Detection of membrane receptors on per tumor cell by nonimmobilized cell capillary electrophoresis and a mathematic model.

Folate receptors (FRs) are a class of valuable therapeutic target which is highly expressed on a variety of cancers. The accurate detection of the expression of FRs in different cells is conducive to improve the accuracy of FR targeted tumor therapy. Herein, a method based on nonimmobilized cell capillary electrophoresis (NICCE) combined with a mathematic model to quantify FRs on each single tumor cell was developed. At first, we studied the interactions between FA and A549, HT-29, HepG2, and U87MG cells by NICCE respectively, and calculated the kinetic parameters (Ka, k', ka, and kd). Next, we established a mathematic model to accurately determine the number of moles of FRs on per A549, HT-29, HepG2, and U87MG cell for the first time, that were (10.44 ± 0.53) × 10-19 mol, (34.32 ± 1.33) × 10-19 mol, (337.14 ± 10.11) × 10-19 mol, and (37.31 ± 2.13) × 10-19 mol. Then, these re-sults were proved to be consistent with the results of enzyme-linked immunosorbent assay (ELISA). Therefore, this method is simple, rapid, sensitive, and without protein separation or purification, which is expected to achieve clinical detection of cell membrane receptor expression level of cell membrane receptors on a single cell, which may be greatly beneficial to further clinical diagnosis and therapy.

Biomimetic cell-cell adhesion capillary electrophoresis for studying Gu-4 antagonistic interaction between cell membrane receptor and ligands.

We report a new method: biomimetic cell-cell adhesion capillary electrophoresis (BCCACE) to screen drugs targeting interactions between cell membrane receptors and ligands under an environment close to physiological conditions, in which the cell membrane receptors/ligands can maintain their natural conformations and bioactivity without being isolated and purified. Firstly, we screened twenty-one lactose derivatives by cell-immobilized capillary electrophoresis and obtained Gu-4 with the best activity (K = 3.58 ±â€¯0.22 × 104) targeting macrophage antigen-1 (Mac-1). Then, BCCACE was performed as follows: HEK 293 cells overexpressed with receptor (intercellular adhesion molecules-1, ICAM-1) were cultured and immobilized on the inner wall of capillaries as stationary phase, which simulated the endothelial cells lining on the inner surface of blood vessels. HEK 293 cells overexpressed with ligand Mac-1 as samples were used to simulate the neutrophils cells in blood vessels. And Gu-4 added into the running buffer solution as the antagonist was used to simulate the drug in blood. The results showed that Gu-4 (40 µM) could selectively inhibit cell-cell adhesion by targeting the interaction between Mac-1 and ICAM-1. Finally, the pharmaceutical efficacy assays of Gu-4 at cellular and animal levels were carried out using the concentration of 40 µM and the dose of 20 mg kg-1 respectively, which showed the anti-cancer metastasis activity of Gu-4 and the validity of the method. This method simulated a complete three-dimensional vascular model, which can easily obtain the suitable blood concentration of drugs. This system simulated the interaction between leukocytes and vascular endothelial cells in the bloodstream antagonized by drugs, and obtained the effective concentration of the antagonist. It can be used as an accuracy and efficient drug screening method and will be expected to become a new method to screen drugs targeting cell-cell adhesion.

Rapid screening of anti-tumor metastasis drugs targeting integrin macrophage antigen-1 using immobilized cell capillary electrophoresis.

In this research a method called immobilized cell capillary electrophoresis (ICCE) was established under approximately physiological conditions for rapid screening of anti-tumor metastasis drugs targeting integrin macrophage antigen-1 (MAC-1). In this method, separation and purification of the target receptors on cell membranes was unnecessary, thus, maintaining their natural conformation and bioactivity. MAC-1-, CD11b-, or CD18-overexpressing HEK293 cells (human embryonic kidney) were cultured and immobilized on the inner wall of capillaries as stationary phase, and their interactions with lactosyl derivative Gu-4 (positive control)/dimethylsulfoxide (DMSO; negative control) were studied using ICCE. Using this method, 29 phenylethanoid glycosides from Cistanches Herba were screened, and the binding kinetic parameters (K, ka, kd, and k') of active compounds were calculated, and the specific subunits of MAC-1 were determined. Then, molecular docking studies were performed to discover the direct interaction sites between active compounds and MAC-1, and the order of Glide-calculated Emodel value obtained from the molecular docking study is consistent with that of the binding constants obtained using ICCE. Finally, pharmaceutical efficacy assays in vitro and in vivo were carried out to show that the anti-tumor metastasis activity of the active compound had better pharmaceutical efficacy and lower toxic side effects. The method was verified to be valid and practical for further use, and it is expected that it will be transferred to capillary array electrophoresis for use in high-throughput drug screening.

Separation and simultaneous quantitation of PGF2α and its epimer 8-iso-PGF2α using modifier-assisted differential mobility spectrometry tandem mass spectrometry.

