Genetic Determinants of Symptoms on Viral DNA Satellites

Begomovirus-DNA-β disease complexes induce different symptom phenotypes in their hosts. To investigate the genetic determinants of the phenotypic differences, Nicotiana spp. and tomato plants were inoculated with infectious clones of Tobacco curly shoot virus (TbCSV)/TbCSV DNA-β (TbCSB) and Tomato yellow leaf curl China virus (TYLCCNV)/TYLCCNV DNA-β (TYLCCNB) pseudorecombinants and showed that TYLCCNB induced characteristic vein-thickening and enation symptoms, while TbCSB only slightly exacerbated the leaf-curling symptoms, regardless of the helper virus being used. The roles of DNA-β-encoded βC1 and a 430-nucleotide fragment containing the A-rich region and the putative βC1 promoter region of the βC1 gene (referred to as AP) in symptom development were further investigated by constructing hybrid satellites in which the βC1 coding region or AP was exchanged between the two satellite molecules. A TYLCCNB hybrid with TbCSB βC1 lost the ability to elicit the vein-thickening and enation phenotypes. TbCSB hybrids containing the TYLCCNB βC1 or AP fragment failed to induce the characteristic vein thickening and enations. A TYLCCNB hybrid having the TbCSB AP fragment produced the enations, but the number of enations was less and their sizes were reduced. Differently from the phloem-specific pattern of the TYLCCNB promoter, a full-length fragment upstream of the TbCSB βC1 gene confers a constitutive β-glucuronidase expression pattern in transgenic tobacco plants. The above results indicate that the DNA-β-encoded βC1 protein is the symptom determinant, but the promoter of the βC1 gene has influence on symptom production.Geminiviruses are small plant viruses with circular single-stranded DNA (ssDNA) genomes that are encapsidated in unique twinned (geminate) particles. Members of the genus Begomovirus are transmitted by whiteflies (Bemisia tabaci) and infect dicotyledonous plants (42). Begomoviruses have either one or two circular ssDNA genomic components (DNA-A and DNA-B). The DNA-A component is capable of autonomous replication and encapsidation, whereas the DNA-B component encodes two proteins (BC1 and BV1) involved in movement (14). Recently, some monopartite begomoviruses have been found in association with a novel satellite DNA molecule, referred to as DNA-β and now known as a betasatellite (2, 5, 20, 22, 38, 45). DNA-β is approximately half the size of the viral genomic DNA, and apart from a nonanucleotide sequence (TAATATTAC), it has little sequence identity with viral genomic DNA. DNA-β depends on the helper virus for replication and encapsidation and, in turn, is required for the induction of bona fide disease symptoms. DNA-β bears a βC1 open reading frame (ORF) on the complementary-sense strand, which is conserved among distinct betasatellites in terms of position and size. Mutational analyses and constitutive expression have shown that βC1 is a strong pathogenicity/symptom determinant (7, 34, 39).Begomovirus-DNA-β disease complexes are associated with a wide range of plant species and induce different sets of symptom phenotypes in their natural hosts (25). However, the contributions of the helper virus and the satellite molecule to symptom development are not clear. Tomato yellow leaf curl China virus (TYLCCNV) and Tobacco curly shoot virus (TbCSV) are monopartite begomoviruses associated with DNA-β, but they differ in the symptom phenotypes induced in Nicotiana spp. and Solanum lycopersicum (7, 22). In the present work, we report that the symptom differences between TYLCCNV/TYLCCNV DNA-β (TYLCCNB) and TbCSV/TbCSV DNA-β (TbCSB) are determined by DNA-β and the DNA-β-encoded βC1 protein is the symptom determinant, but the promoter of the βC1 gene has influence on symptom production.     

Structural insights into the catalytic mechanism of aldehyde-deformylating oxygenases

The fatty alk(a/e)ne biosynthesis pathway found in cyanobacteria gained tremendous attention in recent years as a promising alternative approach for biofuel production. Cyanobacterial aldehyde-deformylating oxygenase (cADO), which catalyzes the conversion of Cn fatty aldehyde to its corresponding Cn-1 alk(a/e)ne, is a key enzyme in that pathway. Due to its low activity, alk(a/e)ne production by cADO is an inefficient process. Previous biochemical and structural investigations of cADO have provided some information on its catalytic reaction. However, the details of its catalytic processes remain unclear. Here we report five crystal structures of cADO from the Synechococcus elongates strain PCC7942 in both its iron-free and iron-bound forms, representing different states during its catalytic process. Structural comparisons and functional enzyme assays indicate that Glu144, one of the iron-coordinating residues, plays a vital role in the catalytic reaction of cADO. Moreover, the helix where Glu144 resides exhibits two distinct conformations that correlates with the different binding states of the di-iron center in cADO structures. Therefore, our results provide a structural explanation for the highly labile feature of cADO di-iron center, which we proposed to be related to its low enzymatic activity. On the basis of our structural and biochemical data, a possible catalytic process of cADO was proposed, which could aid the design of cADO with improved activity.     

