Inhibitory effects of the O - ( 1 - ethoxvl - butyl ) berbarmine (EBB) on cultured fibroblast was studied by observating calmodulin (CaM ) content of cultured fibroblast with ELISA and DNA content at each phase of cell cycle with flow - cvtometerv. The CaM con-tent in the test group . compared to control . decreased markedly and DNA content increased significantly in the G0+Gi phase but reduced in the S phase. These results suggested that inhibitory mechanism of EBB on fibroblast proliferation may be closely related to CaM decrease in cells.

Objective To investigate the expression of serotonin transporter (5-HTT) and serotonin 1A treceptor (5-HT1 A R) located in the chronic unpredictable stress (CUS)-relative brain areas (mPFC,VTA,NAc) in high and low CUS susceptibility rats,thus to unveil the possible mechanism lead to the different CUS susceptibility.Methods One hundred and fifty male Sprague-Dawley rats were randomly assigned into experiment group (n =120) and control group (n =30).Rats in experiment group were trained according to established CUS procedure.OFT and FST were used to assess the different susceptibility to CUS:high susceptibility group (H group)and low susceptibility group (L group).After the model was established,rats were scarified and cardio-perfused,and the brains were removed and sliced up coronarily.The sections including ventral tegmental area (VTA),nucleus accumben (NAc),medial prefrontal cortex (mPFC) were selected.The mRNA levels of 5-HTT and 5-HT1AR in the regions were estimated with in situ hybridization.Results The expression of 5-HTT in H group were significantly lower than that of in the control and L group in all regions (mPFC:169.20 ± 8.23 vs 143.53 ±5.31 ; Nac:177.41 ± 5.68 vs 158.65 ± 5.24 ; VTA:174.16 ± 5.61 vs 158.65 ± 4.85),and the difference between the H and L group was significant(P＜0.01) ;however,the expression of 5-HT1AR in H group were significantly higher than that of in the control and L group in all regions (mPFC:113.98 ± 7.46 vs 125.90 ± 3.30 ; Nac:112.11± 5.50 vs 125.06 ± 3.97 ;VTA:103.11 ± 6.05 vs 115.57 ± 3.19),and the difference between the H and L group was significant (P＜ 0.01).Conclusion The overexpression of 5-HT1AR and down regulation of 5-HTT in the circuit of VTA-NAc-mPFC may be the basis of the high susceptibility to CUS.

By using a yeast two-hybrid system, a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins, and its effects on the growth of yeast cells and the activation of reporter genes were investigated. Total mRNA extracted from Hela cells was reversely transcribed into cDNA. Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector. The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector were transformed into the yeast cell AH109, respectively. After they were cultured respectively in YPDA liquid medium and nutrition-deficient culture medium, their toxicity and transcriptional activation were tested by both the phenotype assay and the color assay. The bait plasmid HPV18 E6 was successfully obtained. After being cultured in YPDA liquid medium for 16h, the A (600 nm) values of two yeast fluids were 0.98+/-0.03 and 0.99+/-0.02, respectively. The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector could grow to white colonies on SD/-Trp/X-alpha-gal plates, while no colony could survive on SD/-His/-Trp/X-alpha-gal, SD/-Ade/-Trp/X-alpha-gal plates, indicating that the bait plasmid pGBKT7-HPV18 E6 was constructed successfully and expressed correctly, and could not activate the transcription of reporter gene alone. The yeast two-hybrid GAL4 system 3 can be utilized to find HPV18 E6 interacting proteins.

The aim of the present study was to examine the effects of suppression of EphB4 and/or mTOR on the biological behaviors of ovarian cancer cells, and the potential regulatory pathways. Antisense EphB4 vectors and shRNA vectors targeting mammalian target of rapamycin (mTOR) were constructed and transfected into A2780 and SKOV3 cells (two ovarian cancer cell lines). The effects of the antisense EphB4 vectors and the shRNA vectors on the proliferation, apoptosis and invasion of ovarian cancer cells were measured, and the expression of EphB4, mTOR and Akt detected. The results showed that transfection with mTOR shRNA could inhibit growth, induce apoptosis, and reduce invasive ability of ovarian cancer cells, which was accompanied by downregulation of EphB4, mTOR and Akt. The inhibitory effects on cell growth caused by mTOR shRNA alone were weaker than those by antisense pEGFP-C1-EphB4. In the antisense pEGFP-C1-EphB4-transfected cells, it was found that EphB4 knockdown could decrease the mTOR expression and slightly reduce the Akt phosphorylation. Significant suppressive effects on cell growth were observed in cells co-transfected with antisense pEGFP-C1-EphB4 and mTOR shRNA. In co-transfection group, the expression levels of EphB4, mTOR and Akt were distinctly lower than those in other groups. It was concluded that suppression of EphB4 may inhibit the growth of ovarian cancer cells by downregulation of the PI3K/Akt/mTOR pathway, and reverse Akt phosphorylation induced by mTOR shRNA. Inhibition of EphB4 and mTOR combined may cooperatively suppress the biological behaviors of ovarian cancer cells.

