The role of TMEM26 in disrupting tight junctions and activating NF-κB signaling to promote epithelial-mesenchymal transition in esophageal squamous cell carcinoma

Abstract Objectives Metastasis is one of the biggest challenges in the management of Esophageal Squamous Cell Carcinoma (ESCC), of which molecular mechanisms remain elusive. The present study aimed to explore the roles and underlying mechanisms of Transmembrane protein 26 (TMEM26) in ESCC. Method TMEM26 expressions in tumorous and adjacent tissues from patients with ESCC and in normal esophageal epithelial and ESCC cell lines were detected by immunostaining and western blotting, respectively. The Epithelial-Mesenchymal Transition (EMT), a critical process during metastasis, was investigated by wound healing and Transwell assays, and EMT-related proteins were examined after the TMEM26 alteration in ESCC cell lines. NF-κB signaling activation and Tight Junction (TJ) protein expression were analyzed by western blotting and immunofluorescence, respectively. In vivo verification was performed on the liver metastatic murine model. Results Compared with non-cancerous esophageal tissues and cells, the TMEM26 expression level was higher in ESCC samples and cell lines, where the plasma membrane localization of TMEM26 was observed. The EMT-related processes of ESCC cells were suppressed by RNAi depletion of TMEM26 but aggravated by TMEM26 overexpression. Mechanistically, TMEM26 promoted NF-κB signaling to accelerate EMT in ESCC cells. The plasma membrane presentation and assembly of TJ proteins were impaired by TMEM26. Conclusion Overall, TMEM26 acts as a critical determinant for EMT in ESCC cells by disrupting TJ formation and promoting NF-κB signaling, which may be a potential therapeutic target for treating metastatic ESCC.

Retention Time and Ion Abundance Ratio of 8 Common Drugs (Poisons) by LC-MS/MS Qualitative Analysis / 法医学杂志

Objective To investigate the maximum allowable deviations of retention time and ion abundance ratio of the 8 common drugs (poisons) from 3 categories, poisons (methamphetamine, morphine, ketamine), benzodiazepines (estazolam, midazolam, diazepam, clonazepam) and barbiturates (phenobar-bital) in blood, by liquid chromatograpy-tandem mass spectrometry (LC-MS/MS) in forensic toxicology analysis. Methods The deviations of retention time and ion abundance ratio at 7 low mass concentrations, limit of detection (LOD), 2LOD, limit of quantitation (LOQ), 1.5LOQ, 2LOQ, 4LOQ and 6LOQ, were tested by LC-MS/MS after liquid-liquid extraction under the conditions of two chromatographic columns and three chromatographs. Results The deviation of absolute retention time of 98.11% of 8 drugs (poisons) in the blood samples was within the range of ± 0.05 min, and that of the relative retention time of 96.21% was within the range of±0.4%. The maximum deviation of the ion abundance ratio was highly correlated with the mass concentration. When the mass concentration of drugs (poisons) was LOQ or above, more than 95% of the absolute deviation and relative deviation of the ion abundance ratio were in the range of±25% and±40%, respectively; when the mass concentration was below LOQ, the range could be expanded to±35% and±50%, respectively. Conclusion It is recommended for the determination range of the absolute retention time deviation of 8 common drugs (poisons) to be±0.1 min and that of the relative retention time deviation to be±1.0%. The determination range of absolute deviation of the ion abundance ratio should be±25% when the mass concentration is LOQ or above, and the relative deviation should be±40%. When the mass concentration is below LOQ, the deviation determination range can be expanded to±35% and±50%, respectively.

Ion Abundance Ratios of Qualitative Analysis by GC-MS of 4 Common Drugs （Poisons / 法医学杂志

Objective To investigate the maximum allowable deviation of ion abundance ratios of characteristic fragment ions in common drugs （poisons） in blood by gas chromatography-mass spectrometry （GC-MS） method. Methods Four common drugs （poisons） （dichlorvos, phorate, diazepam and estazolam） were detected by GC-MS full scan mode after liquid-liquid extraction in two laboratories and under three chromatographic conditions. The deviations of ion abundance ratios of the four common drugs （poisons） in marked blood samples with concentrations of 0.5, 1.0, 2.0, 5.0 and 10.0 μg/mL were analyzed. At the same time, the false negative rates of ion abundance ratios were analyzed when the mass concentration was limit of detection （LOD）, 2LOD, limit of quantitation （LOQ） and 2LOQ, and the false positive rates of ion abundance ratios were analyzed with blank blood samples. Results Under the two laboratories, four common drugs （poisons） and three kinds of chromatography conditions, the differences in deviations of the ion abundance ratios of marked blood samples were not statistically significant （P>0.05）. More than 95% of the absolute deviations of the ion abundance ratios of the marked blood samples were within the range of ±10%, and more than 95% of the relative deviations were within the range of ±25%. In cases of low concentration （concentration less than 2LOQ） or low signal to noise ratio （3-15）, the false negative rate was less than 5% and the false positive rate was 0% when the relative deviation was greater than 50%. Conclusion The absolute deviations of ion abundance ratios of four common drugs （poisons） in marked blood samples are advised to have a determination range within ±10%, and the determination range of relative deviations within ±25%.

