Inhibited Expression of α<Subscript>4</Subscript>β<Subscript>2</Subscript> Nicotinic Acetylcholine Receptor in Blood Leukocytes of Chinese Patients with Vascular Dementia and in Blood Leukocytes as Well as the Hippocampus of Brain from Ischemic Rats

Our present aim was to investigate whether changes in the expression of α₄β₂ nicotinic acetylcholine receptor (nAChR) in patients with vascular dementia (VaD) and ischemic rats are related to cognitive scores. Blood leukocytes for 59 Chinese patients with VaD (diagnosed on the basis of clinical guidelines) and 31 cases as age-matched controls were examined, and the animal model established employing Pulsinelli’s four-vessel occlusion. The levels of α₄ and β₂ subunit mRNA in leukocytes and the hippocampus were analyzed by real-time PCR, and the protein level in the hippocampus by Western blotting. The mini-mental state examination was utilized to characterize the intellectual capacity of the patients with reference to the DSM IV diagnosis and Hachinski Ischemic Scale score, and the Morris Water Maze test to assess the ability of learning and memory of the rats. In patients, the level of α₄ mRNA, but not β₂, in blood leukocytes was clearly lowered, which was significantly correlated to their clinical cognitive test scores. Smoking exerted no impact on the level of α₄ mRNA in the present study. In the blood leukocytes and the hippocampus of the brains of the ischemic rats, the levels of both α₄ and β₂ mRNA were lowered, and the proteins of these subunits in the hippocampus were decreased. The changes of α₄ and β₂ mRNA in blood leukocytes, and their protein levels in the hippocampus were significantly correlated with impaired learning and memory. These findings indicate that alterations in expression of the α₄β₂ subtype of nAChR may be involved in the molecular mechanism(s) underlying the cognitive deficit associated with VaD.     

Ecdysteroids from the Aerial Parts of Paris verticillata

Two new ecdysteroids 14-epi-polypodine B ( 1 ) and 22-oxo-hydroxyecdysterone ( 2 ), along with nine known compounds, polypodine B ( 3 ), viticosterone E ( 4 ), 20-hdroxyecdysone-2-acetate ( 5 ), 22-oxo-20-hydroxyecdysone ( 6 ), 5-hydroxyecdysone ( 7 ), pinnatasterone ( 8 ), 3-epi-20-hydroxyecdysone ( 9 ), ecdysterone ( 10 ) and stachysterone B ( 11 ), were isolated from the aerial parts of Paris verticillata. The structures of all compounds were elucidated by extensive spectroscopic analysis, quantum chemical calculations and ANN-PRA/DP4⁺ probability analysis. Among them, the absolute configuration of compound 1 and 2 was unambiguous determined by ECD. Also, the isolated compounds were assessed for their cytotoxic activities. Compounds 2 , 3 and 7 exhibited significant cytotoxic activities against PC12, LN299 and SMCC7721 cells.     

miR-181a/b-5p regulates human umbilical vein endothelial cell angiogenesis by targeting PDGFRA

Type 2 diabetes mellitus (T2DM) is a growing burden in low-and middle-income countries. Changing lifestyles and lack of physical activity are some of the reasons contributing to this epidemic increase. Co-morbidities associated with T2DM are largely due to the complications which arise as a consequence of endothelial dysfunction. Platelet derived growth factor-alpha (PDGFRA) is a protein responsible for cell proliferation, angiogenesis, migration and invasion. Increased levels of PDGFRA have been reported in T2DM. This study assessed the epigenetic regulation of PDGFRA through microRNAs (miR-181a/b-5p).Using a bioinformatics-based approach, we assessed the binding of miR-181a/b-5p to PDGFRA. Experimentally, this binding was confirmed using a dual luciferase reporter assay. Further, we overexpressed miR-181a/b-5p in Human umbilical vein endothelial cells (HUVECs) and the influence of over-expression on cell proliferation, migration and angiogenesis was assessed using in-vitro approaches. The influence of miR-181a/b-5p over expression on cellular apoptosis was ascertained using a TUNEL assay with concomitant changes being observed in the levels of Bcl-2 and cleaved Caspase-3.In HUVECs, PDGFRA is a direct target for miR-181a/b-5p. Over expression of miR-181a/b-5p decreased cellular proliferation, migration, invasion, and tube formation—a surrogate marker for angiogenesis. miR-181a/b-5p may be used as a therapeutic intervention to restrict uncontrolled levels of PDGFRA and thereby rescue the phenotypes of increased cell proliferation, migration, invasion and tube formation. miR-181a/b negatively regulates PDGFRA levels. Significance of the study : T2DM and its associated complications emerge from endothelial dysfunction. The associated phenotypes are regulated by a number of proteins, one such member being, PDGFRA. PDGFRA is in turn regulated by miR-181a/b-5p. Complementation with miR-181a/b-5p resulted in reversion of phenotypes. Thus, miR-181a/b-5p-mediated suppression of PDGFRA may be used as a therapeutic intervention in the management of type 2 diabetes.     

