Adenovirus-mediated p53 gene transfer sensitizes hepatocellular carcinoma cells to heavy-ion radiation

Background. The purpose of this study was to investigate whether adenovirus-mediated p53 transfer could sensitize hepatocellular carcinoma to heavy-ion irradiation. Methods. HepG2 cells were preexposed to a C-12(6+) beam, and then infected with replication-deficient adenovirus recombinant vectors containing human wild-type p53 (AdCMV-p53) (C-12(6+) irradiation + AdCMV-p53 infection). The survival fraction was determined by clonogenic assay. The cell cycle, cell apoptosis, and p53 expression were monitored by flow cytometric analysis. Results. p53 expression in C-12(6+) irradiation + AdCMV-p53 infection groups was markedly higher than that in C-12(6+) irradiation only groups (P < 0.05), suggesting that the preexposure to the C-12(6+) beam promoted the expression of exogenous p53 in HepG2 cells infected with AdCMV-p53 only. The G(1)-phase arrest and cell apoptosis in the C-12(6+) irradiation + AdCMV-p53 infection groups were significantly more than those in the C-12(6+) irradiated groups (P < 0.05). The survival fractions of the C-12(6+) irradiation + AdCMV-p53 infection groups decreased by 30%-49% compared with those of the C-12(6+) beam-irradiated only groups (P < 0.05). Conclusions. Adenovirus-mediated p53 gene transfer can promote G(1)-phase arrest and cell apoptosis, thus sensitizing hepatocellular carcinoma cells to heavy-ion irradiation.

High-efficiency transfer and expression of AdCMV-p53 in human hepatocellular carcinoma cells induced by low-dose carbon-ion radiation

Objective To investigate whether the irradiation with C-beam could enhance adenovirus-mediated transfer and expression of p53 in human hepatocellular carcinoma. Materials and methods HepG2 cells were exposed to C-beam or gamma-ray and then infected with replicationdeficient adenovirus recombinant vectors containing human wild-type p53 or green fluorescent protein, respectively. The transfer efficiency and expression level of the exogenous gene were detected by flow cytometric analysis. Cell survival fraction was detected by clonogenic assay. Results The transfer frequency in C-beam or gamma-irradiated groups increased by 50-83% and 5.7-38.0% compared with the control, respectively (P < 0.05). Compared with C-beam alone, p53 alone, and gamma-ray with p53, the percentages of p53 positive cells for 1 Gy C-beam with p53 increased by 56.0-72.0%, 63.5-82.0%, and 31.3-72.5% on first and third day after the treatments, respectively (P < 0.05). The survival fractions for the 2Gy C-bearn and AdCMV-p53 infection groups decreased to similar to 2%. Conclusion C-beam irradiation could significantly promote AdCMV-green fluorescent protein transfer and expression of p53.

萤火虫的荧光素酶基因克隆表达及发光行为的初步研究

本研究围绕萤火虫的荧光素酶克隆表达和发光行为两方面来进行。首先对萤火虫的发光体系和发光行为进行了概述，然后对萤火虫荧光素酶cDNA进行了克隆表达分析，探讨了日行性和夜行性萤火虫荧光素酶的差异。最后通过萤火虫的饲养和发光行为观察研究，对卵发光的意义进行了探讨。本研究克隆了三种云南萤火虫的荧光素酶基因，并成功表达出两种有活性的荧光素酶。其中日行性萤火虫云南窗萤(Pyrocoelia pygidialis)的荧光素酶基因完成了首次克隆，并在大肠杆菌中成功表达出有功能的荧光素酶。测序结果表明：P. pygidialis荧光素酶的cDNA序列有1647个碱基，编码548个氨基酸残基。系统发育分析表明云南窗萤与同属三种发光很亮的萤火虫不形成单系，在一定程度上印证了P. pygidialis以及同属微弱发光的日行性种类与持续发出很亮荧光的夜行性种类构成窗萤属两个分支的观点；并且从发光器官的形态差异初步推断这两个分支之间是独立进化的。荧光素酶的结构模拟分析表明，云南窗萤荧光素酶的N端和C端结构域之间的裂沟处存在着五个多肽环，这是已知荧光素酶推测的活性位点。日行性的云南窗萤和三种夜行性萤火虫的荧光素酶有13个不同氨基酸，位于模拟的分子结构表面。这些氨基酸的差异，有利于进一步分析日行性和夜行性萤火虫荧光素酶结构的不同。对栉角雪萤(Diaphanes pectinealis)的荧光素酶cDNA也完成了克隆表达。结果表明其cDNA序列有1647个碱基，编码548个氨基酸残基，比从基因组中获得基因序列多3个碱基。体外表达产物在底物存在时发出黄绿色光。对雌光萤科Rhagophthalmus sp.的荧光素酶基因进行了克隆和氨基酸序列比对。得出其荧光素酶基因与来自同一科的荧光素酶基因有最高的一致性为90.7％，和鞘翅目另外三个科的物种的一致性在65％以下，显示出雌光萤科独特的分类地位。通过对采自昆明郊区的云南窗萤和一种扁萤属萤火虫的饲养和发光行为的观察，得出萤火虫的卵和蛹阶段持续发微弱的光的结论，且首次发现了卵发光在发育过程中的变化。本文对卵发光行为进行了描述，并在此基础上对萤火虫卵和蛹两阶段发光的原因和意义进行了探讨。

