共表达H5N1流感病毒M1和HA基因的DNA疫苗与腺病毒载体疫苗的小鼠免疫评价

为评价在小鼠体内表达流感病毒M1和HA基因诱导的免疫反应,制备共表达H5N1亚型禽流感病毒 (A/Anhui/1/2005) 全长基质蛋白1 (M1) 基因和血凝素 (HA) 基因的重组DNA疫苗pStar-M1/HA和重组腺病毒载体疫苗Ad-M1/HA,将其按初免-加强程序免疫BALB/c小鼠,共免疫4次,每次间隔14 d。第1、3次用DNA疫苗,第2、4次用重组腺病毒载体疫苗,每次免疫前及末次免疫后14 d采集小鼠血清用于检测体液免疫应答,末次免疫后14 d采集小鼠脾淋巴细胞用于检测细胞免疫应答。血凝     

Synthesis, biological evaluation, and molecular modeling of cinnamic acyl sulfonamide derivatives as novel antitubulin agents

A series of novel cinnamic acyl sulfonamide derivatives (9a-16e) have been designed and synthesized and their biological activities were also evaluated as potential tubulin polymerization inhibitors. Among all the compounds, 10c showed the most potent growth inhibitory activity against B16-F10 cancer cell line in vitro, with an IC(50) value of 0.8μg/mL. Docking simulation was performed to insert compound 10c into the crystal structure of tubulin at colchicine binding site to determine the probable binding model. Based on the preliminary results, compound 10c with potent inhibitory activity in tumor growth may be a potential anticancer agent.     

基于叶片尺度观测数据的气孔导度模型评价

气孔导度(g)是控制冠层与大气之间能量和水分交换的重要因素。空气湿度是控制植物叶片气孔导度的一个关键环境因子。在过去的十几年中,普遍得到应用的是Ball-Woodrow-Berry(BWB)模型和Leuning模型中气孔导度与湿度的关系。本研究使用一个诊断变量f(H),基于农田叶片水平的光合-气孔导度观测数据,对BWB模型、Leuning模型以及新发展的power-h模型和power-D模型进行了气孔导度模拟效果的比较和评价。结果表明:BWB模型描述的是g和相对湿度(hs)之间的一种线性关系,当空气较为湿润时,模拟结果存在较大的低估;Leuning模型中反映的是g与饱和水汽压差(Ds)的非线性函数,降低了模拟结果的误差,但仍然不能很好地描述g在较湿状况下的显著升高;相比之下,两个新的模型,即Ds的指数函数和(1-hs)的指数函数形式模型能提高模拟结果的精度。这个研究结果也表明基于Ds的模型模拟效果要好于基于hs的模型。     

Enhancement of excretory production of an exoglucanase from Escherichia coli with phage shock protein A (PspA) overexpression

Production of recombinant proteins by excretory expression has many advantages over intracellular expression in Escherichia coli. Hyperexpression of a secretory exoglucanase, Exg, of Cellulomonas fimi was previously shown to saturate the SecYEG pathway and result in dramatic cell death of E. coli. In this study, we demonstrated that overexpression of the PspA in the JM101(pM1VegGcexL-pspA) strain enhanced excretion of Exg to 1.65 U/ml using shake-flask cultivation, which was 80% higher than the highest yield previously obtained from the optimized JM101(pM1VegGcexL) strain. A much higher excreted Exg activity of 4.5 U/ml was further achieved with high cell density cultivation using rich media. Furthermore, we showed that the PspA overexpression strain enjoyed an elevated critical value (CV), which was defined as the largest quotient between the intracellular unprocessed precursor and its secreted mature counterpart that was still tolerable by the host cells prior to the onset of cell death, improving from the previously determined CV of 20/80 to the currently achieved CV of 45/55 for Exg. The results suggested that the PspA overexpression strain might tolerate a higher level of precursor Exg making use of the SecYEG pathway for secretion. The reduced lethal effect might be attributable to the overexpressed PspA, which was postulated to be able to reduce membrane depolarization and damage. Our findings introduce a novel strategy of the combined application of metabolic engineering and construct optimization to the attainment of the best possible E. coli producers for secretory/excretory production of recombinant proteins, using Exg as the model protein.     

