全基因组重测序揭示青藏高原东缘及东南缘东方蜜蜂遗传多样性与适应性进化

【目的】对青藏高原东缘和东南缘及邻近地区东方蜜蜂Apis cerana样本进行群体遗传多样性与适应性进化研究,为进一步解析青藏高原东方蜜蜂的遗传资源多样性、种群扩散规律以及适应高原生境的分子进化机制提供参考。【方法】对青藏高原东缘和东南缘及邻近地区77群东方蜜蜂样本进行全基因组重测序;运用群体遗传学方法,基于群体结构、主成分分析、系统进化树、遗传分化指数、线粒体基因组单倍型以及选择信号分析对东方蜜蜂这77群及GenBank数据库下载的90群的全基因组重测序原始数据进行分析。【结果】这167群东方蜜蜂区分出来自川西高原、藏南高原、滇北高原和川北高原的4个高原型群,分属于两个进化支,平均遗传分化指数(Fst=0.1178)高于非高原型群的(Fst=0.0411)。基于种群间最小遗传距离分析,东南缘的藏南高原群、滇北高原群与滇南群具有更近的遗传关系;东缘的川西高原群、川北高原群分别与川西山地和秦巴群具有更近的遗传关系。结合线粒体基因组单倍型分析,初步推断出青藏高原东缘及东南缘高原型群体祖先单倍型及来源。选择性分析鉴定到了潜在的参与脂肪酸代谢、光转导、温度适应、卵巢发育等信号通路相关的潜在受选基因。在藏南高原和滇北高原群体中发现两个共同受选择基因ISL-1和FOXO,主要参与胰岛素分泌以应对细胞压力,暗示它们在东方蜜蜂适应青藏高原东南缘生境中发挥着重要作用。【结论】青藏高原东方蜜蜂遗传多样性丰富,东南缘的藏南高原和滇北高原群体以及东缘的川西高原和川北高原群体在遗传分化上明显区分,这4个高原群是由邻近地区非高原群扩散后的地理隔离形成了种群分化;初步筛选获得东方蜜蜂高原环境适应性潜在基因。本研究为进一步探析青藏高原东方蜜蜂适应高原生境下的分子进化机制奠定了基础。     

Comparative transcriptome analysis of developmental stages of the Limonium bicolor leaf generates insights into salt gland differentiation

With the expansion of saline land worldwide, it is essential to establish a model halophyte to study the salt‐tolerance mechanism. The salt glands in the epidermis of Limonium bicolor (a recretohalophyte) play a pivotal role in salt tolerance by secreting excess salts from tissues. Despite the importance of salt secretion, nothing is known about the molecular mechanisms of salt gland development. In this study, we applied RNA sequencing to profile early leaf development using five distinct developmental stages, which were quantified by successive collections of the first true leaves of L. bicolor with precise spatial and temporal resolution. Specific gene expression patterns were identified for each developmental stage. In particular, we found that genes controlling salt gland differentiation in L. bicolor may evolve in a trichome formation, which was also confirmed by mutants with increased salt gland densities. Genes involved in the special ultrastructure of salt glands were also elucidated. Twenty‐six genes were proposed to participate in salt gland differentiation. Our dataset sheds light on the molecular processes underpinning salt gland development and thus represents a first step towards the bioengineering of active salt‐secretion capacity in crops.     

拟南芥HyPRP蛋白AZI1在大肠杆菌中的表达及其抗真菌活性分析

AZI1(AZELAIC ACID INDUCED 1)基因位于拟南芥4号染色体上,编码产物是脂质转移蛋白(lipid transfer protein, LTP)家族的一个成员。该基因在系统获得抗性(systemic acquired resistance, SAR)中具有重要功能,名称来自它可以被壬二酸(azelaic acid, AzA)诱导。已有的研究结果显示,在拟南芥中由AzA和甘油-3-磷酸(glycerol-3-phosphate, G3P)诱导的SAR反应需要AZI1和DIR1,这两个脂质转移蛋白有助于G3P的积累。为了确定AZI1蛋白是否具有抗真菌活性,本工作构建了原核表达载体pET28a-AZI1,利用大肠杆菌BL21(DE3)受体细胞制备了没有信号肽的AZI1重组蛋白。Western免疫印迹分析发现通过半乳糖苷类似物IPTG诱导表达的AZI1重组蛋白主要以包涵体的形式存在。体外抑菌实验以及激光共聚焦显微观察结果表明,用镍离子亲和层析树脂纯化的AZI1重组蛋白对灰霉菌、赤霉菌、棉花枯萎病菌和酿酒酵母细胞的生长/分裂均具有抑制作用。     