Retention Time of Four Common Poisons (Drugs) by GC-MS Qualitative Analysis / 法医学杂志

Objective To investigate the maximum allowable deviation of retention time (RT) or relative retention time (RRT) between the common poisons (drugs) and standard solvent by gas chromatography-mass spectrometry (GC-MS).Methods After pretreatment with liquid-liquid extraction, four common poisons (drugs) —dichlorvos, phorate, diazepam and estazolam—were detected by full scan mode GC-MS.RT and RRT were analyzed according to combined uncertainty and expanded uncertainty. Results The expanded uncertainty of RT and RRT were 6.0× lO-4-14.1×l0-3 and 2.5 ×l0-6-5.9× lO-5 (k=3), respectively.The RT of poisons (drugs) was relatively stable in blood samples with different mass concentrations.Among dichlorvos, phorate, diazepam and estazolam, the absolute deviation and relative deviation of RT were≤0.03 min and≤0.4％, respectively, and those of RRT were≤0.003 min and≤0.3％, respectively.Conclusion The maximum allowable deviations of RT and RRT for common poisons (drugs) in blood samples are recommended to be±0.05 min and±0.5％.

Rapid screening of 3 synthetic cannabinoids in blood by direct analysis in real time mass spectrometry / 中国法医学杂志

Objective To establish a method for rapid detection of JWH-018, JWH-250 and AM-2201 in blood by direct analysis in real-time coupled with tandem mass spectrometry. Methods These samples were extracted by acetonitrile-methanol (4:1), using DART 12Dip-it automatic sampling system. They were analyzed by positive ion and MRM mode. Results The detection limits of JWH-018, JWH-250 and AM-2201 were in good linearity in the range of 0.02-5.00μg/mL. The correlation coefficients were above 0.99 and the detection limits were 0.016μg/mL, 0.003μg/ mL and 0.017μg/mL, respectively. The intra-day and inter-day RSD were less than 15%. Conclusion The method has the advantages of high sensitivity and good accuracy. The sample processing is simple and can be analyzed in short time. This method is suitable for the analysis of JWH-018, JWH-250 and AM-2201 in practical cases.

Protective mechanism of potentilla anserina polysaccharide on mice with d-galactosamine-induced acute liver injury / 第二军医大学学报

Objective To explore the protective mechanism of Potentilla anserina polysaccharide (PAP) on acute liver injury induced by D-galactosamine (D-GlaN) in mice. Methods Sixty Kunming mice were randomly divided into normal control group, model group, bifendate group (positive control) and PAP (50, 100, 200 mg/kg) treated groups, with 10 mice in each group. After treatment with normal saline, bifendate and PAP (50, 100, 200 mg/kg) for 7 days, themice in normal control group were injected intraperitoneally with normal saline, and those in the other 5 groups were injected intraperitoneally with D-GlaN to establish acute liver injury models. All the animals were sacrificed 24 h after model establishment, and the levels of hepatic superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH Px) were determined. Results Compared with normal control group, the model group had significantly increased hepatic MDA level (P<0. 05) and significantly decreased activities of hepatic SOD, GSH-Px and level of GSH (P<0. 05, P<0. 01). Compared with the model group, bifendate and 50, 100, 200 mg/kg PAP significantly decreased hepatic MDA level (P<0. 05), increased the activities of hepatic SOD, GSH-Px and level of GSH (P<0. 05, P<0. 01) in mouse acute liver injury model. Conclusion PAP can protect the liver of mice with acute liver injury induced by D-GlaN, which is probably through scavenging free radicals, protecting cell membranes and inhibiting lipid peroxidation.

Improved synthesis of imatinib mesylate / 药学实践杂志

Objective To obtain the improved synthesis of imatinib mesylate .Methods 4-[(4-methyl-piperazin-1-yl) methyl〗benzoyl chloride dihydrochloride and 4-methyl-N3-[4-(3-pyridyl) pyrimidin-2-yl]-1,3-phenylenediamine were used as starting raw mate-rial to perform a condensation reaction in the aqueous phase to give imatinib free base , which was then neutralized with methanesulfonic acid to obtain the novel antineoplastic tyrosinase inhibitor imatinib mesylate by the use of a two -step reaction .Results The improved syn-thesis was considered to have the advantages of low cost , easy post-processing, high purity, high yield and environmental pollution with the HPLC purity≥99.7%and the single impurity<0.1%.Conclusion The total yield of the novel method was 81.5%, having an e-nough broad industrial production prospect .The targeted compound structure was confirmed by 1 H NMR and ESI-MS.

Synthesis and multidrug resistance reversal activity of 1-akyl-2-acetyl-1,2,3, 4-tetrahydroisoquinoline derivatives / 中国药科大学学报

To study the multidrug resistance activity of 1-alkyl-2-acetyl-1, 2, 3, 4-tetrahydroisoquinoline derivatives. Methods: A series of novel tetrahydroisoquinoline derivatives bearing at C-1 position a carbon chain derived from fatty acids were prepared through the Bischler-Napieralski cyclization reaction. Their multidrug resistance (MDR) reversal cancerous multidrug resistance activities were evaluated against K562 and K562/DOX cell lines in vitro by MTT assay with verapamil as a control. Results and Conclusion: The structures of these tetrahydroisoquinolines were confirmed by extensive spectroscopic methods(1H NMR, MS, IR and elemental ana-lyses). MDR results showed that compounds 7 and 10 exhibited moderate reversal activities, and were slightly less potent than those of verapamil against K562 cell line. It is believed that compounds 7 and 10 have MDR activity.