空心莲子草性可塑性影响下的克隆整合

植物的生活史由其有性生殖构件和营养体构件相互作用共同完成,克隆整合作为克隆植物的重要特征,其与有性生殖特征的相互作用关系却所知很少。该研究通过同质园种植实验,分析了空心莲子草的分株表型、生理、性别等与克隆整合的关系。结果表明:(1)克隆整合以及分株间是否连接对空心莲子草的表型特征、气体交换等生理性状和性别特征均有显著影响。(2)克隆整合显著缩小了雌雄同花和雄蕊心皮化两种性别植株间表型特征的差距,后代的性别特征与营养体表型特征显著相关。(3)在贫瘠的沙土基质中克隆整合明显增加了空心莲子草的营养体生长特征和气体交换等光合生理指标,但这种增加在富含有机质的塘泥基质中不明显。(4)居于不同土壤基质分株间的联系会减少分株表型特征和气体交换对生长环境的响应,并保持母体性别特征不受环境的影响,但单独居于沙土或塘泥单一土壤基质的分株性别特征却因受到环境影响而改变。因此,克隆整合有利于空心莲子草性别特征的稳定。     

Sonic hedgehog signaling regulates Gli3 processing, mesenchymal proliferation, and differentiation during mouse lung organogenesis

Lack of Sonic hedgehog (Shh) signaling, mediated by the Gli proteins, leads to severe pulmonary hypoplasia. However, the precise role of Gli genes in lung development is not well established. We show Shh signaling prevents Gli3 proteolysis to generate its repressor forms (Gli3R) in the developing murine lung. In Shh(-/-) or cyclopamine-treated wild-type (WT) lung, we found that Gli3R level is elevated, and this upregulation appears to contribute to defects in proliferation and differentiation observed in the Shh(-/-) mesenchyme, where Gli3 is normally expressed. In agreement, we found Shh(-/-);Gli3(-/-) lungs exhibit enhanced growth potential. Vasculogenesis is also enhanced; in contrast, bronchial myogenesis remains absent in Shh(-/-);Gli3(-/-) compared with Shh(-/-) lungs. Genes upregulated in Shh(-/-);Gli3(-/-) relative to Shh(-/-) lung include Wnt2 and, surprisingly, Foxf1 whose expression has been reported to be Shh-dependent. Cyclins D1, D2, and D3 antibody labelings also reveal distinct expression patterns in the normal and mutant lungs. We found significant repression of Tbx2 and Tbx3, both linked to inhibition of cellular senescence, in Shh(-/-) and partial derepression in Shh(-/-); Gli3(-/-) lungs, while Tbx4 and Tbx5 expressions are less affected in the mutants. Our findings shed light on the role of Shh signaling on Gli3 processing in lung growth and differentiation by regulating several critical genes.     

Multiple strategies to improve sensitivity,speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection

Background  

In the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for the applications in rapid pathogen detection, especially at point-of-care diagnostics. Here, we describe and explore several strategies to improve one of the isothermal technologies, helicase-dependent amplification (HDA).     

整合REV LTR序列的MDV弱毒株的分离鉴定与生物学特性

【目的】从非免疫健康三黄鸡中分离到一株马立克氏病病毒(MDV),命名为MDV GD06株,本工作系统研究了其生物学特性。【方法】PCR扩增GD06株Meq基因及短末端重复区(RS)序列,进行序列分析,并用间接免疫荧光试验进行血清型特异性鉴定;通过接种SPF鸡和鸡胚成纤维细胞(CEF)来判定GD06株体内外的增殖能力;为确定GD06株的致病性,用1日龄SPF鸡进行攻毒试验。【结果】GD06株为MDV血清I型病毒,其基因组中自然整合禽网状内皮增殖症病毒(REV)的长末端重复序列(LTR),Meq基因富含脯氨酸的结构域比参考强毒株Md5多59个氨基酸,与MD商品化疫苗CVI988/Rispens和814株相符,具有弱毒株的特征。在接种CEF细胞96、120、144、168、192 h,GD06株病毒滴度分别为1.9×105、3.9×105、6.1×105、6.5×105、5.8×105PFU,明显比CVI988/Rispens(病毒滴度分别为1.3×105、3.5×105、5.0×105、5.7×105、4.7×105PFU)高(P0.05);接种SPF鸡21、28天,GD06株在鸡体内的病毒滴度分别为740和350 PFU,明显比CVI988/Rispens株(病毒滴度分别为460、216 PFU)高(P0.05),结果表明,GD06株在CEF细胞和鸡体内具有比CVI988/Rispens更快的增殖能力。人工接种攻毒实验表明,GD06株对SPF鸡没有致病性,不引免疫抑制。【结论】研究结果表明,MDV GD06株为国内首次分离的自然整合有REV LTR序列的重组MDV弱毒株。     

