassays, Affinity Ultrafiltration LC-MS/MS, Metabolomics Tools, and In Silico Prediction.

The prevalence of gout and the adverse effects of current synthetic anti-gout drugs call for new natural and effective xanthine oxidase (XOD) inhibitors to target this disease. Based on our previous finding that an edible seaweed Pterocladiella capillacea extract inhibits XOD, XOD-inhibitory and anti-inflammatory activities were used to evaluate the anti-gout potential of different P. capillacea extract fractions. Through affinity ultrafiltration coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS), feature-based molecular networking (FBMN), and database mining of multiple natural products, the extract's bioactive components were traced and annotated. Through molecular docking and ADMET analysis, the possibility and drug-likeness of the annotated XOD inhibitors were predicted. The results showed that fractions F4, F6, F4-2, and F4-3 exhibited strong XOD inhibition activity, among which F4-3 reached an inhibition ratio of 77.96% ± 4.91% to XOD at a concentration of 0.14 mg/mL. In addition, the P. capillacea extract and fractions also displayed anti-inflammatory activity. Affinity ultrafiltration LC-MS/MS analysis and molecular networking showed that out of the 20 annotated compounds, 8 compounds have been previously directly or indirectly reported from seaweeds, and 4 compounds have been reported to exhibit anti-gout activity. Molecular docking and ADMET showed that six seaweed-derived compounds can dock with the XOD activity pocket and follow the Lipinski drug-like rule. These results support the value of further investigating P. capillacea as part of the development of anti-gout drugs or related functional foods.

Exogenous Iron Induces NADPH Oxidases-Dependent Ferroptosis in the Conidia of Aspergillus flavus.

Aspergillus flavus is saprophytic soil fungus that contaminates seed crops with the carcinogenic secondary metabolite aflatoxin, posing a significant threat to humans and animals. Ferrous sulfate is a common iron supplement that is used to the treatment of iron-deficiency anemia. Here, we identified an unexpected inhibitory role of ferrous sulfate on A. flavus. With specific fluorescent dyes, we detected several conidial ferroptosis hallmarks in conidia under the treatment of 1 mM Fe2+, including nonapoptosis necrosis, iron-dependent, lipid peroxide accumulation, and ROS burst. However, unlike traditional ferroptosis in mammals, Fe2+ triggered conidial ferroptosis in A. flavus was regulated by NADPH oxidase (NOXs) activation instead of Fenton reaction. Transcriptomic and some other bioinformatics analyses showed that NoxA in A. flavus might be a potential target of Fe2+, and thus led to the occurrence of conidial ferroptosis. Furthermore, noxA deletion mutant was constructed, and both ROS generation and conidial ferroptosis in ΔnoxA was reduced when exposed to Fe2+. Taken together, our study revealed an exogenous Fe2+-triggered conidial ferroptosis pathway mediated by NoxA of A. flavus, which greatly contributes to the development of an alternative strategy to control this pathogen.

Inverse molecular docking reveals a novel function of thymol: Inhibition of fat deposition induced by high-dose glucose in Caenorhabditis elegans.

As a natural product isolated from thyme oil in thyme, thymol (2-isopropyl-5-methylphenol) harbors antiviral, antioxidant, and other properties, and thus could be potentially used for the treatment of various diseases. However, the function of thymol has not been comprehensively studied. Here, we applied an inverse molecular docking approach to identify unappreciated functions of thymol. Potential targets of thymol in humans were identified by the server of DRAR-CPI, and targets of interest were then assessed by GO and KEGG pathway analysis. Subsequently, homologous proteins of these targets in Caenorhabditis elegans were identified by Blast tool, and their three-dimensional structures were achieved using Swiss-Model workspace. Interaction between thymol and the targeted proteins in worms was verified using AutoDock 4.0. Analyses of the targets revealed that thymol could be potentially involved in the glycolysis/gluconeogenesis and fatty acid degradation pathways. To verify the activity of thymol on lipid deposition in vivo, the C. elegans model was established. The lipid content of nematodes induced by high-dose glucose was determined by Oil Red O and Nile Red staining, and gene expression was assessed by qRT-PCR. The results showed that thymol might lead to the acceleration of ß-oxidation by upregulating cpt-1, aco, fabp, and tph-1, causing the descent of lipid content in nematodes. Our findings indicated that thymol could be potentially used for the treatment of chronic metabolic diseases associated with increased fatty acid deposition.

