比较不同血清型AAV携带HBV基因组建立乙肝小鼠模型效果

近年来,用8型腺相关病毒携带1.3拷贝HBV(Hepatitis B virus)基因组建立的HBV持续感染小鼠模型受到越来越多的关注。本研究比较了除AAV8之外的其他4种血清型重组腺相关病毒(Recombinant adeno-associated virus,rAAV)建立乙肝小鼠模型效果。首先,将携带1.3拷贝ayw亚型HBV基因组的1型、2型、5型、8型、9型腺相关病毒分别以1×10~(11) vg/只(Viral genome,vg)的剂量尾静脉注射C57BL/6J小鼠;利用ELISA方法监测小鼠血清中HBeAg和HBsAg表达水平;用定量PCR方法检测小鼠血清和肝脏中HBV DNA拷贝数;用免疫组化方法检测小鼠肝脏中HBc Ag的表达;用HE染色检测小鼠肝脏病理变化。结果显示,在持续8周中,5组小鼠血清中都检测到HBeAg和HBsAg的表达,血清和肝脏中均检测到HBV DNA的存在。HBeAg、HBsAg、HBV DNA表达水平高低依次为AAV8AAV9AAV1AAV5AAV2。5组小鼠用免疫组化方法都检测到肝脏中HBcAg表达,HE染色病理检测均观察到不同程度的肝损伤。本研究扩大了能用于建立乙肝小鼠持续感染模型可选择的AAV载体种类,发现虽然AAV1、2、5、9的建模效果不如AAV8,但它们都可以介导建立持续感染的乙肝小鼠模型,建模效果依次为AAV8AAV9AAV1AAV5AAV2。其中AAV9介导的建模效果与AAV8载体最为接近,可以替代AAV8载体用于有效地建立HBV持续感染的小鼠模型。     

MET18 Connects the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Active DNA Demethylation in Arabidopsis

DNA demethylation mediated by the DNA glycosylase ROS1 helps determine genomic DNA methylation patterns and protects active genes from being silenced. However, little is known about the mechanism of regulation of ROS1 enzymatic activity. Using a forward genetic screen, we identified an anti-silencing (ASI) factor, ASI3, the dysfunction of which causes transgene promoter hyper-methylation and silencing. Map-based cloning identified ASI3 as MET18, a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Affinity purification followed by mass spectrometry indicated that ROS1 physically associates with MET18 and other CIA components. Yeast two-hybrid and split luciferase assays showed that ROS1 can directly interact with MET18 and another CIA component, AE7. Site-directed mutagenesis of ROS1 indicated that the conserved iron-sulfur motif is indispensable for ROS1 enzymatic activity. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation.     

哈尔滨黄土炭屑记录的中晚更新世以来古火活动及其驱动机制

炭屑是探索古火活动和人类用火及其驱动机制的重要指标,为研究古环境和古气候的演变提供了一种新的途径。我国东北地区的炭屑研究较为薄弱,仅有的少数研究集中在全新世,缺少更长时间的沉积记录。为此,我们对哈尔滨黄土炭屑和总有机碳(TOC)进行分析,以重建中晚更新世以来松嫩平原东部的古火活动与古植被-古气候间的关系,并进一步探讨古火活动的驱动机制。结果表明,哈尔滨黄土炭屑形态揭示研究区主要以木本植物为主,炭屑粒度特征记录本地区为区域性古火事件。哈尔滨黄土-古土壤炭屑浓度曲线与TOC曲线比较吻合,古土壤炭屑浓度高,在弱古土壤层L1S1呈现出逐渐增加的趋势,表明温度的上升和生物量增多导致古火活动较为增强,揭示出古火活动主要受控于生物量。通过与Rb/Sr比值、全球CO_2S浓度以及深海氧同位素的对比,发现古火还受温度条件的限制。温度作为触发因素,对植物的生长和古火的发生起到了积极的作用,即温度影响木本植物生长,较高的温度导致更密集的蒸发,较低的温度会导致较高的有效湿度,从而对火灾的发生和植物的生长产生影响,这也指示了古火-植被-气候之间的复杂性。     

Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA‐directed DNA methylation

Argonaute (AGO) family proteins are conserved key components of small RNA‐induced silencing pathways. In the RNA‐directed DNA methylation (RdDM) pathway in Arabidopsis, AGO6 is generally considered to be redundant with AGO4. In this report, our comprehensive, genomewide analyses of AGO4‐ and AGO6‐dependent DNA methylation revealed that redundancy is unexpectedly negligible in the genetic interactions between AGO4 and AGO6. Immunofluorescence revealed that AGO4 and AGO6 differ in their subnuclear co‐localization with RNA polymerases required for RdDM. Pol II and AGO6 are absent from perinucleolar foci, where Pol V and AGO4 are co‐localized. In the nucleoplasm, AGO4 displays a strong co‐localization with Pol II, whereas AGO6 co‐localizes with Pol V. These patterns suggest that RdDM is mediated by distinct, spatially regulated combinations of AGO proteins and RNA polymerases. Consistently, Pol II physically interacts with AGO4 but not AGO6, and the levels of Pol V‐dependent scaffold RNAs and Pol V chromatin occupancy are strongly correlated with AGO6 but not AGO4. Our results suggest that AGO4 and AGO6 mainly act sequentially in mediating small RNA‐directed DNA methylation.     

H1亚型猪流感病毒血凝素基因在昆虫细胞中的表达及其间接

根据GenBank发表的H1亚型猪流感HA基因序列设计引物,扩增出HA基因片段.将其克隆到pFastBacGP67B杆状病毒载体上,筛选阳性重组转座载体pFastBacGP67B-H1,转化含有杆状病毒穿梭载体(bacmid)的DH10Bac感受态细胞,构建杆状病毒表达载体获得重组转座子(rBacmid-H1),在脂质体介导下转染sf9昆虫细胞,获得重组杆状病毒(rBV-H1),再感染细胞,收获目的蛋白.通过血凝试验、免疫印迹法、免疫组化分析表明该蛋白得到表达,且具有良好的生物学活性.利用表达的蛋白作为猪流感间接ELISA的抗原,初步建立H1亚型猪流感的间接ELISA检测方法,并对内蒙古、辽宁和黑龙江等地送检的93份猪血清进行了检测,阳性率为31.18%,为研制开发快速、准确、简便的H1亚型猪流感鉴别诊断试剂盒奠定基础.     

Characteristics of cytosine methylation status and methyltransferase genes in the early development stage of cauliflower (Brassica oleracea L. var. botrytis)

DNA methylation is one of the most important epigenetic modifications involved in the development and differentiation in plants. Hypocotyl and cotyledon are the two major tissues of cauliflower (Brassica oleracea L. var. botrytis) seedlings. Both tissues show significantly different tissue specificity and regenerative abilities in vitro. However, the characteristics of DNA methylation modification and its roles in regulating the organ development in cauliflower remain largely unknown. In the present study, the DNA methylation status between the hypocotyl and cotyledon of cauliflower seedlings were analyzed. The results indicated that although the hypocotyl and cotyledon of cauliflower seedlings share the same genome, the genomic DNA methylation levels and patterns at CCGG sites were different. Compared with the cotyledon, the hypocotyl showed higher DNA methylation level, and more loci showing methylation pattern adjustments were also discovered. Twelve loci with changes of DNA methylation patterns were further explored. The quantitative expression analysis indicated that eight out of twelve sequenced fragments showed differential expression between the hypocotyl and cotyledon, of which the expression of six sequences was identified to be negative correlation with their DNA methylation status. In addition, three main DNA methyltransferase genes MET1, CMT3 and DRM were first explored in cauliflower. The results indicated that the expression of these three genes was closely associated with the different DNA methylation status in the hypocotyl and cotyledon. These findings provided more information to further explore the roles of DNA methylation modification in tissue differentiation and development of cauliflower.     

Proteomics reveals different pathological processes of adipose tissue,liver, and skeletal muscle under insulin resistance

Type 2 diabetes mellitus is the most common type of diabetes, and insulin resistance (IR) is its core pathological mechanism. Proteomics is an ingenious and promising Omics technology that can comprehensively describe the global protein expression profiling of body or specific tissue, and is widely applied to the study of molecular mechanisms of diseases. In this paper, we focused on insulin target organs: adipose tissue, liver, and skeletal muscle, and analyzed the different pathological processes of IR in these three tissues based on proteomics research. By literature studies, we proposed that the main pathological processes of IR among target organs were diverse, which showed unique characteristics and focuses. We further summarized the differential proteins in target organs which were verified to be related to IR, and discussed the proteins that may play key roles in the emphasized pathological processes, aiming at discovering potentially specific differential proteins of IR, and providing new ideas for pathological mechanism research of IR.