蜗牛酶对原生质体游离的影响

用10科11种双子叶植物的营养器官,在含1mg/L 2,4—D的MS固体培养基上诱导形成愈伤组织。取其生长旺盛的区域进行原生质体游离,并采用未经处理的相应植物的新鲜叶或茎作对照。研究结果表明,蜗牛酶对愈伤组织的原生质体游离具有明显的促进作用,而对照却无此效应。还研究了两种植物愈伤组织的继代天数和不同的酶组合对原生质体游离的影响。     

Engineered Pentafunctional Minicellulosome for Simultaneous Saccharification and Ethanol Fermentation in Saccharomyces cerevisiae

Several yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and β-glucosidases in Saccharomyces cerevisiae. These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast.     

宏基因组文库中的组成型启动子在大肠杆菌中的克隆

启动子是决定基因表达水平的重要因素之一。组成型表达启动子被认为是工业上表达重要蛋白质的理想启动子。本研究利用蔗糖为唯一碳源的基本培养基对榨糖废水浸润的土壤微生物宏基因组文库进行筛选,获得两个阳性克隆。对其中一个克隆的柯斯质粒进行亚克隆,利用在线启动子预测和序列比对工具对其中一个亚克隆子进行分析,获得一个启动子序列。然后,利用PCR方法将该启动子和地衣芽孢杆菌的α-淀粉酶基因一起克隆到T载体上。结果表明该启动子在不加诱导剂的条件下能够在大肠杆菌中启动外源基因的高效表达。本研究结果为在生物领域中组成型启动子的应用研究提供了基础。     

Cellular repressor of E1A-stimulated genes regulates vascular endothelial cell migration by the ILK/AKT/mTOR/VEGF(165) signaling pathway

The migration of vascular endothelial cells plays a critical role in a variety of vascular physiological and pathological processes, such as embryonic development, angiogenesis, wound healing, re-endothelialization, and vascular remodeling. This study clarified the role and mechanism of a new vascular homeostasis regulator, Cellular repressor of E1A-stimulated genes (CREG), in the migration of primary human umbilical vein endothelial cells (HUVECs). A wound healing assay and transwell migration model showed that upregulation of CREG expression induced HUVEC migration and it was positively correlated with the expression of vascular endothelial growth factor. Furthermore, wild type integrin-linked kinase reversed the poor mobility of CREG knock-down HUVECs; in contrast, kinase-dead integrin-linked kinase weakened the migration of HUVECs. We also studied the effect of CREG on HUVEC migration by the addition of an mTOR inhibitor, recombinant vascular endothelial growth factor₁₆₅, neutralizing antibody of vascular endothelial growth factor₁₆₅ and AKT siRNA, and we concluded that CREG induces endothelial cell migration by activating the integrin-linked kinase/AKT/mTOR/VEGF₁₆₅ signaling pathway.     

The enhancement of radiosensitivity in human esophageal squamous cell carcinoma cells by zoledronic acid and its potential mechanism

Esophageal squamous cell carcinoma (ESCC) has a low 5-year patient survival rate. Radiotherapy, as a preoperative or postoperative treatment of surgery, has a crucial role in improving local control and survival of ESCC. Various chemotherapeutic and biologic agents have been used as radio-sensitizers in combination with radiotherapy. Here, we demonstrate that zoledronic acid (ZOL) has a radio-sensitizing effect on ESCC cells. Exposure of ESCC cancer cells to ZOL plus radiation resulted in increased cell death through arresting the cell cycle between S and G2/M phases. ZOL appeared to inhibit proliferation, tube formation and invasion of endothelial cells. These anti-angiogenetic effects were more marked concurrently with irradiation. In addition, synergistic suppressive effects on VEGF expression were observed after combined treatment. Our data suggest that the combination of ZOL and radiation is a promising therapeutic strategy to enhance radiation therapy for ESCC patients.     

病毒功能性受体的筛选新策略及其应用

病毒通过与靶细胞表面的特异性受体结合,进而吸附、入侵靶细胞,劫持细胞的复制机器完成自身的复制周期。细胞受体作为病毒入侵宿主的门户,细胞受体的结构与功能及其介导病毒入侵的机制一直是病毒学研究的热点之一。对细胞受体的研究有助于了解病毒的致病机制并为病毒性疾病的防控提供科学依据。近年来,利用功能缺失性基因组学筛选病毒功能性受体的进展极大地拓展了人类对病毒入侵机制的认识和理解。目前,应用较为广泛的病毒功能性受体筛选策略包括RNA干扰技术、利用单倍体细胞系随机插入逆转录病毒致突变技术以及基于CRISPR/Cas9系统的新型编辑技术。本文系统介绍并比较了这三种病毒功能性受体筛选新策略,同时对其应用及优缺点进行了归纳总结,可为相关研究者提供一定的参考。