Thymol Induces Conidial Apoptosis in Aspergillus flavus via Stimulating K+ Eruption.

Aspergillus flavus is a notorious foodborne fungus, posing a significant risk to humans in the form of hepatocellular carcinoma or aspergillosis. Thymol, as a food preservative, could efficiently kill conidia of A. flavus. However, the underlying mechanisms by which thymol kills A. flavus are not completely understood. With specific fluorescent dyes, we detected several apoptotic hallmarks, including chromatin condensation, phosphatidylserine externalization, DNA damage, mitochondrial depolarization, and caspase 9 activation in conidia exposed to 200 µg/mL of thymol, indicating that thymol induced a caspase-dependent conidial apoptosis in A. flavus. Chemical-protein interactome (CPI) and autodock analyses showed that KCNAB, homologue to the ß-subunit of the voltage-gated potassium channel (Kv) and aldo-keto reductase, was the potential target of thymol. Following studies demonstrated that thymol could activate the aldo-keto reductase activity of KCNAB in vitro and stimulate a transient K+ efflux in conidia, as determined using a Port-a-Patch. Blocking K+ eruption by 4-aminopyridine (a universal inhibitor of Kv) could significantly alleviate thymol-mediated conidial apoptosis, indicating that activation of Kv was responsible for the apoptosis. Taken together, our results revealed a K+ efflux-mediated apoptotic pathway in A. flavus, which greatly contributed to the development of an alternative strategy to control this pathogen.

The design of a mechanical wave-like DNA nanomachine for the fabrication of a programmable and multifunctional molecular device.

We report a novel mechanical wave-like DNA nanomachine. A successive stem-loop structure is involved, which will rearrange successively from one side to the opposite side upon binding with an input activator just like the motion of a mechanical wave, and thereby achieving diverse functions.

A one-pot strategy for the detection of proteins based on sterically and allosterically tunable hybridization chain reaction.

In this work, we report a facile one-pot strategy for protein detection based on sterically and allosterically tunable hybridization chain reaction (HCR). In our strategy, DNA hairpins H1 and H2 are dual-labeled with pyrene moieties through a six-carbon-atom spacer at each end; and a single-stranded DNA primer is designed to contain two small molecules near each end. In the absence of target protein, the primer can trigger HCR events between alternating H1 and H2 hairpins to form a nicked double-helix. As a result, the pyrene excimers are formed to emit at approximately 485nm. On the contrary, upon binding of the specific target protein onto the primer through the protein-small molecule interaction, the HCR will be inhibited due to the steric and allosteric effect. The changes of the fluorescent signals of pyrene excimers are in response to the concentration of target protein, so that the detection of protein can be realized. We have demonstrated the feasibility of this strategy by using streptavidin (SA) and folate receptor (FR) as model targets. Results show that both of them can be well detected with a detection limit of 1.07nM and 2.7nM, respectively. The developed method for protein assay is flexible, so we infer that the one-pot strategy holds great potential for the detection of other proteins.

Rapid detection of acute myocardial infarction-related miRNA based on a Capture-interCalation-electroCatalysis (3C) strategy.

Acute myocardial infarction (AMI) is one of the most urgent and serious diseases that may cause cardiac death in a few hours. Rapid diagnosis of AMI is the pre-requisite for timely interventions. Recently, several specific circulating miRNAs have been proven to have high correlation with AMI. To adopt miRNA as a biomarker may improve the diagnostic accuracy. However, it is a pity that the current available methods for the detection of miRNA usually require a few hours, which is too long for the diagnosis of AMI. In this paper, by adopting a capture DNA, an electrochemical active intercalator and an unimmobilized enzyme, we develop a Capture-interCalation-electroCatalysis (3C) strategy for the rapid detection of AMI-related miRNA. The whole detection process can be completed in 35 min, which is much shorter than most current methods and is acceptable for the diagnosis of AMI. This strategy also shows favorable sensitivity and selectivity, thus provides an alternative for the detection of miRNA. Most importantly, this effort may promote miRNA to work as an effective biomarker in the diagnosis of AMI.

Detection of microRNA SNPs with ultrahigh specificity by using reduced graphene oxide-assisted rolling circle amplification.

Here we report a reduced graphene oxide-assisted rolling circle amplification for the detection of miRNA SNPs. The difference of the signal of a miRNA SNP reaches 100 fold, a value over 10 times larger than some current methodologies, which allows the discrimination of a SNP even with the naked eye.