A simple chip free-flow electrophoresis for monosaccharide sensing via supermolecule interaction of boronic acid functionalized quencher and fluorescent dye.

Here, a simple micro free-flow electrophoresis (µFFE) was developed for fluorescence sensing of monosaccharide via supermolecule interaction of synthesized boronic acid functionalized benzyl viologen (ο-BBV) and fluorescent dye. The µFFE contained two open electrode cavities and an ion-exchange membrane was sandwiched between two polymethylmethacrylate plates. The experiments demonstrated the following merits of developed µFFE: (i) up to 90.5% of voltage efficiency due to high conductivity of ion-exchange membrane; (ii) a strong ability against influence of bubble produced in two electrodes due to open design of electrode cavities; and (iii) reusable and washable separation chamber (45 mm × 17 mm × 100 µm, 77 µL) avoiding the discard of µFFE due to blockage of solute precipitation in chamber. Remarkably, the µFFE was first designed for the sensing of monosaccharide via the supermolecule interaction of synthesized ο-BBV, fluorescent dye, and monosaccharide. Under the optimized conditions, the minimum concentration of monosaccharide that could be detected was 1 × 10(-11) M. Finally, the developed device was used for the detection of 0.3 mM glucose spiked in human urine. All of the results demonstrated the feasibility of monosaccharide detection via the µFFE.

Vacuolating Cytotoxin A Triggers Mitophagy in Helicobacter pylori-Infected Human Gastric Epithelium Cells.

Helicobacter pylori (H. pylori)-derived vacuolating cytotoxin A (VacA) causes damage to various organelles, including mitochondria, and induces autophagy and cell death. However, it is unknown whether VacA-induced mitochondrial damage can develop into mitophagy. In this study, we found that H. pylori, H. pylori culture filtrate (HPCF), and VacA could activate autophagy in a gastric epithelial cell line (GES-1). VacA-caused mitochondrial depolarization retards the import of PINK1 into the damaged mitochondria and evokes mitophagy. And, among mass spectrometry (LC-MS/MS) identified 25 mitochondrial proteins bound with VacA, Tom20, Tom40, and Tom70, TOM complexes responsible for PINK1 import, were further identified as having the ability to bind VacA in vitro using pull-down assay, co-immunoprecipitation, and protein-protein docking. Additionally, we found that the cell membrane protein STOM and the mitochondrial inner membrane protein PGAM5 also interacted with VacA. These findings suggest that VacA captured by STOM forms endosomes to enter cells and target mitochondria. Then, VacA is transported into the mitochondrial membrane space through the TOM complexes, and PGAM5 aids in inserting VacA into the inner mitochondrial membrane to destroy the membrane potential, which promotes PINK1 accumulation and Parkin recruitment to induce mitophagy. This study helps us understand VacA entering mitochondria to induce the mitophagy process.

Parthenolide and arsenic trioxide co-trigger autophagy-accompanied apoptosis in hepatocellular carcinoma cells.

Although arsenic trioxide (ATO) shows a strong anti-tumor effect in the treatment of acute promyelocytic leukemia, it does not benefit patients with hepatocellular carcinoma (HCC). Thus, combination therapy is proposed to enhance the efficacy of ATO. Parthenolide (PTL), a natural compound, selectively eradicates cancer cells and cancer stem cells with no toxicity to normal cells. In this study, we chose PTL and ATO in combination and found that nontoxic dosage of PTL and ATO co-treatment can synergistically inhibit the in vitro and in vivo proliferation activity of HCC cells through suppressing stemness and self-renewal ability and inducing mitochondria-dependent apoptosis. More importantly, USP7-HUWE1-p53 pathway is involved in PTL enhancing ATO-induced apoptosis of HCC cell lines. Meanwhile, accompanied by induction of apoptosis, PTL and ATO evoke autophagic activity via inhibiting PI3K/Akt/mTOR pathway, and consciously controlling autophagy can improve the anti-HCC efficacy of a combination of PTL and ATO. In short, our conclusion represents a novel promising approach to the treatment of HCC.

Mid-scale free-flow electrophoresis with gravity-induced uniform flow of background buffer in chamber for the separation of cells and proteins.

