Formation of Adeno-Associated Virus Circular Genomes Is Differentially Regulated by Adenovirus E4 ORF6 and E2a Gene Expression

A central feature of the adeno-associated virus (AAV) latent life cycle is persistence in the form of both integrated and episomal genomes. However, the molecular processes associated with episomal long-term persistence of AAV genomes are only poorly understood. To investigate these mechanisms, we have utilized a recombinant AAV (rAAV) shuttle vector to identify circular AAV intermediates from transduced HeLa cells and primary fibroblasts. The unique structural features exhibited by these transduction intermediates included circularized monomer and dimer virus genomes in a head-to-tail array, with associated specific base pair alterations in the 5′ viral D sequence. In HeLa cells, the abundance and stability of AAV circular intermediates were augmented by adenovirus expressing the E2a gene product. In the absence of E2a, adenovirus expressing the E4 open reading frame 6 gene product decreased the abundance of AAV circular intermediates, favoring instead the linear replication form monomer (Rf_m) and dimer (Rf_d) structures. In summary, the formation of AAV circular intermediates appears to represent a new pathway for AAV genome conversion, which is consistent with the head-to-tail concatemerization associated with latent-phase persistence of rAAV. A better understanding of this pathway may increase the utility of rAAV vectors for gene therapy.     

Multifunctional roles of a bacteriophage phi 29 morphogenetic factor in assembly and infection

Low copy number proteins within macromolecular complexes, such as viruses, can be critical to biological function while comprising a minimal mass fraction of the complex. The Bacillus subtilis double-stranded DNA bacteriophage phi 29 gene 13 product (gp13), previously undetected in the virion, was identified and localized to the distal tip of the tail knob. Western blots and immuno-electron microscopy detected a few copies of gp13 in phi 29, DNA-free particles, purified tails, and defective particles produced in suppressor-sensitive (sus) mutant sus13(330) infections. Particles assembled in the absence of intact gp13 (sus13(342) and sus13(330)) had the gross morphology of phi 29 but were not infectious. gp13 has predicted structural homology and sequence similarity to the M23 metalloprotease LytM. Poised at the tip of the phi 29 tail knob, gp13 may serve as a plug to help restrain the highly pressurized packaged genome. Also, in this position, gp13 may be the first virion protein to contact the cell wall in infection, acting as a pilot protein to depolymerize the cell wall. gp13 may facilitate juxtaposition of the tail knob onto the cytoplasmic membrane and the triggering of genome injection.     

‘Green tides’ are overwhelming the coastline of our blue planet: taking the world’s largest example

A broad spectrum of events that come under the category of green tide are recognized world-wide as a response to elevated levels of seawater nutrients in coastal areas. Green tides involve a wide diversity of sites, macroalgal species, consequences, and possible causes. Here we review the effect of natural and man-induced environmental fluctuations on the frequency and apparent spread of green tides. This article highlights the need for interdisciplinary research aimed at shedding light on the basic mechanisms governing the occurrence and succession of green algae in coastal seas. This will result in more effective management and mitigation of the effects of green tides, thus safeguarding the intrinsic and commercial value of coastal marine ecosystems.     

Sunitinib Reverse Multidrug Resistance in Gastric Cancer Cells by Modulating Stat3 and Inhibiting P-gp Function

Sunitinib, a small-molecule multi-targeted tyrosine kinase inhibitor, has been applied in phase II clinical trial as second-line treatment for advanced gastric cancer. In this study, we determined the effect of Sunitinib on the multidrug resistance in gastric cancer cells selected by vincristine. Our results showed that Sunitinib significantly enhanced the cytotoxicity of adriamycin, vincristine, etoposide, 5-Fluorouracil, and cisplatin in multidrug-resistant gastric cancer cells (SGC7901/VCR). Sunitinib significantly increased the intracellular accumulation and retention of rhodamine 123 in the SGC7901/VCR cells. However, Sunitinib, at a concentration that reverses MDR, had no significant effect on P-gp protein or mRNA expression levels. In addition, the present study revealed that Sunitinib inhibited Stat3 and down-regulated Bcl-2 in SGC7901/VCR cells, which might also contribute to the reversal of MDR. In conclusion, Sunitinib reverses multidrug resistance in gastric cancer cells by inhibiting P-gp transporter function and modulating Stat3 and Bcl-2. Further study with Sunitinib may be helpful for developing combination therapeutic strategy or circumventing gastric cancer MDR to other conventional anti-cancer drugs.     

