Objective To evaluate effects of simultaneous inhibition of mammalian target of rapamycin complex 1(mTORC1)kinase and glycogen synthase kinase-3β(GSK-3β)on phosphorylation of 4E-binding protein-1(4EBP1), cap-dependent translation, as well as survival and apoptosis of melanoma cells. Methods Cultured A375 cells were classified into several groups to be treated with dimethyl sulfoxide (DMSO group), the mTORC1 kinase inhibitor everolimus at a concentration of 5 nmol/L (everolimus group), the GSK-3β kinase inhibitor AR-A014418 at a concentration of 10 μmol/L (AR-A014418 group), or 5 nmol/L everolimus and 10 μmol/L AR-A014418(combined treatment group). After additional culture, Western-blot analysis was performed to measure protein expressions of phosphorylated 4EBP1 (p4EBP1)and survivin in A375 cells, m7GTP pull down assay to estimate interaction between eukaryotic initiation factor-4E (eIF4E)and eIF4G, cell counting kit 8 (CCK8)assay to evaluate cell proliferation, and flow cytometry to detect cell apoptosis. Results Both everolimus and AR-A014418 had inhibitory effects on 4EBP1 phosphorylation and survivin expression. The expressions of p4EBP1-65 and survivin were both significantly decreased in the everolimus group (0.74 ± 0.05 and 0.71 ± 0.06 respectively), AR-A014418 group (0.62 ± 0.06 and 0.58 ± 0.07 respectively)and combined treatment group (0.14 ± 0.04 and 0.09 ± 0.05 respectively)compared with the DMSO group (1.00 ± 0.07 and 1.00 ± 0.06, respectively, all P < 0.001), with the most significant decrease observed in the combined treatment group. As m7GTP pull-down assay showed, the everolimus group, AR-A014418 group and combined treatment group all showed significantly lower relative expression levels of eIF4G(0.72 ± 0.04, 0.67 ± 0.05 and 0.12 ± 0.05 vs. 1.00 ± 0.06, all P < 0.001), but significantly higher relative expression levels of 4EBP1 (1.98 ± 0.16, 2.32 ± 0.17 and 7.58 ± 0.25 vs. 1.00 ± 0.08, all P < 0.001)than the DMSO group, and the combined treatment group showed the lowest eIF4G expression but highest 4EBP1 expression. After 24-hour culture, the proliferation of A375 cells was inhibited by 18.5% ± 1.3% in the everolimus group, 19.8% ± 1.8% in the AR-A014418 group, and 61.2% ± 2.1% in the combined treatment group compared with the DMSO group, with the strongest inhibition noted in the combined treatment group. The inhibitory effects of everolimus and AR-A014418 on cell proliferation increased over time, and showed the same trend at 48 hours. Flow cytometry showed that the apoptosis of A375 cells was accelerated by the 24-hour treatments with everolimus and AR-A014418 alone or in combination, with the apoptosis rate being 14.28% ± 2.18%, 14.57% ± 2.35% and 55.18% ± 6.27% in the everolimus group, AR-A014418 group and combined treatment group respectively, and the combined treatment showed the strongest accelerating effect. Conclusion The combined treatment with everolimus and AR-A014418 can evidently inhibit 4EBP1 phosphorylation and eIF4F complex formation in A375 cells, which then suppress cap-dependent translation and promote apoptosis of melanoma cells.

Objective To study the awareness of perimenopausal hormone replacement therapy (HRT) among a part of the medical care personnel in Guiyang . Methods A survey was conducted among 500 medical staff members in 4 hospitals of Guiyang by cluster random sampling using questionnaire about HRT. Results The survey showed that 74.6% (373/500) medical staff thought that the hormone replacement therapy was necessary to perimenopausal women; 96.7% (87/90) of obstetrics and gynecology doctors believed that it was necessary for perimenopausal women to use HRT,which was significantly higher than the doctors of other specialties 68.6% (166/242) and the nurses group 71.4% (120/168) (χ2=28.509, 23.537, P<0.01). Only 5.8%(29/500) of the medical personnel were willing to recommend HRT. In light of the attitude for recommending HRT, the obstetricians and gynecologists group was more significantly higher than the other specialties doctors group (χ2=86.781, P<0.01). Conclusion The knowledge of hormone replacement therapy in part of Guiyang medical personnel is not sufficient;the recommending rate of HRT was low;the side effects of HRT was still a concern. There are differences between obstetrics and gynecology doctors and doctors other specialties and nurses in HRT knowledge.

Objective:To observe the expression of adenosine kinase (ADK) in the hippocampus of patients with refractory epilepsy, and to explore the role of ADK in the pathogenesis of refractory epilepsy.Methods:Thirteen patients with intractable epilepsy who underwent surgical resection of hippocampal tissue at the First Affiliated Hospital of Harbin Medical University were collected as the epilepsy group; At the same time, 4 cases of relatively normal temporal lobe brain tissue from patients with traumatic brain injury undergoing debridement surgery (without previous history of epileptic seizures) were collected, and these 4 patients served as the control group. The expression of ADK in two groups of specimens was detected at the tissue, gene, and protein levels using methods such as dual fluorescence immunohistochemistry, real-time quantitative polymerase chain reaction (RT Real time PCR), and Western blotting.Results:In the human brain, ADK was mainly expressed in the nucleus of astrocytes. Through histological observation, ADK was weakly expressed in normal brain tissue, while there is significant proliferation of glial cells and excessive expression of ADK in the brain tissue of patients with refractory epilepsy. The percentage of ADK positive glial cells in the epilepsy group was (53.90±17.59)%, and the control group was (23.82±4.18)%, with a statistically significant difference ( P<0.01). At the genetic level, using RT Real time PCR, it was found that the expression level of ADK mRNA in the epilepsy group was higher than that in the control group, with a 2 -△△Cp of 13.36, which was 13.36 times higher than that in the control group. At the protein level, the expression of ADK protein in the epilepsy group was found to be higher than that in the control group using protein immunoblotting ( P<0.01). Conclusions:ADK is weakly expressed in the nucleus of astrocytes in normal human brain tissue. In the brain tissue of patients with refractory epilepsy, astrocytes significantly proliferate and there is excessive expression of ADK. ADK may play an important role in the occurrence and development of refractory epilepsy in humans.