P2X receptors are non-selective cation channels gated by extracellular ATP, and the P2X7 receptor subtype plays a crucial role in the immune and nervous systems. Altered expression and dysfunctions of P2X7 receptors caused by genetic deletions, mutations, and polymorphic variations have been linked to various diseases, such as rheumatoid arthritis and hypertension. Despite the availability of crystal structures of P2X receptors, the mechanism of competitive antagonist action for P2X receptors remains controversial. Here, we determine the crystal structure of the chicken P2X7 receptor in complex with the competitive P2X antagonist, TNP-ATP. The structure reveals an expanded, incompletely activated conformation of the channel, and identified the unique recognition manner of TNP-ATP, which is distinct from that observed in the previously determined human P2X3 receptor structure. A structure-based computational analysis furnishes mechanistic insights into the TNP-ATP-dependent inhibition. Our work provides structural insights into the functional mechanism of the P2X competitive antagonist.

BackgroundAccumulating evidence supports the hypothesis that cancer stem cells (CSCs) are essential for cancer initiation, metastasis and drug resistance. However, the functional association of gastric CSC markers with stemness and epithelial-mesenchymal transition (EMT) signature genes is unclear.MethodsqPCR was performed to measure the expression profiles of stemness and EMT signature genes and their association with putative CSC markers in gastric cancer tissues, cancer cell lines and sphere cells. Western blot analysis was used to confirm the results of the transcript analysis. Cell proliferation, cell migration, drug resistance and sphere cell growth assays were conducted to measure the expansion and invasion abilities of the cells. Tumor xenograft experiments were performed in NOD/SCID mice to test cell stemness in vivo. Flow cytometry and immunofluorescence staining were used to analyze cell subpopulations.ResultsThe expression of LGR5 was strikingly up-regulated in sphere cells but not in cancer tissues or parental adherent cells. The up-regulation of LGR5 was also positively associated with stemness regulators (NANOG, OCT4, SOX2, and AICDA) and EMT inducers (PRRX1, TWIST1, and BMI1). In addition, sphere cells exhibited up-regulated vimentin and down-regulated E-cadherin expression. Using gene-specific primers, we found that the NANOG expression primarily originates from the retrogene NANOGP8. Western blot analysis showed that the expression of both LGR5 and NANOG is significantly higher in sphere cells. LGR5 over-expression significantly enhanced sphere cell growth, cell proliferation, cell migration and drug resistance in MGC803 cells. Tumor xenografts in nude mice showed that sphere cells are at least 10 times more efficient at tumor initiation than adherent cells. Flow cytometry analysis showed that ~20% of sphere cells are LGR5+/CD54+, but only ~3% of adherent cells are Lgr5+/CD54+. Immunofluorescence staining supports the above results.ConclusionThe LGR5-expressing fraction of CD54+ cells represents gastric cancer CSCs, in which LGR5 is closely associated with stemness and EMT core genes, and NANOG expression is mainly contributed by the retrogene NANOGP8. Sphere cells are the best starting materials for the characterization of CSCs.

Renal SNX5 positively regulates IDE expression and function. This study is the first to demonstrate the novel and crucial role of renal SNX5 in insulin and glucose metabolism.

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Expression of HMGA2 was closely associated with occurrence of epithelial-to-mesenchymal transition. Expression of HMGA2, loss of E-cadherin and expression of vimentin may indicate high degree malignancy of bladder cancer. Loss of E-cadherin expression and positive expression of vimentin may predict recurrence of bladder cancer.

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BackgroundAccumulating evidence demonstrated that NANOG1, the key transcription factor for embryonic stem cells, is associated with human cancers. NANOGP8, one of the pseudogenes in NANOG gene family, contains an intact open reading frame and also said to be expressed in cancer tissues. Therefore, a systematic study is greatly needed to address the following questions: among NANOG1 and NANOGP8, which gene is the main contributor for NANOG expression in cancer cells and which one is the key regulator responsible for stemness, epithelial-mesenchymal transition (EMT), metastasis, chemoresistance and other malignant phenotypes. Here we try to explore these issues with gastric adenocarcinoma cell lines in vitro using variety of molecular and cellular techniques.MethodsSpecial primers were designed to distinguish PCR products from NANOG1 and NANOGP8. Sphere-forming cells were cultured with serum-free and selective medium. A stable cell line was established with infection of lentivirus containing NANOGP8. qPCR was performed to measure NANOGP8 expression and its association with stemness, EMT and CSC markers in adherent cells and sphere-forming cells. Western blot analysis was deployed to confirm results of the transcript analysis. Experiments of cell proliferation, migration, invasion, clonogenic assay, sphere cell growth assays, cell cycle analysis, β-catenin accumulation and translocation in nucleus, and drug resistance were conducted to measure the impact of NANOGP8 on malignant statuses of gastric cancer cells. Immunofluorescence staining was used to analyze cell subpopulations with different markers.ResultsNANOGP8 is mainly responsible for NANOG expression in sphere-forming (stem cell-like) cells derived from gastric cancer cell lines regardless their differentiation status. Ectopic expression of NANOGP8 significantly up-regulates stemness transcription factors, EMT inducers, and cancer stem cell markers (CSC) including Lgr5. NANOGP8 also promotes expression of the signature genes vimentin and N-caderin for mesenchymal cells and down-regulates the signature gene E-caderin for epithelial cells whereby confer the cells with mesenchymal cell phenotype. In NANOGP8 over-expressed adherent and sphere-forming cells, Lgr5+ cells are significantly increased. Ectopic expression of NANOGP8 endows gastric cells with enhanced proliferation, migration, invasion, sphere-forming and clonogenic capacity, and chemoresistance. NANOGP8 expression also enhances β-catenin accumulation in nucleus and strengthens Wnt signal transduction.ConclusionNANOGP8 is the main regulator of gastric cancer stem cells. It is closely associated with EMT, stemness, and CSC marker as well as Wnt signal pathway. NANOGP8 is correlated with cell proliferation, migration, invasion, clonogenic capacity, β-catenin accumulation in nucleus, and chemoresistance in gastric cancer. NANOGP8 is a promising molecular target for clinical intervention of gastric cancer.

AAV9 vector provides efficient gene transfer in all segments of the renal nephron, with minimum expression in non-renal cells, when administered retrogradely via the ureter. It is important to restrict the transgene expression to the desired cell type within the kidney, so that the physiological endpoints represent the function of the transgene expressed in that specific cell type within kidney. We hypothesized that segment-specific gene expression within the kidney can be accomplished using the highly efficient AAV9 vectors carrying the promoters of genes that are expressed exclusively in the desired segment of the nephron in combination with administration by retrograde infusion into the kidney via the ureter. We constructed AAV vectors carrying eGFP under the control of: kidney-specific cadherin (KSPC) gene promoter for expression in the entire nephron; Na+/glucose co-transporter (SGLT2) gene promoter for expression in the S1 and S2 segments of the proximal tubule; sodium, potassium, 2 chloride co-transporter (NKCC2) gene promoter for expression in the thick ascending limb of Henle's loop (TALH); E-cadherin (ECAD) gene promoter for expression in the collecting duct (CD); and cytomegalovirus (CMV) early promoter that provides expression in most of the mammalian cells, as control. We tested the specificity of the promoter constructs in vitro for cell type-specific expression in mouse kidney cells in primary culture, followed by retrograde infusion of the AAV vectors via the ureter in the mouse. Our data show that AAV9 vector, in combination with the segment-specific promoters administered by retrograde infusion via the ureter, provides renal nephron segment-specific gene expression.