Electrochemical assay of the relationship between the inhibition of phosphatidylinositol 3-kinase pathway and estrogen receptor expression in breast cancer

Breast cancer has become one of the most threatening diseases to women throughout the world. Emerging evidence implies that estrogen receptor (ER) and phosphatidylinositol 3-kinase (PI3K) pathways play central roles in both breast cancer progression and response to therapy. In this work, we have probed into ER expression related to the PI3K pathway at the protein level with an electrochemical technique based on the detection of ER proteins in nuclear extracts with an Exonuclease III protection-based strategy. Experimental results show that an increased number of ER proteins can be detected upon PI3K inhibition, demonstrating the reversal effect of the PI3K inhibitor on ER expression. Moreover, treatment with different concentrations of the PI3K inhibitor NVP-BEZ235 can result in a dose-dependent alteration of ER protein levels, implying an intimate link between ER and PI3K pathways. This work may be a great help to understand the mysteries underlying PI3K-related endocrine resistance and to evaluate the effect of therapeutic interventions in the future.     

Novel moving reaction boundary-induced stacking and separation of human hemoglobins in slab polyacrylamide gel electrophoresis

We developed a novel polyacrylamide gel electrophoresis (PAGE) method to stack and separate human hemoglobins (Hbs) based on the concept of moving reaction boundary (MRB). This differs from the classic isotachophoresis (ITP)-based stacking PAGE in the aspect of buffer composition, including the electrode buffer (pH 8.62 Tris–Gly), sample buffer (pH 6.78 Tris–Gly), and separation buffer (pH 8.52 Tris–Gly). In the MRB-PAGE system, a transient MRB was formed between alkaline electrode buffer and acidic sample buffer, being designed to move toward the anode. Hbs carried partial positive charges in the sample buffer due to its pH below pI values of Hbs, resulting in electromigrating to the cathode. Hbs would carry negative charges quickly when migrated into the alkaline electrode buffer and be transported to the anode until meeting the sample buffer again. Thus, Hbs were stacked within a MRB until the transient MRB reached the separation buffer and then separated by zone electrophoresis with molecular sieve effect of the gel. The experimental results demonstrated that there were three clear and sharp protein zones of Hbs (HbA_1c, HbA₀, and HbA₂) in MRB-PAGE, in contrast to only one protein zone (HbA₀) in ITP-PAGE for large-volume loading (≥15 μl), indicating high stacking efficiency, separation resolution, and good sensitivity of MRB-PAGE. In addition, MRB-PAGE was performed in a conventional slab PAGE device, requiring no special device. Thus, it could be widely used in separation and analysis of diluted protein in a standard laboratory.

Simultaneous quantitation of seven alkaloids in processed Fuzi decoction by rapid resolution liquid chromatography coupled with tandem mass spectrometry

A rapid analytical method based on rapid resolution LC coupled with MS/MS was first established to quantify seven alkaloids in processed Fuzi decoction. The chromatographic method was optimized to allow simultaneous analysis of all analytes in 5 min and demonstrated good linearity (r > 0.9995), repeatability (RSD < 4.36%), intra‐ and interday precisions (RSD < 5.07%) with good accuracies (97.76–105.08%) and good recovery (95.0–107.5%) of seven alkaloids, namely higenamine, benzoylhypaconine, benzoylmesaconine, benzoylaconine, aconitine, hypaconitine, and mesaconitine. The LODs for these markers were in the range of 2.30–17.00 pg/mL. Quantitative analysis of the seven alkaloids in Baifupian decoction and Heishunpian decoction showed that the content of the seven marker chemicals varied significantly and concluded that the quality of Fuzi was greatly affected by different processed methods. The developed method could be used as a rapid, sensitive, and reliable approach for assessment of the quality of processed Fuzi and related decoction.     

An improved SPE method for fractionation and identification of phospholipids

This work reports an efficient and universal SPE method developed for separation and identification of phospholipids derived from complex biological samples. For the separation step, sequential combination of silica gel‐aminopropyl‐silica gel SPE cartridges is applied. This setup enables separation of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, phosphatidylserine, cardiolipin, and sphingomyelin into four fractions according to the polarity of their headgroups. Sample acquisition of the SPE fractions is performed by a high‐resolution LC‐MS system consisting of a hybrid linear IT Fourier transform ion cyclotron resonance mass spectrometer coupled to RP‐HPLC. The unequivocal advantage of our SPE sample preparation setup is avoidance of analyte peak overlapping in the determination step done by RP‐HPLC. Overlapping phospholipid signals would otherwise exert adverse ion suppression effects. An additional benefit of this method is the elimination of polar and nonpolar (e.g. neutral lipids) contaminants from the phospholipid fractions, which highly reduces contamination of the LC‐MS system. The method was validated with fermentation samples of organic waste, where 78 distinct phospholipid and sphingomyelin species belonging to six lipid classes were successfully identified.     

