Discovery and Pharmacophore Studies of Novel Pyrazole‐Based Anti‐Melanoma Agents

Due to the rising incidence and lack of effective treatments, malignant melanoma is the most dangerous form of skin cancer, so that new treatment strategies are urgently needed. Several recent developments indicate that the V600E mutant BRAF (BRAF^V600E) is a validated target for antimelanoma‐drug development. Based on in silico screening results, a series of novel pyrazole derivatives has been designed, synthesized, and evaluated in vitro for their inhibitory activities against BRAF^V600E melanoma cells. Compound 3d exhibited the most potent inhibitory activity with an IC₅₀ value of 0.63 μM for BRAF^V600E and a GI₅₀ value of 0.61 μM for mutant BRAF‐dependent cells. Furthermore, the QSAR modeling and the docking simulation of inhibitor analogs provide important pharmacophore clues for further structural optimization.     

Three New Isomeric Indole Alkaloids from Nauclea officinalis

Three new isomeric monoterpene indole alkaloids, naucleofficines I–III ( 1 – 3 , resp.) were isolated from the stems (with bark) of Nauclea officinalis. Their structures were elucidated on the basis of spectroscopic methods and single‐crystal diffraction analyses. The cytotoxic activities of 1 – 3 against human colon cancer, human gastric cancer, and human hepatoma cells were also investigated.     

New Metabolites from Endolichenic Fungus Pleosporales sp.

Eight new metabolites were obtained from the culture of an endolichenic fungus, Pleosporales sp. Their structures were determined as three terphenyl derivatives, cucurbitarins A–C ( 1 – 3 , resp.), two structurally related compounds, cucurbitarins D and E ( 4 and 5 , resp.), two benzocoumarins, 3,10‐dihydroxy‐4,8‐dimethoxy‐6‐methylbenzocoumarin ( 6 ) and 3,8,10‐trihydroxy‐4‐methoxy‐6‐methylbenzocoumarin ( 7 ), as well as one cyclohexenone, (5R)‐5‐hydroxy‐2,3‐dimethylcyclohex‐2‐en‐1‐one ( 8 ), based on the spectroscopic data.     

骨形成蛋白2/珍珠层粉/壳聚糖支架制备及生物性能研究

目的:制备骨形成蛋白2/珍珠层粉/壳聚糖复合多孔支架,观察支架生物性能。方法:采用冷冻干燥法制备骨形成蛋白2/珍珠层粉/壳聚糖多孔支架。用光学显微镜和扫描电子显微镜观察支架表面形貌及孔径大小,用比重瓶法检测支架孔隙率,热重分析探讨支架的热稳定性,用微力试验机进行压缩性能测试,并将支架与兔骨髓间充质干细胞共培养检测细胞黏附性能,将支架埋置大鼠皮下观察其炎症反应。结果与结论:制备的骨形成蛋白2/珍珠层粉/壳聚糖支架孔径大小为100~300μm,孔隙率为91.64%,压缩应力达3.37MPa,与细胞共培养贴附较好,有良好的组织相容性,提示该支架可做为组织工程支架材料应用于临床上骨组织缺损的修复。     

小鼠脑微血管内皮细胞与新生隐球菌GXM作用后基因表达谱分析

目的探索新生隐球菌GXM能否影响脑微血管内皮细胞基因的表达,为进一步研究隐球菌嗜中枢性的分子机制提供线索。方法使用Roche Nimble Gen 12×135K小鼠基因表达谱芯片,筛选小鼠脑微血管内皮细胞系b End.3与不同浓度新生隐球菌GXM作用后差异表达的基因;结合基因本体论(Gene Ontology),使用top GO进行差异基因GO分析,结合GO语义挖掘与隐球菌侵袭中枢神经系统能力相关的差异表达基因的信息;采用荧光实时定量PCR对重要基因PIK3C2G和ADAMDEC1的表达水平变化加以验证。结果 b End.3细胞与新生隐球菌GXM作用前后基因表达对比发现,实验组GXM(90μg/m)组总共有402个基因表达上调,296个基因表达下调,GXM(180μg/m)组总共有421个基因表达上调,564个基因表达下调,细胞膜、细胞骨架、磷酸肌醇-3-激酶活化、1-磷酸肌醇-3-激酶活化、细胞紧密连接等生物过程差异表达基因较为富集;对PIK3C2G基因和ADAMDEC1基因表达水平变化进行荧光定量PCR验证,结果与芯片结果一致,基因表达水平不同程度上升,且与GXM浓度正相关。结论新生隐球菌GXM能够影响脑微血管内皮细胞基因表达,PIK3C2G基因、ADAMDEC1基因表达上调可能和隐球菌穿越血脑屏障有关。     

