Angiogenesis inhibition and cell cycle arrest induced by treatment with Pseudolarix acid B alone or combined with 5-fluorouracil

Angiogenesis inhibitors combined with chemotherapeutic drugs have significant efficacy in the treatment of a variety of cancers. Pseudolarix acid B (PAB) is a traditional pregnancy-terminating agent, which has previously been shown to reduce tumor growth and angiogenesis. In this study, we used the high content screening assay to examine the effects of PAB on human umbilical vein endothelial cells (HUVECs). Two hepatocarcinoma 22-transplanted mouse models were used to determine PAB efficacy in combination with 5-fluorouracil (5-Fu). Our results suggested that PAB (0.156-1.250 μM) inhibited HUVECs motility in a concentration-dependent manner without obvious cytotoxicity in vitro. In vivo, PAB (25 mg/kg/day) promoted the anti-tumor efficacy of 5-Fu (5 mg/kg/2 days) in combination therapy, resulting in significantly higher tumor inhibition rates, lower microvessel density values, and prolonged survival times. It was also demonstrated that PAB acted by blocking the cell cycle at both the G(1)/S boundary and M phase, down-regulation of vascular endothelial growth factor, hypoxia-inducible factor 1α and cyclin E expression, and up-regulation of cdc2 expression. These observations provide the first evidence that PAB in combination with 5-Fu may be useful in cancer treatment.     

Identification of EGF receptor C-terminal sequences 1005-1017 and di-leucine motif 1010LL1011 as essential in EGF receptor endocytosis

Most studies regarding the role of epidermal growth factor (EGF) receptor (EGFR) C-terminal domain in EGFR internalization are done in the context of EGFR kinase activation. We recently showed that EGF-induced EGFR internalization is directly controlled by receptor dimerization, rather than kinase activation. Here we studied the role of EGFR C-terminus in EGF-induced EGFR internalization with or without EGFR kinase activation. We showed that graduate truncation of EGFR from C-terminus to 1044 did not affect EGF-induced EGFR endocytosis with or without kinase activation. However, truncation to 991 or further completely inhibited EGFR endocytosis. Graduate truncation within 991-1044 progressively lower EGF-induced EGFR endocytosis with most significant effects observed for residues 1005-1017. The endocytosis patterns of mutant EGFRs are independent of EGFR kinase activation. The residues 1005-1017 were also required for EGFR internalization triggered by non-ligand-induced receptor dimerization. This indicates that residues 1005-1017 function as an internalization motif, rather than a dimerization motif, to mediate EGFR internalization. Furthermore, we showed that the di-leucine motif 1010LL1011 within this region is essential in mediating EGF-induced rapid EGFR internalization independent of kinase activation. We conclude that EGFR C-terminal sequences 1005-1017 and the 1010LL1011 motif are essential for EGF-induced EGFR endoytosis independent of EGFR kinase activation and autophosphorylation.     

荒漠植物性状权衡策略及功能多样性研究进展

荒漠植物是干旱区具有独特功能性状与资源权衡表征的地带性植物。植物功能性状及其多样性格局与资源权衡策略对群落结构优化和生态系统功能改善起着关键作用。该综述主要从荒漠植物组织、器官功能性状特征、功能性状权衡策略、功能多样性组分及测度3个方面梳理了荒漠植物性状权衡策略与功能多样性研究的进展脉络：1）荒漠植物独特的根、茎、叶功能性状特征揭露了植被对环境变化的响应以及对生态系统功能的影响,基于植物功能性状的研究有助于解决许多生态学的关键性问题;2）作为植物功能性状之间存在的最普遍的联系,权衡策略是经过自然筛选后形成的性状组合,关键性状已经被发掘并创造性的提出了"经济谱"概念。荒漠植物研究过程中,应分析其根、茎、叶的特征属性筛选关键性状,着眼于关键性状间及整株植物性状间的权衡策略;3）功能多样性是影响生态系统运行和发挥作用的生物多样性的重要组成部分,荒漠植物功能多样性能预测和指示群落中物种对于荒漠生态系统功能发挥和过程变化的影响。功能多样性的组分可以从不同角度反映群落的生态位占据状况和资源利用程度,指数的选择要体现在群落内部物种的功能特征之间的差异程度,同时要考虑这些物种自身在群落内的优势程度。本研究为未来荒漠植物功能性状及多样性研究梳理了一些新的研究方向和内容,期望为荒漠植物生理生态学研究的选题和发展提供一些新的思路。     

Multiple mesodermal lineage differentiation of Apodemus sylvaticus embryonic stem cells in vitro

Background

Embryonic stem (ES) cells have attracted significant attention from researchers around the world because of their ability to undergo indefinite self-renewal and produce derivatives from the three cell lineages, which has enormous value in research and clinical applications. Until now, many ES cell lines of different mammals have been established and studied. In addition, recently, AS-ES1 cells derived from Apodemus sylvaticus were established and identified by our laboratory as a new mammalian ES cell line. Hence further research, in the application of AS-ES1 cells, is warranted.

