G-quadruplex inducer/stabilizer pyridostatin targets SUB1 to promote cytotoxicity of a transplatinum complex.

Pyridostatin (PDS) is a well-known G-quadruplex (G4) inducer and stabilizer, yet its target genes have remained unclear. Herein, applying MS proteomics strategy, we revealed PDS significantly downregulated 22 proteins but upregulated 16 proteins in HeLa cancer cells, of which the genes both contain a number of G4 potential sequences, implying that PDS regulation on gene expression is far more complicated than inducing/stabilizing G4 structures. The PDS-downregulated proteins consequently upregulated 6 proteins to activate cyclin and cell cycle regulation, suggesting that PDS itself is not a potential anticancer agent, at least toward HeLa cancer cells. Importantly, SUB1, which encodes human positive cofactor and DNA lesion sensor PC4, was downregulated by 4.76-fold. Further studies demonstrated that the downregulation of PC4 dramatically promoted the cytotoxicity of trans-[PtCl2(NH3)(thiazole)] (trans-PtTz) toward HeLa cells to a similar level of cisplatin, contributable to retarding the repair of 1,3-trans-PtTz crosslinked DNA lesion mediated by PC4. These findings not only provide new insights into better understanding on the biological functions of PDS but also implicate a strategy for the rational design of novel multi-targeting platinum anticancer drugs via conjugation of PDS as a ligand to the coordination scaffold of transplatin for battling drug resistance to cisplatin.

Baicalin Targets HSP70/90 to Regulate PKR/PI3K/AKT/eNOS Signaling Pathways.

Baicalin is a major active ingredient of traditional Chinese medicine Scutellaria baicalensis, and has been shown to have antiviral, anti-inflammatory, and antitumor activities. However, the protein targets of baicalin have remained unclear. Herein, a chemical proteomics strategy was developed by combining baicalin-functionalized magnetic nanoparticles (BCL-N3@MNPs) and quantitative mass spectrometry to identify the target proteins of baicalin. Bioinformatics analysis with the use of Gene Ontology, STRING and Ingenuity Pathway Analysis, was performed to annotate the biological functions and the associated signaling pathways of the baicalin targeting proteins. Fourteen proteins in human embryonic kidney cells were identified to interact with baicalin with various binding affinities. Bioinformatics analysis revealed these proteins are mainly ATP-binding and/or ATPase activity proteins, such as CKB, HSP86, HSP70-1, HSP90, ATPSF1ß and ACTG1, and highly associated with the regulation of the role of PKR in interferon induction and the antiviral response signaling pathway (P = 10-6), PI3K/AKT signaling pathway (P = 10-5) and eNOS signaling pathway (P = 10-4). The results show that baicalin exerts multiply pharmacological functions, such as antiviral, anti-inflammatory, antitumor, and antioxidant functions, through regulating the PKR and PI3K/AKT/eNOS signaling pathways by targeting ATP-binding and ATPase activity proteins. These findings provide a fundamental insight into further studies on the mechanism of action of baicalin.

Open-flow microperfusion combined with mass spectrometry for in vivo liver lipidomic analysis.

At present, conventional microdialysis (MD) techniques cannot efficiently sample lipids in vivo, possibly due to the high mass transfer resistance and/or the serious adsorption of lipids onto the semi-permeable membrane of a MD probe. The in vivo monitoring of lipids could be of great significance for the study of disease development and mechanisms. In this work, an open-flow microperfusion (OFM) probe was fabricated, and the conditions for sampling lipids via OFM were optimized. Using OFM, the recovery of lipid standards was improved to more than 34.7%. OFM is used for the in vivo sampling of lipids in mouse liver tissue with fibrosis, and it is then combined with mass spectrometry (MS) to perform lipidomic analysis. 156 kinds of lipids were identified in the dialysate collected via OFM, and it was found that the phospholipid levels, including PC, PE, and SM, were significantly higher in a liver suffering from fibrosis. For the first time, OFM combined with MS to sample and analyze lipids has provided a promising platform for in vivo lipidomic studies.

In Situ Visualization of Proteins in Single Cells by Time-of-Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags.