鄱阳湖水利枢纽工程建设对草鱼江湖洄游潜在的影响

鄱阳湖是长江四大家鱼索饵、育肥的重要场所,近年来鄱阳湖出现了枯水季水位严重降低、枯水期延长、湿地面积缩小的现象。为解决鄱阳湖水资源、水文、水生态等问题,建议在鄱阳湖入江水道兴建控制闸水利枢纽。然而,拟建的水利枢纽工程将打破鄱阳湖与长江的天然连通性,可能会对四大鱼类洄游过程产生影响。通过构建二维和三维水动力模型,分析鄱阳湖水利枢纽建设后入江水道与枢纽洄游通道的水动力学特征,结合实验和文献获得的草鱼幼鱼和成鱼游泳能力参数,阐明了枢纽建设对草鱼洄游的影响。结果表明:在设计调度模式下,草鱼幼鱼入湖期间,湖口段适宜通过天数达到83.74%以上,说明湖口及入江水道的水动力条件对洄游的影响较小,同时,枢纽工程处在过鱼高峰期仍能保持较高的过闸效率;草鱼成鱼出湖期间,丰、平水年闸前水动力条件对洄游的影响较小,仅在枯水年闸前流速几乎静止,草鱼适宜出湖天数偏低。在该调度模式下,水利枢纽建设运行后鄱阳湖整体水动力条件能够满足草鱼洄游需求。目前设计的鱼道在高、低水位时期均出现局部流速过大的现象,不满足过鱼条件。从四大家鱼江湖洄游的角度为鄱阳湖水利枢纽工程设计和运行提供科学参考。     

The diagnostic value of serum DSA-TRF in hepatocellular carcinoma

TRF is a glycoprotein mainly secreted by hepatocytes, The aim of this study was to explore the diagnostic value of aberrant glycosylated serum transferrin (TRF) especially containing multi-antennary glycans in hepatocellular carcinoma (HCC).A total of 581 subjects including HCC patients, liver cirrhosis (LC) patients, chronic hepatitis (CHB) patients and healthy controls (HC) were recruited. All the subjects were randomly assigned to training group (n?=?411) and validation group (n?=?170). We firstly analyzed the serum protein N-glycome profiling of HCC, LC, and HC by DNA sequencer–assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) technology. We established a lectin-antibody sandwich ELISA (Lectin-ELISA) method to detect multi-antennary glycans-contained TRF (DSA-TRF) in serum, in which Datura stramonium Agglutinin (DSA) was used for specific recognition. Levels of serum DSA-TRF and TRF were analyzed respectively. The diagnostic efficacies of DSA-TRF and TRF of differentiating HCC patients from CHB, LC patients and HC within the training group were evaluated using receiver operating characteristic (ROC) curve and tested in the validation group.The result found that in training group, serum TRF and DSA-TRF levels differed significantly between HCC (1.86?±?0.50, g/L, 0.285?±?0.06), CHB?+?LC (2.39?±?0.74, g/L, 0.189?±?0.07) and HC (1.92?±?0.69, g/L, 0.249?±?0.09) (HCC vs. CHB?+?LC, P?<?0.001; HCC vs. HC, P?<?0.001; CHB?+?LC vs. HC, P?<?0.001). The area under the ROC curve (AUC) of DSA-TRF was significantly superior to AFP (0.880, 95%CI: 0.834–0.925 vs. 0.776, 95%CI: 0.725–0.827, P?=?0.003) in differentiating HCC from CHB?+?LC. The AUC of diagnostic model GlycoTRF1 (0.981, 95%CI: 0.969–0.993) was higher than DSA-TRF and AFP alone (P<0.001) in differentiating HCC from CHB?+?LC, which was verified in validation group.The results indicated that the serum DSA-TRF might serve as a potential glycan biomarker for distinguishing HCC from CHB and LC.