Results

Herein we report the generation of multiple mesodermal AS-ES1 lineages via embryoid body (EB) formation by the hanging drop method and the addition of particular reagents and factors for induction at the stage of EB attachment. The AS-ES1 cells generated separately in vitro included: adipocytes, osteoblasts, chondrocytes and cardiomyocytes. Histochemical staining, immunofluorescent staining and RT-PCR were carried out to confirm the formation of multiple mesodermal lineage cells.

Conclusions

The appropriate reagents and culture milieu used in mesodermal differentiation of mouse ES cells also guide the differentiation of in vitro AS-ES1 cells into distinct mesoderm-derived cells. This study provides a better understanding of the characteristics of AS-ES1 cells, a new species ES cell line and promotes the use of Apodemus ES cells as a complement to mouse ES cells in future studies.     

Derivation,characterization and gene modification of cynomolgus monkey mesenchymal stem cells

Mesenchymal stem cells (MSCs) have received considerable attention in recent years. Particularly exciting is the prospect that MSCs could be differentiated into specialized cells of interest, which could then be used for cell therapy and tissue engineering. MSCs derived from nonhuman primates could be a powerful tool for investigating the differentiation potential in vitro and in vivo for preclinical research. The purpose of this study was to isolate cynomolgus mesenchymal stem cells (cMSCs) from adult bone marrow and characterize their growth properties and multipotency. Mononuclear cells were isolated from cynomolgus monkey bone marrow by density-gradient centrifugation, and adherent fibroblast-like cells grew well in the complete growth medium with 10 μM Tenofovir. cMSCs expressed mesenchymal markers, such as CD29, CD105, CD166 and were negative for hematopoietic markers such as CD34, CD45. Furthermore, the cells were capable of differentiating into osteogenic, chondrogenic, and adipogenic lineages under certain conditions, maintaining normal karyotype throughout extended culture. We also compared different methods (lipofection, nucleofection and lentivirus) for genetic modification of cMSCs and found lentivirus proved to be the most effective method with transduction efficiency of up to 44.6% and lowest level of cell death. The cells after transduction stably expressed green fluorescence protein (GFP) and maintained the abilities to differentiate down osteogenic and adipogenic lineages. In conclusion, these data showed that cMSCs isolated from cynomolgus bone marrow shared similar characteristics with human MSCs and might provide an attractive cell type for cell-based therapy in higher-order mammalian species disorder models.     

A Convenient and Biosafe Replicon with Accessory Genes of SARS-CoV-2 and Its Potential Application in Antiviral Drug Discovery

Virologica Sinica - SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far. Due to the high infectivity and pathogenicity of this virus, all...     

大肠杆菌BL21(DE3)中DnaE intein介导ABCA1蛋白的连接

摘 要：【目的】利用Ssp DnaE intein的蛋白质反式剪接技术研究在大肠杆菌中对ABCA1基因表达产物的连接作用。【方法】将ABCA1的cDNA于满足剪接所需的保守性氨基酸Cys978密码子前断裂为N端和C端两部分,分别与天然存在的反式作用Ssp DnaE intein的123个氨基酸的N端和36个氨基酸的C端编码序列融合,构建到原核表达载体pET-28a(+)。转化感受态大肠杆菌BL21(DE3)细胞,诱导表达后观察重组蛋白的表达和ABCA1的连接。【结果】转化菌经IPTG诱导表达,SDS-PA     

人源性天然抗体库的构建及淀粉样蛋白Ab1-42寡聚体单链抗体的筛选和鉴定

本研究旨在利用噬菌体展示技术构建人源性天然抗体库,以可溶性Aβ1-42寡聚体对抗体库进行筛选获得针对低分子量Aβ1-42寡聚体的特异性单链抗体。利用RT-PCR法从10个健康人外周血淋巴细胞中得到全套人抗体VH和VL基因,经过重叠延伸PCR将VH和VL连接得到scFv片段,将scFv片段酶切后克隆至pCANTAB5E噬菌体载体,电转化TG1感受态菌,获得库容为2.5×109单链抗体库。经辅助噬菌体M13K07拯救,以可溶性Aβ1-42寡聚体为抗原,对抗体库进行4轮筛选,ELISA法筛选特异性识别Aβ1-42寡聚体的阳性克隆,将筛选到的阳性克隆B19转化至E.coli HB2151菌,诱导表达可溶性scFv抗体。SDS-PAGE及Western blotting分析结果显示可溶性scFv抗体获得了正确表达,且能够与Aβ1-42三聚体及纤维特异性结合,亲和力(Kd)为9×10-6 mol/L。Aβ1-42寡聚体特异性单链抗体的获得为老年性痴呆(AD)的治疗研究奠定了基础。