Benzophenanthridine Alkaloid Chelerythrine Elicits Necroptosis of Gastric Cancer Cells via Selective Conjugation at the Redox Hyperreactive C-Terminal Sec498 Residue of Cytosolic Selenoprotein Thioredoxin Reductase.

Targeting thioredoxin reductase (TXNRD) with low-weight molecules is emerging as a high-efficacy anti-cancer strategy in chemotherapy. Sanguinarine has been reported to inhibit the activity of TXNRD1, indicating that benzophenanthridine alkaloid is a fascinating chemical entity in the field of TXNRD1 inhibitors. In this study, the inhibition of three benzophenanthridine alkaloids, including chelerythrine, sanguinarine, and nitidine, on recombinant TXNRD1 was investigated, and their anti-cancer mechanisms were revealed using three gastric cancer cell lines. Chelerythrine and sanguinarine are more potent inhibitors of TXNRD1 than nitidine, and the inhibitory effects take place in a dose- and time-dependent manner. Site-directed mutagenesis of TXNRD1 and in vitro inhibition analysis proved that chelerythrine or sanguinarine is primarily bound to the Sec498 residue of the enzyme, but the neighboring Cys497 and remaining N-terminal redox-active cysteines could also be modified after the conjugation of Sec498. With high similarity to sanguinarine, chelerythrine exhibited cytotoxic effects on multiple gastric cancer cell lines and suppressed the proliferation of tumor spheroids derived from NCI-N87 cells. Chelerythrine elevated cellular levels of reactive oxygen species (ROS) and induced endoplasmic reticulum (ER) stress. Moreover, the ROS induced by chelerythrine could be completely suppressed by the addition of N-acetyl-L-cysteine (NAC), and the same is true for sanguinarine. Notably, Nec-1, an RIPK1 inhibitor, rescued the chelerythrine-induced rapid cell death, indicating that chelerythrine triggers necroptosis in gastric cancer cells. Taken together, this study demonstrates that chelerythrine is a novel inhibitor of TXNRD1 by targeting Sec498 and possessing high anti-tumor properties on multiple gastric cancer cell lines by eliciting necroptosis.

Protocol for assaying irreversible inhibitors of thioredoxin reductase 1.

Selenoprotein thioredoxin reductase 1 (TXNRD1) is a promising therapeutic target, with several inhibitors reported to inhibit TXNRD1 activity. These inhibitors have the potential for applications such as anti-tumor medications. Here, we present a protocol for assessing irreversible inhibitors of TXNRD1. We describe four assays covering cellular TXNRD activity measurement, recombinant enzyme-based activity determination, differential scanning fluorimetry (DSF), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This protocol will facilitate the screening and development of potential small-molecule inhibitors of TXNRD1.

Guiding bar motif of thioredoxin reductase 1 modulates enzymatic activity and inhibitor binding by communicating with the co-factor FAD and regulating the flexible C-terminal redox motif.

Thioredoxin reductase (TXNRD) is a selenoprotein that plays a crucial role in cellular antioxidant defense. Previously, a distinctive guiding bar motif was identified in TXNRD1, which influences the transfer of electrons. In this study, utilizing single amino acid substitution and Excitation-Emission Matrix (EEM) fluorescence spectrum analysis, we discovered that the guiding bar communicates with the FAD and modulates the electron flow of the enzyme. Differential Scanning Fluorimetry (DSF) analysis demonstrated that the aromatic amino acid in guiding bar is a stabilizer for TXNRD1. Kinetic analysis revealed that the guiding bar is vital for the disulfide reductase activity but hinders the selenocysteine-independent reduction activity of TXNRD1. Meanwhile, the guiding bar shields the selenocysteine residue of TXNRD1 from the attack of electrophilic reagents. We also found that the inhibition of TXNRD1 by caveolin-1 scaffolding domain (CSD) peptides and compound LCS3 did not bind to the guiding bar motif. In summary, the obtained results highlight new aspects of the guiding bar that restrict the flexibility of the C-terminal redox motif and govern the transition from antioxidant to pro-oxidant.

A review on the synthesis of metal oxide nanomaterials by microwave induced solution combustion.

With the increasing awareness of environmental protection and health, the preparation of metal oxide nanomaterials by environmentally friendly methods is favored by more and more researchers both at home and abroad. The preparation of metal oxide nanomaterials by a microwave-induced solution combustion synthesis method can significantly reduce the reaction time, energy consumption, and the use of toxic chemicals, which is an energy-saving, environmentally friendly method for nanomaterials synthesis. In addition, the microwave-induced solution combustion synthesis (MISCS) method has many advantages such as fast reaction speed, high selectivity, small product size, and homogeneous composition. This paper briefly describes the mechanism of the MISCS and its advantages in the synthesis of metal oxide nanomaterials, the related studies on microwave-induced solution combustion synthesis of metal oxide nanomaterials, and the effects of process parameters on the microstructure and properties of metal oxide nanomaterials synthesized by MISCS. Furthermore, some technical difficulties facing the synthesis of metal oxide nanomaterials by MISCS are summarized, and the future direction has also been prospected.

The molecular characteristics of avian influenza viruses (H9N2) derived from air samples in live poultry markets.