Ectopic expression of CC chemokine receptor 7 promotes prostate cancer cells metastasis via Notch1 signaling

There currently exists no satisfactory treatment for patients with prostate cancer with local evolution and distant metastasis. Previous studies have confirmed the importance of CC chemokine receptor 7 (CCR7) in the invasion and metastasis of prostate cancer. And increasing evidence prove that Notch1 can play diametrically opposite roles in the development and progression of different tumors. To demonstrate the correlation between CCR7 and Notch1, PC-3 cells were transfected with pcDNA3.1-CCR7 or CCR7 si-RNA, respectively. Then Western blot analysis was used to detect the expressions of Notch1, ERK, P38, JNK, NF-κB, MMP-9, and epithelial-mesenchymal transition (EMT)-related proteins. Moreover, matrigel invasion assays were performed to assess the migratory and invasive activities of PC-3 cells. PcDNA3.1-CCR7 increased the expression of Notch1, phospho-MAPK, phospho-P65, MMP-9, N-cadherin, and Snail in PC-3 cells, but decreased the expression of E-cadherin. PcDNA3.1-CCR7 also promoted the migration and invasion of PC-3 cells. However, CCR7 si-RNA reversed the effect of pcDNA3.1-CCR7 in PC-3 cells. And MAPK and NF-κB pathway inhibitors were used to testify that activation of Notch1 induces EMT through MAPK and NF-κB pathway. All these results indicate that upregulation of Notch1 by CCR7 can accelerate the evolution of EMT and develop the invasion and metastasis in prostate cancer cells by activating MAPK and NF-κB signaling pathways in prostate cancer cells, which provides a new molecular evidence for targeted therapy in metastatic prostate cancer.     

Adipose tissue deletion of Gpr116 impairs insulin sensitivity through modulation of adipose function

G protein-coupled receptor 116 (GPR116) is a novel member of the G protein-coupled receptors and its function is largely unknown. To investigate the physiological function of GPR116 in vivo, we generated adipose tissue specific conditional Gpr116 knockout mice (CKO) and fed them on standard chow or high fat diets. Selective deletion of Gpr116 in adipose tissue caused a pronounced glucose intolerance and insulin resistance in mice, especially when challenged with a high fat diet. Biochemical analysis revealed a more severe hepatosteatosis in CKO mice. Additionally, we found that CKO mice showed a lowered concentration of circulating adiponectin and an increased level of serum resistin. Our study suggests that GPR116 may play a critical role in controlling adipocyte biology and systemic energy homeostasis.     

Sample size determination for testing nonequality under a three‐treatment two‐period incomplete block crossover trial

To reduce the lengthy duration of a crossover trial for comparing three treatments, the incomplete block design has been often considered. A sample size calculation procedure for testing nonequality between either of the two experimental treatments and a placebo under such a design is developed. To evaluate the performance of the proposed sample size calculation procedure, Monte Carlo simulation is employed. The accuracy of the sample size calculation procedure developed here is demonstrated in a variety of situations. As compared with the parallel groups design, a substantial proportional reduction in the total minimum required sample size in use of the incomplete block crossover design is found. A crossover trial comparing two different doses of formoterol with a placebo on the forced expiratory volume is applied to illustrate the use of the sample size calculation procedure.     

Competence-Independent Activity of Pneumococcal Enda Mediates Degradation of Extracellular DNA and Nets and Is Important for Virulence

Membrane surface localized endonuclease EndA of the pulmonary pathogen Streptococcus pneumoniae (pneumococcus) is required for both genetic transformation and virulence. Pneumococcus expresses EndA during growth. However, it has been reported that EndA has no access to external DNA when pneumococcal cells are not competent for genetic transformation, and thus, unable to degrade extracellular DNA. Here, by using both biochemical and genetic methods, we demonstrate the existence of EndA-mediated nucleolytic activity independent of the competence state of pneumococcal cells. Pneumococcal mutants that are genetically deficient in competence development and genetic transformation have extracellular nuclease activity comparable to their parental wild type, including their ability to degrade neutrophil extracellular traps (NETs). The autolysis deficient ΔlytA mutant and its isogenic choline-treated parental wild-type strain D39 degrade extracellular DNA readily, suggesting that partial cell autolysis is not required for DNA degradation. We show that EndA molecules are secreted into the culture medium during the growth of pneumococcal cells, and contribute substantially to competence-independent nucleolytic activity. The competence-independent activity of EndA is responsible for the rapid degradation of DNA and NETs, and is required for the full virulence of Streptococcus pneumoniae during lung infection.