Enhanced Biological Hydrogen Production from Escherichia coli with Surface Precipitated Cadmium Sulfide Nanoparticles

The precipitation of cadmium sulfide nanoparticles is induced on the surface of Escherichia coli , and the biological hydrogen production efficiency under visible light (VL) irradiation is investigated. When endogenous [Ni–Fe]‐hydrogenase is anaerobically induced, an additional 400 µmol of hydrogen gas is generated within 3 h from the hybrid system suspension (50 mL) under VL irradiation (2000 W m^?2), corresponding to an increase in hydrogen production of ≈30%. The apparent quantum efficiencies of the hybrid system under 470 and 620 nm VL irradiation are 7.93% and 9.59%, respectively, which are higher than those of many photoheterotrophic bacteria. Furthermore, the mechanism of the enhanced hydrogen evolution is investigated. The interaction between photogenerated electrons and cells of E. coli is confirmed by heat‐treatment, electron‐scavenger, and separation studies. The acceleration of pyruvate generation, inhibition of lactate fermentation, increase of formate concentration, stimulation of hydrogenase activity, and elevation of nicotinamide adenine dinucleotide (NAD)H/NAD ratio in the hybrid system are responsible for the enhanced hydrogen production. A feasibility study is also conducted using wastewater and natural sunlight for the hydrogen production by the hybrid system. An additional 120 µmol of hydrogen is generated from the hybrid system within 3 h under these conditions using natural resources.     

静脉注射41BBLGFP融合表达质粒体内表达及其生物学活性

通过RTPCR方法扩增小鼠41BBLcDNA,以pEGFPN1为载体,构建融合蛋白41BBLGFP重组表达质粒p41BBLGFP.采用基于流体力学原理建立的裸DNA体内转染技术,从小鼠尾静脉快速(15s)注射质粒p41BBLGFP进行体内转染.荧光显微镜观察组织切片,见小鼠肝、脾、肾及肺中均有报告基因GFP表达,尤以肝细胞中荧光最强.进一步用Western印迹和免疫组织化学染色法确定肝细胞表面表达41BBL,用Hsp70H22细胞抗原肽皮下免疫小鼠,同时尾静脉注射质粒p41BBLGFP,检测血清中IL2和IFNγ的分泌.结果显示,质粒注射联合免疫组小鼠血清IL2和IFNγ的浓度分别较生理盐水对照组增加了3倍和4倍;脾细胞对H22细胞的杀伤率则由单独免疫组的45.74%±3.27%增至86.74%±9.36%.结果表明,体内(主要在肝脏)转染质粒p41BBLGFP可以成功表达,表达产物具有41BBL的生物学活性,为进一步研究体内转染41BBL用于基因治疗奠定了基础.     

五唇兰雌配子体发育和胚胎发生的研究

五唇兰的胚珠倒生型,具薄珠心,两层珠被。胚囊发育为双孢子葱型,成熟胚囊８核。从传粉到受精约５０ｄ,正常双受精。胚具５－６细胞的胚柄,种子成熟时胚柄及胚乳核消失,成熟种子只具单层细胞的种皮和一个未分化的珠珠形胚。     

新城疫病毒非编码序列的遗传演化趋势

【目的】探讨新城疫病毒全基因序列中非编码序列的分子演化规律。【方法】结合本研究室2012年自产蛋下降鸭群中分离测序的一株鸭源新城疫病毒全序列,从GenBank下载35株不同基因型新城疫病毒全长cDNA序列,获取非编码序列,分别绘制引导序列、尾随序列、F-HN及HN-L基因间隔序列(IGS)的遗传进化树,比较编码基因内5’及3’UTR序列核苷酸序列替代特点。【结果】非编码序列的长度及位置高度保守,而其核苷酸基因序列在不断发生变异,且变异趋势与编码基因序列相一致。【结论】新城疫病毒在整个基因组上编码和非编码序列同步发生变异。