过表达Lin28a/Lin28b对let-7家族活性的影响

研究在HeLaS3细胞中过表达Lin28a/Lin28b对let-7家族miRNA表达水平和活性的影响。首先,构建Lin28a和Lin28b的表达载体pAAV2neo-Lin28a和pAAV2neo-Lin28b,分别转染HeLaS3细胞并筛选获得Lin28a和Lin28b的稳定表达细胞株HeLaS3/pAAV2neo-Lin28a和HeLaS3/pAAV2neo-Lin28b。然后,以pAAV2neo-Gluc-(Fluc)为基本骨架,构建并获得检测let-7家族miRNA活性的8种质粒型载体,并包装为相应的重组腺相关病毒(Recombinant adeno-associated virus,rAAV),作为检测miRNA靶序列介导的转录后抑制活性的传感器,命名为Asensor。在此基础上,以HeLaS3细胞为对照,用Western blot检测HeLaS3/pAAV2neo-Lin28a和HeLaS3/pAAV2neo-Lin28b细胞中Lin28a和Lin28b表达水平,用QRT-PCR测定let-7家族各成员表达水平,用Asensor检测let-7家族各成员活性。Western blot结果显示,HeLaS3/pAAV2neo-Lin28a和HeLaS3/pAAV2neo-Lin28b均能有效地表达Lin28a和Lin28b蛋白;QRT-PCR检测结果显示,相比于HeLaS3细胞,HeLaS3/pAAV2neo-Lin28a细胞中let-7家族各成员表达水平都下降(除let-7e外),但不同成员下降幅度存在差异;Asensor检测结果显示,let-7家族所有成员活性水平都下降,但不同成员下降幅度也存在差异,且同一成员活性水平与表达水平及其下降趋势也不一致。相比于HeLaS3细胞,HeLaS3/pAAV2neo-Lin28b细胞中let-7家族成员的表达和活性水平均明显下降,但表达水平的下降幅度比HeLaS3/pAAV2neo-Lin28a细胞大,而活性的下降幅度却与之相近。本研究建立了一种检测和比较miRNA靶序列介导的转录后抑制活性的方法,首次研究了过表达Lin28a和Lin28b对细胞中的let-7家族miRNA活性影响,并发现Lin28a和Lin28b对let-7家族miRNA表达水平的影响和对其相应活性的影响不一致性,说明在检测miRNA表达水平的同时检测miRNA活性能更全面揭示miRNA的调节功能,为进一步研究let-7家族的调控机制奠定了基础。     

沙利度胺通过诱导G1阻滞和凋亡抑制血管内皮细胞增殖

沙利度胺是一种抗血管生成药物,临床上用于治疗多种肿瘤,但其抗肿瘤血管生成机制尚不十分清楚. 本文采用MTT法观察沙利度胺对体外培养的血管内皮细胞增殖的影响. 结果发现,沙利度胺能够抑制血管内皮细胞的增殖,其IC50为16.47 μg/mL;然后采用Hoechst染色和流式细胞仪检测细胞凋亡和细胞周期,发现沙利度胺能够诱导内皮细胞凋亡,并干扰细胞的周期,出现G0/G1期阻滞. 最后,通过Western印迹方法分析沙利度胺对血管内皮细胞Bcl-2蛋白表达的影响,发现抗凋亡的Bcl-2蛋白表达水平随沙利度胺浓度增大而降低. 初步结果提示,沙利度胺可能通过阻遏抗凋亡分子Bcl-2表达,激活诱导G1期阻滞的信号通路而抑制内皮细胞新生,从而抑制肿瘤生长. 诱导内皮细胞凋亡及G1期阻滞的具体分子机制正在研究中.     

巢蛋白基因沉默通过激活细胞周期依赖性激酶（cdk5）促进大鼠神经胶质瘤细胞C6的迁移和增殖

中间纤维蛋白巢蛋白(nestin)在各种胚胎前体细胞及成熟组织中均有表达.近年一些研究显示,巢蛋白的表达上调和一些恶性肿瘤的病理特征有相关性.但是,巢蛋白在干细胞分化及肿瘤发生中的作用还不为人知.在本研究中,我们运用短发卡状的RNA为工具,以大鼠神经胶质瘤细胞系C6为模型,对巢蛋白的功能进行了研究.划痕实验和迁移实验的结果均显示,巢蛋白基因沉默可以促进C6细胞的迁移.同时,BrdU渗入实验显示,此过程伴随着细胞增殖的增加.进一步研究显示,细胞周期依赖性激酶cdk5的活性在此过程中有显著的增加.此外,巢蛋白基因沉默所引起的迁移改变可以被cdk5特异性抑制剂roscovitine所回复, 而对细胞增殖则没有显著影响.综上所述,本研究揭示了巢蛋白基因沉默与神经胶质瘤细胞的迁移和增殖相关,而cdk5是此过程的重要调节因子.     