Because many therapeutic agents are contaminated by epimeric impurities or form epimers as a result of metabolism, analytical tools capable of determining epimers are increasingly in demand. This article is a proof-of-principle report of a novel DMS-MS/MS method to separate and simultaneously quantify epimers, taking PGF2α and its 8-epimer, 8-iso-PGF2α, as an example. Good accuracy and precision were achieved in the range of 10-500 ng/mL with a run time of only 1.5 min. Isopropanol as organic modifier facilitated a good combination of sensitivity and separation. The method is the first example of the quantitation of epimers without chromatographic separation.

Simultaneous quantitation of the diastereoisomers of scholarisine and 19-epischolarisine, vallesamine, and picrinine in rat plasma by supercritical fluid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study.

Dengtaiye tablet has been used to treat chronic bronchitis cough. Scholarisine, 19-epischolarisine, vallesamine, and picrinine are the representative constituents of Dengtaiye. A rapid and sensitive assay based on supercritical fluid chromatography with tandem mass spectrometry has been developed and validated for the determination of the diastereoisomers of scholarisine and 19-epischolarisine, vallesamine, and picrinine in rat plasma using lamotrigine as internal standard. The analysis in a run time of only 6 min was performed on an ACQUITY UPC(2) Trefoil(TM) BEH 2-EP column (3.0 × 150 mm, 2.5 µm) at 50ºC. The mobile phase consisting of carbon dioxide and methanol (2 mM ammonium formate) was performed as follows: 15% methanol (2 mM ammonium formate) maintained at 0-2 min, 15-19% methanol (2 mM ammonium formate) at 2-4 min, 19-15% methanol (2 mM ammonium formate) at 4-6 min. The flow rate was 1.50 mL/min. The assay was linear over the concentration ranges 50-10000 pg/mL for scholarisine, 19-epischolarisine, vallesamine, and picrinine with corresponding lower limits of quantitation of 50 pg/mL. Intra- and interday precisions were in the range 1.42-12.85% with accuracies in the range -11.71-2.48%. The method was successfully applied to a pharmacokinetic study involving a single oral administration of 108 mg/kg Dengtaiye tablet to rats.

Protective Immune Responses Elicited by Fusion Protein Containing PsaA and PspA Fragments.

Streptococcus pneumoniae is an important pathogen accounting for a large number of deaths worldwide. Due to drawbacks of the current polysaccharide-based vaccine, the most promising way to generate an improved vaccine may be to utilize protection-eliciting pneumococcal proteins. Pneumococcal surface adhesin A (PsaA) and pneumococcal surface protein A (PspA) are two vaccine candidates which have been evaluated against S. pneumoniae infection in animal models or human clinical trials with encouraging results. In this study, the efficacy of the fusion protein PsaA-PspA, which includes PsaA part and PspA part, in inducing immunoprotective effects against fatal pneumococcal challenge was evaluated in an animal model. PspA part of PsaA-PspA fusion protein contains both family1 N-terminal region and family 2 N-terminal clade-defining region of PspA. Immunization with the PsaA-PspA fusion protein induced high levels of antibodies against both PsaA and PspA, which could bind to intact S. pneumoniae strains bearing different PspAs. Ex vivo stimulation of splenocytes from mice immunized with PsaA-PspA induced IL-17A secretion. Mice immunized with PsaA-PspA showed reduced S. pneumoniae levels in the blood and lungs compared with the PBS group after intranasal infection. Finally, mice immunized with PsaA-PspA fusion proteins were protected against fatal challenge with pneumococcal strains expressing different PspAs regardless of the challenge route. These results support the PsaA-PspA fusion protein as a promising vaccine strategy, as demonstrated by its ability to enhance the immune response and stimulate production of high titer antibodies against S. pneumoniae strains bearing heterologous PspAs, as well as confer protection against fatal challenge with PspA family 1 and family 2 strains.

Dithiothreitol-capped fluorescent gold nanoclusters: an efficient probe for detection of copper(II) ions in aqueous solution.

We report here a Green method for the synthesis of fluorescent gold nanoclusters using dithiothreitol (DTT) as both a capping agent and reducing agent at 22 °C and pH 8. The physical and chemical properties of the synthesized AuNCs@DTT were studied by TEM and UV-vis absorption, fluorescence, and X-ray photoelectron spectroscopy. AuNCs@DTT recognizes cupric ions with high selectivity and sensitivity, which allows this material to act as a copper(II) sensor in aqueous solution. A linear relationship was observed between the fluorescence intensity of the DTT capped gold nanoclusters and the concentration of copper(II) ions, in the range of 0-60 µM with a detection limit of 80 nM. The copper content in serum was also analyzed by using this copper sensor. It was shown that data obtained using the proposed method was comparable to values obtained by the traditional colorimetric method. This technique represents an alternative method for the determination of serum copper in clinical diagnosis especially for those laboratories which lack expensive analytical facilities.