Kv1.3 deletion biases T cells toward an immunoregulatory phenotype and renders mice resistant to autoimmune encephalomyelitis

Increasing evidence suggests ion channels have critical functions in the differentiation and plasticity of T cells. Kv1.3, a voltage-gated K(+) channel, is a functional marker and a pharmacological target for activated effector memory T cells. Selective Kv1.3 blockers have been shown to inhibit proliferation and cytokine production by human and rat effector memory T cells. We used Kv1.3 knockout (KO) mice to investigate the mechanism by which Kv1.3 blockade affects CD4(+) T cell differentiation during an inflammatory immune-mediated disease. Kv1.3 KO animals displayed significantly lower incidence and severity of myelin oligodendrocyte glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis. Kv1.3 was the only K(V) channel expressed in MOG 35-55-specific CD4(+) T cell blasts, and no K(V) current was present in MOG-specific CD4(+) T cell-blasts from Kv1.3 KO mice. Fewer CD4(+) T cells migrated to the CNS in Kv1.3 KO mice following disease induction, and Ag-specific proliferation of CD4(+) T cells from these mice was impaired with a corresponding cell-cycle delay. Kv1.3 was required for optimal expression of IFN-γ and IL-17, whereas its absence led to increased IL-10 production. Dendritic cells from Kv1.3 KO mice fully activated wild-type CD4(+) T cells, indicating a T cell-intrinsic defect in Kv1.3 KO mice. The loss of Kv1.3 led to a suppressive phenotype, which may contribute to the mechanism by which deletion of Kv1.3 produces an immunotherapeutic effect. Skewing of CD4(+) T cell differentiation toward Ag-specific regulatory T cells by pharmacological blockade or genetic suppression of Kv1.3 might be beneficial for therapy of immune-mediated diseases such as multiple sclerosis.     

Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

While epidermal growth factor receptor (EGFR) has been shown to be important in the entry process for multiple viruses, including hepatitis C virus (HCV), the molecular mechanisms by which EGFR facilitates HCV entry are not well understood. Using the infectious cell culture HCV model (HCVcc), we demonstrate that the binding of HCVcc particles to human hepatocyte cells induces EGFR activation that is dependent on interactions between HCV and CD81 but not claudin 1. EGFR activation can also be induced by antibody mediated cross-linking of CD81. In addition, EGFR ligands that enhance the kinetics of HCV entry induce EGFR internalization and colocalization with CD81. While EGFR kinase inhibitors inhibit HCV infection primarily by preventing EGFR endocytosis, antibodies that block EGFR ligand binding or inhibitors of EGFR downstream signaling have no effect on HCV entry. These data demonstrate that EGFR internalization is critical for HCV entry and identify a hitherto-unknown association between CD81 and EGFR.     

Complete Genome Sequences of Highly Divergent Torque Teno Virus Type II from Swine Herds

Through routine and nested PCR amplifications, four complete genome sequences of porcine Torque teno virus (TTV) type II were obtained from swine herds. By comparison with the TTV genome sequences deposited in GenBank, we found the most divergent types so far described. The level of genetic diversity between these genomes is higher than would be expected within a single virus species. A nucleotide and amino acid phylogenetic tree was constructed.     

MicroRNA-494 is required for the accumulation and functions of tumor-expanded myeloid-derived suppressor cells via targeting of PTEN

Myeloid-derived suppressor cells (MDSCs) potently suppress the anti-tumor immune responses and also orchestrate the tumor microenvironment that favors tumor angiogenesis and metastasis. The molecular networks regulating the accumulation and functions of tumor-expanded MDSCs are largely unknown. In this study, we identified microRNA-494 (miR-494), whose expression was dramatically induced by tumor-derived factors, as an essential player in regulating the accumulation and activity of MDSCs by targeting of phosphatase and tensin homolog (PTEN) and activation of the Akt pathway. TGF-β1 was found to be the main tumor-derived factor responsible for the upregulation of miR-494 in MDSCs. Expression of miR-494 not only enhanced CXCR4-mediated MDSC chemotaxis but also altered the intrinsic apoptotic/survival signal by targeting of PTEN, thus contributing to the accumulation of MDSCs in tumor tissues. Consequently, downregulation of PTEN resulted in increased activity of the Akt pathway and the subsequent upregulation of MMPs for facilitation of tumor cell invasion and metastasis. Knockdown of miR-494 significantly reversed the activity of MDSCs and inhibited the tumor growth and metastasis of 4T1 murine breast cancer in vivo. Collectively, our findings reveal that TGF-β1-induced miR-494 expression in MDSCs plays a critical role in the molecular events governing the accumulation and functions of tumor-expanded MDSCs and might be identified as a potential target in cancer therapy.