Application of supercritical fluid extraction coupled with counter–current chromatography for extraction and online isolation of unstable chemical components from Rosa damascena

Supercritical fluid extraction (SFE) coupled with high‐speed counter‐current chromatography (HSCCC) was successfully used for the extraction and online isolation of the unstable compounds from Rosa damascene in a single extraction and separation operation in two stages. The solvent systems of SFE/HSCCC were optimized with the help of multiexponential function model. At the first stage, the upper phase of the solvent system of n‐butanol–tert‐butyl methyl ether–acetonitrile–0.1% aqueous TFA (1.7:1.0:0.8:4.0, v/v/v/v) was used as both the SFE entrainer and the HSCCC stationary phase, and the target compounds were eluted with the corresponding lower phase to separate the hydrophobic compounds. At the second stage, the upper phase of the solvent system of n‐hexane–ethyl acetate–methanol–water (3.2:1.0:2.8:2.6, v/v/v/v) was used as both the SFE entrainer and the HSCCC stationary phase, followed by elution with the corresponding lower phase to separate the moderate hydrophobic compounds. Six compounds including formononetin, delphinidin, cyaniding, 5,6,4′‐trihydroxy‐7,8‐dimethoxy flavone, 5,3′‐dihydroxy‐7,8‐dimethoxy flavone, and 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxy flavone were successfully separated in one extraction–separation operation within 300 min. The targeted compounds were identified by MS and NMR spectroscopy. This research has opened up great prospects for industrial application of SFE/HSCCC to the extraction and separation of unstable compounds.     

A rapid and convenient method for preparative separation of three indissolvable polyphenols from Euphorbia pekinensis by the flexible application of solvent extraction combined with counter‐current chromatography

A rapid and convenient method was established to preparatively isolate the three ellagic acid types of compounds, which were the main polyphenols in Euphorbia pekinensis, by flexibly applying solvent extraction combined with counter‐current chromatography (CCC). The total extract (extracted using 95% ethanol) of E. pekinensis was pretreated by two simple steps before CCC isolation, following the procedure: the total extract was extracted by classical solvent extraction using petroleum ether and ethyl acetate, respectively, and then the ethyl acetate extract was suspended using 95% ethanol, after being allowed to stand overnight, the sediment was obtained. Partial sediment (100 mg) was then directly separated by CCC with a two‐phase solvent system composed of chloroform‐95% ethanol‐water‐85% formic acid (50:50:50:5, v/v/v/v). About 22 mg of 3,3′‐dimethoxy ellagic acid (1), 12 mg of 3,3′‐di‐O‐methyl‐4‐O‐(β‐d ‐xylopyranosyl)ellagic acid (2), and 35 mg of ellagic acid (3) with purities of 96.0, 95.2, and 95.4% were obtained respectively in one step within 4 h. After being purified by washing with methanol, the purities of the three compounds obtained were all above 98%. The purities were determined by HPLC and their chemical structures were further identified by ¹H and ¹³C NMR spectroscopy. The recoveries were calculated as 84.6, 85.7, and 89.5%, respectively. The result demonstrated that the present isolation method was rapid, economical and efficient for the preparative separation of polyphenols from E. pekinensis.     

Rapid determination of catecholamines in urine samples by nonaqueous microchip electrophoresis with LIF detection

A method was developed for the rapid separation of catecholamines by nonaqueous microchip electrophoresis (NAMCE) with LIF detection, A homemade pump‐free negative pressure sampling device was used for rapid bias‐free sampling in NAMCE, the injection time was 0.5 s and the electrophoresis separation conditions were optimized. Under the optimized conditions, the samples were separated completely in <1 min. The average migration times of the epinephrine (E), dopamine (DA), and norepinephrine (NE) were 34.26, 43.81, and 50.07 s, with an RSD of 1.05, 1.26, and 0.89% (n = 7), respectively. The linearity of the method ranged from 0.0125 to 2.0 mg/L for E and 0.025～4.0 mg/L for DA and NE, with correlation coefficients ranging between 0.9978 and 0.9986. The detection limits of E, DA, and NE were 2.5, 5.0, and 5.0 μg/L, respectively. The recoveries of E, DA, and NE in spiked urine samples were between 86 and 103%, with RSDs of 4.5～6.8% (n = 5). The proposed NAMCE with LIF detection combined with a pump‐free negative pressure sampling device is a simple, inexpensive, energy efficient, miniaturized system that can be successfully applied for the determination of catecholamines in urine samples.     

Impact of photogenerated charge behaviors on luminescence of Eu3+-incorporated microporous titanosilicate ETS-10

A series of Eu³⁺-incorporated ETS-10 samples were successfully prepared based on the traditional ion exchange method. The relationship between photogenerated charge behaviors and luminescent properties has been investigated in detail. It has been demonstrated that as a result of the charge transfer from the titanate quantum wires to Eu³⁺ crystal field states, the host matrix ETS-10 functions as the sensitizer of Eu³⁺ to enhance the red luminescence, while Eu³⁺ cations contribute to the recombination of photogenerated charges. The behavior of photogenerated charges has significant impact on the luminescent properties of Eu³⁺-incorporated ETS-10 materials.