     

A novel recombinant immunotoxin with the smallest ribosome‐inactivating protein Luffin P1: T‐cell cytotoxicity and prolongation of allograft survival

In the creation of stable tolerance to MHC‐incompatible allografts, reducing the large mass of donor‐reactive cells via apoptosis is often required. Apoptosis induction by immunotoxins targeting surface molecules specifically presented on donor‐reactive cytopathic T effector (T_eff) cells is a promising strategy. Traditionally, the toxin moieties are bacterial exotoxins or plant‐derived ribosome‐inactivating proteins (RIPs) with large molecular size and strong immunogenicity, hence causing the problems of tissue penetration, host immune reaction and quick clearance. We have identified a novel class of small molecule RIPs (<10 kD) from the seeds of the plant Luffa cylindrica. The smallest member of this family, Luffin P1, has a molecular weight of 5226.8 Da, yet possessing a highly potent inhibitory activity on cell‐free protein synthesis with IC50 of 0.88 nM. We now report a recombinant hIL‐2‐Luffin P1 immunotoxin, which strongly inhibited T‐cell proliferation in mixed lymphocyte reaction and ConA response with IC50 of 1.8–10 nM. In vivo, hIL‐2‐Luffin P1 significantly prolonged the survival of major MHC‐mismatched skin and kidney allografts in animal models. Thus, we demonstrate for the first time the efficacy of the smallest immunotoxin that could be further combined with other pharmacological and immunological reagents for synergistic control of pathogenic lymphocytes in immune‐mediated diseases.     

中国基因技术领域战略规划框架与研发现状分析及建议

文中简要介绍中国政府和中国科学院在基因技术研究领域的科技战略框架,以及在此指导下,中国研究人员取得的卓越进展,并通过文献计量和专利分析的方法揭示中国基因技术研发现状。无论在论文数量和质量,还是专利申请数量方面,中国都有了显著提升,但在国际合作和产学研结合方面仍有待加强。未来中国还需要抓好顶层设计,加强政府引导和监管,引入企业和社会的多方投资,加大科普宣传力度,预防生物安全和生物安保风险等。基因技术领域的创新和突破将为现代化产业的可持续发展提供主要的技术推动力,为中国生物经济发展注入新的活力。     

结核分枝杆菌Rv1168c蛋白的基因表达、纯化及结构分析

【目的】应用原核表达体系对结核分枝杆菌PPE蛋白家族Rv1168c进行高效表达,进一步进行蛋白纯化和结构分析。【方法】以结核分枝杆菌H37Rv基因组为模板,扩增Rv1168c基因,构建pET32a-Rv1168c重组质粒;转化重组质粒到大肠杆菌DH5α并在BL21(DE3)诱导表达,通过十二烷基硫酸钠-聚丙烯酰胺电泳(SDS-PAGE)鉴定Rv1168c在大肠杆菌中的表达情况;Ni-NTAHis﹡Bind Resin纯化重组蛋白Rv1168c;SDS-PAGE和质谱分析测定相对分子量后,用圆二色光谱(CD)和同源模建方法分析和检测重组蛋白Rv1168c的二级和三级结构。【结果】成功克隆了971bp的目的基因Rv1168c,并获得了高纯度的重组蛋白Rv1168c。重组蛋白的分子量为51.5kDa(含载体蛋白)。25℃时重组蛋白Rv1168c的二级结构包括34.4%α螺旋,33.7%β转角,31.9%无规则卷曲,它的三维模型显示为(β/α)5结构。【结论】成功得到高纯度的重组目的Rv1168c蛋白,并初步进行了结构分析,为进一步对Rv1168c结构和功能研究奠定了基础。     

Exosomal Transfer of Vasorin Expressed in Hepatocellular Carcinoma Cells Promotes Migration of Human Umbilical Vein Endothelial Cells

Vasorin (VASN) is a type I transmembrane protein that plays important roles in tumor development and vasculogenesis. In this paper, we showed that VASN could be a key mediator of communication between tumor cells and endothelial cells. We confirmed for the first time that HepG2-derived VASN can be transferred to human umbilical vein endothelial cells (HUVECs) via receptor mediated endocytosis of exosomes, at least in part through HSPGs. The HepG2-derived VASN containing exosomes promote migration of recipient HUVECs cells. Our results identify a novel pathway by which a functional protein expressed in tumor cells affects the biological fate of endothelial cells via exosomes.