Janus激酶1/胞外信号调节激酶通路参与C反应蛋白诱导的心肌细胞MMP-10活性上调

为探讨心肌细胞中Janus 激酶1（JAK1）在C反应蛋白（CRP）诱导的基质金属蛋白酶（MMP）-10活性上调过程中的作用,本研究以炎症细胞因子CRP诱导MMP-10上调的原代乳鼠心肌细胞为研究对象,使用JAK1的特异磷酸化抗体,用Western 印迹技术检查JAK1的磷酸化水平的激活情况.应用Western 印迹技术和酶谱法分别检测胞外信号调节激酶（ERK）的磷酸化水平和MMP-10的活性变化.实验分为4组：对照组,CRP组,CRP+抑制剂组和抑制剂组. 结果显示, 磷酸化的JAK1在CRP刺激后1/12 h即可出现,在1 h达到高峰,而总的JAK1不改变;JAK1抑制剂piceatannol可以使磷酸化的ERK水平减弱,也可以使MMP-10的活性明显降低（P <0.01）. 研究结果提示,JAK1是通过激活ERK,从而上调MMP-10的活性,为心力衰竭提供了一个可能的治疗靶点.     

Mycobacterium vaccae as adjuvant therapy to anti-tuberculosis chemotherapy in never-treated tuberculosis patients: a meta-analysis

Objective

To evaluate the effectiveness and safety of heat-killed M.vaccae added to chemotherapy of never-treated tuberculosis (TB) patients.

Methods

The databases of Medline, Embase, Biosis, Cochrane Central Register of Controlled Trials, SCI, CBM, VIP and CNKI were searched. Randomized controlled trials (RCT) and Controlled clinical trials (CCT) comparing M.vaccae with or without a placebo-control injection as adjuvant therapy in the chemotherapy of never-treated TB patients were included. Two reviewers independently performed data extraction and quality assessment. Data were analyzed using RevMan 5.0 software by The Cochrane Collaboration.

Results

Fifty four studies were included. At the end of the follow-up period, Pooled RR (Risk Ratio) and its 95% CI of sputum smear conversion rate were 1.07 (1.04, 1.10) in TB patients without complications, 1.17 (0.92, 1.49) in TB patients with diabetes mellitus, 1.02 (0.94, 1.10) in TB patients with hepatitis B, and 1.46 (0.21, 10.06) in TB patients with pneumosilicosis. In elderly TB patients the RR was 1.22 (1.13, 1.32). Analysis of each time point during the follow-up period showed that M.vaccae could help to improve the removal of acid-fast bacilli from the sputum, and promote improvement of radiological focal lesions and cavity closure. Compared with the control group, the differences in levels of immunological indicators of Th1 such as IL-2 and TNF-α were not statistical significant (P = 0.65 and 0.31 respectively), and neither was that of IL-6 produced by Th2 (P = 0.52). An effect of M.vaccae of prevention of liver damage was found in TB patients with hepatitis B (RR 0.20 and 95% CI (0.12, 0.33). No systemic adverse events were reported.

Conclusion

Added to chemotherapy, M.vaccae is helpful in the treatment of never-treated TB patients in terms of improving both sputum conversion and X-ray appearances.     

Metabolic patterns and biotransformation activities of resveratrol in human glioblastoma cells: relevance with therapeutic efficacies

Background

Trans-resveratrol rather than its biotransformed monosulfate metabolite exerts anti-medulloblastoma effects by suppressing STAT3 activation. Nevertheless, its effects on human glioblastoma cells are variable due to certain unknown reason(s).

Methodology/Principal Findings

Citing resveratrol-sensitive UW228-3 medulloblastoma cell line and primarily cultured rat brain cells/PBCs as controls, the effect of resveratrol on LN-18 human glioblastoma cells and its relevance with metabolic pattern(s), brain-associated sulfotransferase/SULT expression and the statuses of STAT3 signaling and protein inhibitor of activated STAT3 (PIAS3) were elucidated by multiple experimental approaches. Meanwhile, the expression patterns of three SULTs (SULT1A1, 1C2 and 4A1) in human glioblastoma tumors were profiled immunohistochemically. The results revealed that 100 µM resveratrol-treated LN-18 generated the same metabolites as UW228-3 cells, while additional metabolite in molecular weight of 403.0992 in negative ion mode was found in PBCs. Neither growth arrest nor apoptosis was found in resveratrol-treated LN-18 and PBC cells. Upon resveratrol treatment, the levels of SULT1A1, 1C2 and 4A1 expression in LN-18 cells were more up-regulated than that expressed in UW228-3 cells and close to the levels in PBCs. Immunohistochemical staining showed that 42.0%, 27.1% and 19.6% of 149 glioblastoma cases produced similar SULT1A1, 1C2 and 4A1 levels as that of tumor-surrounding tissues. Unlike the situation in UW228-3 cells, STAT3 signaling remained activated and its protein inhibitor PIAS3 was restricted in the cytosol of resveratrol-treated LN-18 cells. No nuclear translocation of STAT3 and PIAS3 was observed in resveratrol-treated PBCs. Treatment with STAT3 chemical inhibitor, AG490, committed majority of LN-18 and UW228-3 cells but not PBCs to apoptosis within 48 hours.

Conclusions/Significance

LN-18 glioblastoma cells are insensitive to resveratrol due to the more inducible brain-associated SULT expression, insufficiency of resveratrol to suppress activated STAT3 signaling and the lack of PIAS3 nuclear translocation. The findings from PBCs suggest that an effective anticancer dose of resveratrol exerts little side effect on normal brain cells.