Objective: To study the relationship between platelet activation and the degree of bleeding in patients with primary immune thrombocytopenia (ITP) . Methods: 43 patients with ITP were assessed based on ITP-BAT bleeding grading system. Platelet membrane glycoproteins (GP) Ⅰb, GPⅡb/Ⅲa and P-selectin expression were detected by flow cytometry analysis with and without adenosine diphosphate (ADP) stimulation. Association of platelet activation with platelet count, immature platelet fraction (IPF) , bleeding severity were evaluated. Results: GPⅡb/Ⅲa and P-selection expressions on unstimulated platelet in ITP patients were higher than those in healthy controls (65.69±10.73 vs 7.16±0.99, t=4.130, P<0.001; 15.43±1.41 vs 12.55±1.03, t=2.070, P=0.043, respectively) , and GPⅠb expression was lower than that in healthy controls (240.11±24.93 vs 295.11±22.15, t=2.417, P=0.020) . Comparatively to healthy individuals, following ADP stimulation, GPⅡb/Ⅲa expression in ITP patients increased (133.96±12.17 vs 39.67±4.99, t=5.256, P<0.001) , whereas GPⅠb and P-selection expressions decreased (37.09±3.94 vs 109.77±23.66, t=3.901, P<0.001; 149.06±19.14 vs 205.73±21.00, t=2.070, P=0.043, respectively) . ADP-stimulated GPⅠb, ADP-stimulated and unstimulated P-selection, proportion of GPⅠb, P-selection levels with/without ADP stimulation were significantly associated with platelet counts (P<0.05) . ADP-stimulated P-selection and proportion of P-selection levels with/without ADP stimulation were significantly associated with IPF (P<0.05) . There were significant differences in the expressions of unstimulated P-selection, ADP-stimulated P-selection, ADP-stimulated GPⅠb stratified by bleeding grades (P<0.05) . The ratios of P-selection, GPⅡb/Ⅲa and GPⅠb with/without ADP stimulation in ITP patients were significantly different among various bleeding grades (P<0.05) . Higher proportion of GPⅠb with/without ADP stimulation was associated with higher risk of bleeding (OR=3.05, P=0.011) . Lower proportion of GPⅡb/Ⅲa and P-selection with/without ADP was associated with higher risk of bleeding (OR=0.32, P=0.023; OR=0.04, P=0.006, respectively) . Conclusion Platelet activation index could accurately assess the degree of bleeding in patients with ITP, and also be used as the observation index and reference index for treatment.

To screen for novel binding proteins interacting with high-risk HPV 18 E6 oncogene, the strain AH109 was transformed with pGBKT7-HPV18 E6 plasmid, and subsequent transference was utilized to screen for interacting proteins with HPV 18 E6 in human Hela cDNA library. HPVl8 E6 mRNA was expressed in yeast and there was no self-activation and toxicity in AH109. Seven proteins that interacted with HPV18 E6, including transmembrane protein 87B, phosphonoformate im- muno-associated protein 5, vimentin, KM-HN-1 protein, dedicator of cytokinesis 7, vaccinia related kinase 2 and a hypothetical protein, were identified. It was suggested that yeast two-hybrid system is an efficient for screening interacting proteins. The high-risk HPV 18 E6 oncogene may interact with the proteins, which may be associated with signal transduction and transeriptional control, epithelial cell invasion and migration, as well as humoral and cellular immune etc. This investigation provides functional clues for further exploration of potential oncogenesis targets for cancer biotherapy.