OBJECTIVE: To study the molecular characteristics of H9N2-subtype avian influenza viruses (AIVs) isolated from air samples collected in live poultry markets (LPMs) and explore their sequence identities with AIVs that caused human infection. METHODS: Weekly surveillance of H9N2-subtype AIVs in the air of LPMs was conducted from 2015 to 2016. H9-positive samples were isolated from chicken embryos. Whole genome sequences of the isolated AIVs were obtained through high-throughput sequencing. Phylogenetic analysis and key loci variations of the sequences were further analyzed. RESULTS: A total of 327 aerosol samples were collected from LPMs. Nine samples were positive for H9-subtype AIVs based on quantitative real-time reverse transcription polymerase chain reaction (qRRT-PCR). According to the whole genome sequence analysis and phylogenetic analysis, except for the A/Environment/Zhongshan/ZS201505/2015 (ZS201505) strain, 8 gene segments of 8 aerosol H9N2 isolates and 2 H9N2 human isolates in 2015 were located in the same clade. Among key loci variations, except for the ZS201505 strain, H9N2-subtype AIVs had no mutations in eight receptor binding sites of hemagglutinin (HA), and stalks of neuraminidase (NA) proteins exhibited a deletion site of three bases. The PA gene of ZS201503 and ZS201602 exhibited an L336M mutation. The N30D and T215A mutations in the M1 gene and amino acid residues L89V in PB2, P42S in NS1 and S31N in M2 were retained in these 9 strains of H9N2 isolates, which could enhance the virus's virulence. CONCLUSION: Live H9N2 AIVs survived in the aerosol of LPMs in Zhongshan City. The aerosol viruses had a close evolutionary relationship with human epidemic strains, indicating that there might be a risk of AIV transmission from polluted aerosols in LPMs to humans. Mutations in H9N2-subtype AIVs isolated from air samples collected from LPMs suggested their pathogenicity was enhanced to infect humans.

[Study of the bacterial community structure of microbiota in bacterial vaginosis using amplified ribosomal DNA restriction analysis].

OBJECTIVE: To study the bacterial community structure of the microbiota in the vaginal fluid from patients with bacterial vaginosis. METHODS: The composition of bacteria in the samples of vaginal fluid from 3 patients with bacterial vaginosis and 1 normal premenopausal control was investigated by amplified ribosomal DNA restriction analysis(ARDRA). RESULTS: Lactobacillus species were the predominant bacteria in the woman without bacterial vaginosis. Bacterial vaginosis was associated with higher concentrations of a variety of bacterial groups. Women with bacterial vaginosis had greater bacterial diversity, with 31 to 37 OTUs operational taxonomic units detected per sample. The species associated with bacterial vaginosis were Leptotrichia, Prevotella sp. and Megasphaera including several species with no close cultivated relatives. CONCLUSIONS: Women with bacterial vaginosis have complex vaginal infections with many newly recognized species. ARDRA allows rapid analysis of the diversity of microorganisms in the vagina, and is capable of identifying potentially pathogenic bacteria that can not be identified by general culture.

Archaeal diversity in acid mine drainage from Dabaoshan Mine, China.

Three acid mine drainage (AMD) samples collected from Dabaoshan Mine (Guangdong Province, China) were studied. In addition to physicochemical analyses, the diversity and community structures of the archaeal communities in these samples were described at the genetic level by amplified ribosomal DNA restriction analysis (ARDRA). Nine different ARDRA patterns were obtained from 146 clones and were studied as operational taxonomic units (OTUs), which were re-amplified and sequenced. Sequence data and phylogenetic analysis showed that most of the clones belonged to the Thermoplasmatales, and that archaea belonging to the Sulfolobales were absent. Only 1 OTU attributed to Ferroplasma was found and was observed to be abundant in all 3 samples. Eight OTUs were related to 2 new undefined groups in the Thermoplasmatales. Of the 8 OTUs, the clones in 2 similar units were isolated from samples collected from an abandoned sulfide mine (Huelva, Spain) and those in 5 similar units were isolated from samples collected from a closed copper mine (Tonglushan, China). These diversities were characterized by the reciprocal of Simpson's index (1/D) and correlated with the concentrations of ferrous ions and toxic ions in the AMD samples. The high temperature of the sampling sites was one of the factors that could explain why archaea belonging to the Thermoplasmatales were abundant in the analyzed AMD samples while those belonging to the Sulfolobales were absent.

Diversity of bacterial communities in acid mine drainage from the Shen-bu copper mine, Gansu province, China

This study presents bacterial population analyses of microbial communities inhabiting three sites of acid mine drainage (AMD) in the Shen-bu copper mine, Gansu Province, China. These sites were located next to acid-leached chalcopyrite slagheaps that had been abandoned since 1995. The pH values of these samples with high concentrations of metals ranged from 2.0 to 3.5. Amplified ribosomal DNA restriction analysis (ARDRA) was used to characterize the bacterial population by amplifying the 16S rRNA gene of microorganisms. A total of 39 operational taxonomic units (OTUs) were obtained from the three samples and sequenced from 384 clones. Sequence data and phylogenetic analyses showed that two dominant clones (JYC-1B, JYC-1D) in sample JYC-1 represented 69.5 percent of the total clones affiliated with Acidithiobacillus ferrooxidans (gamma-Proteobacteria), and the most dominant clones of JYC-2 and JYC-3 were affiliated with Caulobacter crescentus (alpha-Protebacteria). At the level of bacterial divisions, differences in the relative incidence of particular phylogenetic groups among the three samples and discrepancies in physicochemical characteristics suggested that the physico-chemical characteristics had an influence on phylogenetic diversity. Furthermore, the relationships between the discrepancies of physicochemical characteristics and the diversity of the bacteria communities in the three samples suggested that the biogeochemical properties, pH and concentration of soluble metal, could be key factors in controlling the structure of the bacterial population.