Time-dependent elastohydrodynamic lubrication analysis of total knee replacement under walking conditions

This work is concerned with the lubrication analysis of artificial knee joints, which plays an increasing significant role in clinical performance and longevity of components. Time-dependent elastohydrodynamic lubrication analysis for normal total knee replacement is carried out under the cyclic variation in both load and speed representative of normal walking. An equivalent ellipsoid-on-plane model is adopted to represent an actual artificial knee. A full numerical method is developed to simultaneously solve the Reynolds and elasticity equations using the multigrid technique. The elastic deformation is based on the constrained column model. Results show that, under the combined effect of entraining and squeeze-film actions throughout the walking cycle, the predicted central film thickness tends to decrease in the stance phase but keeps a relatively larger value at the swing phase. Furthermore, the geometry of knee joint implant is verified to play an important role under its lubrication condition, and the length of time period is a key point to influence the lubrication performance of joint components.     

Recombineering BAC transgenes for protein tagging

Protein tagging offers many advantages for proteomic and regulomic research, particularly due to the use of generic and highly sensitive methods that can be applied with reasonable throughput. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. However, these advantages are compromised if the tagged protein is overexpressed, which is usually the case from cDNA expression vectors. BAC (bacterial artificial chromosome) transgenes present a way to express a chosen protein at physiological levels with all regulatory elements in their native configurations, including cell cycle, alternative splicing and microRNA regulation. Recombineering has become the method of choice for modifying large constructs like BACs. Here, we present a method for protein tagging by recombineering BACs, transfecting cells and evaluating tagged protein expression.     

Macrophage migration inhibitory factor induced by dengue virus infection increases vascular permeability

Dengue virus (DENV) infection can cause mild dengue fever or severe dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). Serum levels of the macrophage migration inhibitory factor (MIF) have been shown to be correlated with severity and mortality in DENV patients, but the pathogenic roles of MIF in DHF/DSS are still unclear. Increase in vascular permeability is an important hallmark of DHF/DSS. In this study, we found that DENV infection of the human hepatoma cell line (Huh 7) induced MIF production. Conditioned medium collected from DENV-infected Huh 7 cells enhanced the permeability of the human endothelial cell line (HMEC-1) which was reduced in the presence of a MIF inhibitor, ISO-1 or medium from DENV-infected MIF knockdown Huh 7 cells. To further identify whether MIF can alter vascular permeability, we cloned and expressed both human and murine recombinant MIF (rMIF) and tested their effects on vascular permeability both in vitro and in vivo. Indirect immunofluorescent staining showed that the tight junction protein ZO-1 of HMEC-1 was disarrayed in the presence of rMIF and partially recovered when cells were treated with ISO-1 or PI3K/MEK-ERK/JNK signaling pathway inhibitors such as Ly294002, U0126, and SP600215. In addition, ZO-1 disarray induced by MIF was also recovered when CD74 or CXCR2/4 expression of HMEC-1 were inhibited. Last but not least, the vascular permeabilities of the peritoneal cavity and dorsal cutaneous capillary were also increased in mice treated with rMIF. Taken together; these results suggest that MIF induced by DENV infection may contribute to the increase of vascular permeability during DHF/DSS. Therapeutic intervention of MIF by its inhibitor or neutralizing antibodies may prevent DENV-induced lethality.     

Genetic modification of alternative respiration in Nicotiana benthamiana affects basal and salicylic acid-induced resistance to potato virus X

Background  

Salicylic acid (SA) regulates multiple anti-viral mechanisms, including mechanism(s) that may be negatively regulated by the mitochondrial enzyme, alternative oxidase (AOX), the sole component of the alternative respiratory pathway. However, studies of this mechanism can be confounded by SA-mediated induction of RNA-dependent RNA polymerase 1, a component of the antiviral RNA silencing pathway. We made transgenic Nicotiana benthamiana plants in which alternative respiratory pathway capacity was either increased by constitutive expression of AOX, or decreased by expression of a dominant-negative mutant protein (AOX-E). N. benthamiana was used because it is a natural mutant that does not express a functional RNA-dependent RNA polymerase 1.