A large-scale free-flow electrophoresis (LS-FFE) is often too large for cell separation of lab scale, whereas micro-FFE (µFFE) has great difficulty in cell isolation due to easy blockage by cell accumulation in µFFE. In this study, a mid-scale FFE (MS-FFE) is developed for cell and protein separations. The volume of the separation chamber (70×40×0.1-0.8 mm) is from 280 µL to 2.24 mL, much lower than that in an LS-FFE but higher than that in a µFFE. Gravity is used for uniform flow of the background buffer only via a single pump with 16 channels and the sample is injected via an adjuster originally used for clinical intravenous injection. The experiments reveal that the hydrodynamic and electrohydrodynamic flows are much stable, and the Joule heat can be effectively dispersed without obvious positive or negative deviation as shown by the omega plots. By the device, Escherichia coli and Staphylococcus aureus, which easily accumulate to block µFFE and are separated with difficulty due to their same negative charges carried, can be well isolated under the conditions of 4.5 mM pH 8.5 Tris-boric buffer (4.5 mM Tris, 4.5 mM boric acid) with 0.10 mM ethylene diamine tetraacetic acid and 5% m/v sucrose, 200 µL/min, 800 V, and sample injection via inlet 4. The mid-scale FFE device could also be used for the separation of three model proteins of horse heart cytochrome c, myoglobin and bovine serum albumin. The device has clear significance for mid-scale separation of cells and proteins.

Suppression Of Aberrant Activation Of NF-κB Pathway In Drug-resistant Leukemia Stem Cells Contributes To Parthenolide-potentiated Reversal Of Drug Resistance In Leukemia.

Although many drugs that targeted the specific features of leukemia stem cells (LSCs) have substantial application in the clinical treatment of leukemia, the LSCs relapsed and caused drug-resistant leukemia. Therefore, it is necessary to identify the unique features of LSCs in relapsing and drug-resistant leukemia and also to explore the drugs that directed at these features. Our clinical data have indicated that relapsed patients with acute myeloid leukemia have more abundant proportion of LSCs with enhanced breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) expression when compared to the untreated patients. The results showed that compared with LSCs derived from sensitive K562 cells, LSCs from drug-resistant K562/ADM cells have much higher chemotherapeutic resistance, and so we termed these cells as "drug-resistant LSCs". Subsequently, aberrant activation of NF-κB pathway in drug-resistant LSCs was further using gene chip analysis. Also, parthenolide (PTL), which is a specific NF-κB inhibitor, effectively eliminated drug-resistant LSCs and enhanced the sensitivity of K562/ADM cells to doxorubicin-induced apoptosis by down-regulating NF-κB pathway-mediated P-gp expression. These findings make the research area of LSCs more abundant and provide a potential therapeutic strategy for the treatment of refractory and relapsed leukemia.

Impact of glutathione-HbA1c on HbA1c measurement in diabetes diagnosis via array isoelectric focusing, liquid chromatography, mass spectrometry and ELISA.

Hemoglobin A1c (HbA1c) has been proven to be a key biomarker for diabetes screening, and glutathiolation of HbA1c (viz., GSS-HbA1c) has been identified. However, the impact of GSS-HbA1c on the measurement of HbA1c for diabetes screening has not been quantitatively assessed yet. To address the issue, the micropreparative capillary isoelectric focusing (cIEF) developed in our previous work was used for the high resolution separation and purification of hemoglobin (Hb) species. The main fractions of HbA0, HbA3 and HbA1c extracted from the developed cIEF were identified by validated Mono S method. The proposed GSS-HbA1c fractions in the cIEF were pooled and identified by electrospray ionization mass spectrometry (ESI-MS). The HbA1c enzyme-linked immunosorbent assay (ELISA) kit was employed for further quantitative analysis of GSS-HbA1c. A total of 34 blood samples with HbA1c levels from 4.2% to 13.4% were assessed via the above comprehensive strategy of IEF-HPLC-MS-ELISA. It was demonstrated that the HbA1c levels detected by cation exchange LC were considerably influenced by the glutathiolation of Hb and the range of detected GSS-HbA1c values was between 0.23% and 0.74%. The results and developed cIEF methods have considerable significances for investigation of diabetes and clinical diagnosis.