Summary: Glomerulosclerosis, defined as phenotype transition of mesangial cell and deposition of extracelluar matrix, remains a chronic disease with excessive morbidity and mortality. The molecular mechanism underlying the suppression of mesangial cell activation is not fully understood. Since activation of peroxisome proliferators-activated receptor γ (PPAR1,) has been proposed to decrease the effects of transforming growth factor-β (TGF-β) on glomerulosclerosis, we examined here whether and how telmisartan, an angiotensin Ⅱ type Ⅰ receptor blocker with PPARγ-modulating activity, inhibited TGF-β-induced giomerulosclerosis in rat glomerular mesangial cells. Protein levels of PPARγ were detected by Western blot. Activation of PPARγ response element (PPRE) was analyzed by luciferase assays. Deposition of extracelluar matrix was tested by confocol laser scanning. The results showed that telmisartan, but not valsartan, another angiotensin Ⅱ type Ⅰ receptor blocker,up-regulated PPARγ protein levels in a dose-dependent manner (P<0.05). Activation of PPRE, represented by luciferase activity, was also increased with higher concentration of telmisartan in a dose-dependent manner (P<0.05). Furthermore, telmisartan inhibited TGF-β-induced α-smooth muscle actin expression and collagen IV secretion in mesangial cells. GW9662, an inhibitor of PPAR-γ,blocked the inhibitory effects of telmisartan on TGF-β-induced glomerulosclerosis in mesangial cells. Our study indicates a benefit of telmisartan as a PPARγ agonist against TGF-β-induced mesangial cells activation in renal glomerulus. It may provide possibility that telmisartan works as a potential agent against diabetic nephropathy and hypertensive renal disease.

The aim of the present study was to examine the effects of suppression of EphB4 and/or mTOR on the biological behaviors of ovarian cancer cells, and the potential regulatory pathways. Antisense EphB4 vectors and shRNA vectors targeting mammalian target of rapamycin (mTOR) were constructed and transfected into A2780 and SKOV3 cells (two ovarian cancer cell lines). The effects of the antisense EphB4 vectors and the shRNA vectors on the proliferation, apoptosis and invasion of ovarian cancer cells were measured, and the expression of EphB4, mTOR and Akt detected. The results showed that transfection with mTOR shRNA could inhibit growth, induce apoptosis, and reduce invasive ability of ovarian cancer cells, which was accompanied by downregulation of EphB4, mTOR and Akt. The inhibitory effects on cell growth caused by mTOR shRNA alone were weaker than those by antisense pEGFP-C1-EphB4. In the antisense pEGFP-C1-EphB4-transfected cells, it was found that EphB4 knockdown could decrease the mTOR expression and slightly reduce the Akt phosphorylation. Significant suppressive effects on cell growth were observed in cells co-transfected with antisense pEGFP-C1-EphB4 and mTOR shRNA. In co-transfection group, the expression levels of EphB4, mTOR and Akt were distinctly lower than those in other groups. It was concluded that suppression of EphB4 may inhibit the growth of ovarian cancer cells by downregulation of the PI3K/Akt/mTOR pathway, and reverse Akt phosphorylation induced by mTOR shRNA. Inhibition of EphB4 and mTOR combined may cooperatively suppress the biological behaviors of ovarian cancer cells.
Apoptosis ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; genetics ; Female ; Humans ; Ovarian Neoplasms ; pathology ; physiopathology ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Receptor, EphB4 ; genetics ; metabolism ; Suppression, Genetic ; genetics

Objective To explore the difference of gut microbiota between type 2 diabetes mellitus (T2DM) and non-diabetic population in Beijing. Methods 83 T2DM patients were selected as T2DM group and 64 non-diabetic subjects were selected as control group. Fecal samples were collected from all the subjects. The intestinal flora was detected by metagenome sequencing technology. Results 11 bacterialphyla were detec-ted in the two groups, there were significant differences in species diversity of Actinobacteria (P=0. 013), Firmicutes (P=0. 005), Fusobacteria (P=0. 001), Proteobacteria (P<0. 001) between the two groups. Actinobacteria, Fusobacteria and Proteobacteria were all enriched in the T2DM group, Firmicutes were enriched in the control group. 152 bacterial genera were detected in the two groups with 31 bacterial genera ofsignificant differences. In T2DM group, the levels of Roseburia, Eubacterium and Faecalibacterium decreased, while the levels of Bifidobacterium, Lactobacillus and Escherichia increased. Conclusion There are significant differ-ences in the composition of gut microbiota between T2DM patients and non-diabetic population. Regulation of gut microbiota in T2DM patients may be helpful to improve the condition of T2DM.

Schizophrenia(SZ)is a serious mental illness,and traditional diagnostic methods are prone to missed and misdiagnosis.Using machine learning(ML)algorithms can extract SZ relative features from functional MRI(fMRI)data,hence being helpful for diagnosing and predicting treatment response of SZ.The research progresses of ML based on fMRI for diagnosis and treatment of SZ were reviewed in this article.