In situ visualization of proteins of interest in single cells is attractive in cell biology, molecular biology, and biomedicine fields. Time-of-flight-secondary ion mass spectrometry (ToF-SIMS) is a powerful tool for imaging small organic molecules in single cells, yet difficult to image biomacromolecules such as proteins and DNA. Herein, a universal strategy is reported to image specific proteins in single cells by ToF-SIMS following genetic incorporation of fluorine-containing unnatural amino acids as a chemical tag into the proteins via a genetic code expansion technique. The method was developed and validated by imaging a green fluorescence protein (GFP) in Escherichia coli (E. coli) and human HeLa cancer cells and then utilized to visualize the characteristic polar distribution of chemotaxis protein CheA in E. coli cells and the interaction between high-mobility group box 1 protein and cisplatin-damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for in situ visualization of specific proteins as well as the interactions between proteins and drugs or drug-damaged DNA in single cells.

Uptake and Transformation of Silver Nanoparticles and Ions by Rice Plants Revealed by Dual Stable Isotope Tracing.

Knowledge on the uptake and transformation of silver nanoparticles (AgNPs) and Ag+ ions by organisms is critical for understanding their toxicity. Herein, the differential uptake, transformation, and translocation of AgNPs and Ag+ ions in hydroponic rice ( Oryza sativa L.) is assessed in modified Hewitt (with Cl- ions, HS(Cl)) and Hogland solutions (without Cl- ions, HS) using dual stable isotope tracing (107AgNO3 and 109AgNPs). After coexposure to 107Ag+ ions and 109AgNPs at 50 µg L-1 (as Ag for both) for 14 days, a stimulatory effect was observed on root elongation (increased by 68.8 and 71.9% for HS(Cl) and HS, respectively). Most of the Ag+ ions (from 107Ag+ ions and 109AgNPs) were retained on the root surface, while the occurrence of AgNPs (from 109AgNPs and 107Ag+ ions) was observed in the root, suggesting the direct uptake of AgNPs and/or reduction of Ag+ ions. Higher fractions of Ag+ ions in the shoot suggest an in vivo oxidation of AgNPs. These results demonstrated the intertransformation between Ag+ ions and AgNPs and the role of AgNPs as carriers and sources of Ag+ ions in organisms, which is helpful for understanding the fate and toxicology of Ag.

Enantioselective degradation of alpha-cypermethrin and detection of its metabolites in bullfrog (rana catesbeiana).

Bullfrog, as a kind of amphibians, can be easily exposed to varied pollutants in the environment for the reason of its habitats and highly permeable skin. We investigated the degradation kinetics and residues of α-cypermethrin in bullfrog by two different methods of administration for the environmental monitoring the behavior of one of the most used pesticides in the amphibians. The oral administration and water exposure of α-cypermethrin on bullfrog was studied in this work. α-Cypermethrin and its main metabolites of cis-3-(2',2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DCCA) and 3-phenoxybenzoic acid (3-PBA), which have been determined that having correlation with a number of epidemic diseases, were detected simultaneously. The method for residue analysis in the bullfrog's organs was validated. The average recoveries for α-cypermethrin were ranged from 71.7% to 100.3% and the limit of quantification was 0.005mg/kg. The average recoveries of its metabolites at levels between 0.002 and 0.5mg/kg ranged between 77.9% and 102.4% with a limit of quantification of 0.002mg/kg. Furthermore, the enantiomers of α-cypermethrin were separated on gas chromatograph (GC) equipped with a chiral column of BGB-172 and the metabolites were detected by gas chromatography (GC) after derivatization. After exposure of α-cypermethrin on bullfrog, the enantioselective degradation behavior was observed and its metabolites were detected in bullfrog tissues. The dynamic trends of α-cypermethrin and its metabolites were fitted to a two-compartment model except 3-PBA fitting to one-compartment model in skin. Concentration of α-cypermethrin and its metabolites in bullfrog's organs increased and reached an equilibrium state during water exposure of α-cypermethrin. Liver and kidney were the major organs for α-cypermethrin and its metabolites retention in both experiments.