Cationic Lipid Bilayer Coated Gold Nanoparticles-mediated Transfection of Mammalian Cells

Here, we demonstrated dimethyldioctadecylammonium bromide (DODAB), a cationic lipid, bilayer coated Au nanoparticles (AuNPs) could efficiently deliver two types of plasmid DNA into human embryonic kidney cells (HEK 293) in the presence of serum. The transfection efficiency of AuNPs was about five times higher than that of DODAB. The interaction of AuNPs with DNA was characterized with dye intercalation assay and agarose gel electrophoresis. The morphology of the complex of AuNPs with DNA was observed with scanning electron microscope (SEM). The intracellular trafficking of the complex was monitored with transmission electron microscope (TEM).

Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG

The complex copolymer of hyperbranched polyethylenimine (PEI) with hydrophobic poly(gamma-benzyl L-glutamate) segment (PBLG) at their chain ends was synthesized. This water-soluble copolymer PEI-PBLG (PP) was characterized for DNA complexation (gel retardation assay, particle size, DNA release and DNase I protection), cell viability and in vitro transfection efficiency. The experiments showed that PP can effectively condense pDNA into particles. Size measurement of the complexes particles indicated that PP/DNA tended to form smaller nanoparticles than those of PEI/DNA, which was caused by the hydrophobic PBLG segments compressing the PP/DNA complex particles in aqueous solution. The representative average size of PP/DNA complex prepared using plasmid DNA (pEGFP-N1, pDNA) was about 96 nm. The condensed pDNA in the PP/pDNA complexes was significantly protected from enzymatic degradation by DNase1. Cytotoxicity studies by MTT colorimetric assays suggested that the PP had much lower toxicity than PEI. The in vitro transfection efficiency of PP/pDNA complexes improved a lot in HeLa cells, Vero cells and 293T cells as compared to that of PEI25K by the expression of Green Fluorescent Protein (GFP) as determined by flow cytometry. Thus, the water-soluble PP copolymer showed considerable potential as carriers for gene delivery.

Poly(L-lysine)-graft-chitosan copolymers: Synthesis, characterization, and gene transfection effect

Polypeptide/polysaccharide graft copolymers poly(L-lysine)-graft-chitosan (PLL-g-Chi) were prepared by ring-opening polymerization (ROP) of epsilon-benzoxycarbonyl L-lysine N-carboxyanhydrides (Z-L-lysine NCA) in the presence of 6-O-triphenylmethyl chitosan. The PLL-g-Chi copolymers were thoroughly characterized by H-1 NMR, C-13 NMR, Fourier transform infrared (FT-IR), and gel permeation chromatography (GPC). The number-average degree of polymerization of PLL grafted onto the chitosan backbone could be adjusted by controlling the feed ratio of NCA to 6-O-triphenylmethyl chitosan. The particle size of the complexes formed from the copolymer and calf thymus DNA was measured by dynamic light scattering (DLS). It was found in the range of 120 similar to 340 nm. The gel retardation electrophoresis showed that the PLL-g-Chi copolymers possessed better plasmid DNA-binding ability than chitosan. The gene transfection effect in HEK 293T cells of the copolymers was evaluated, and the results showed that the gene transfection ability of the copolymer was better than that of chitosan and was dependent on the PLL grafting ratio. The PLL-g-Chi copolymers could be used as effective gene delivery vectors.

Factor analysis used to peak position optimization and spectral interference correction in inductively coupled plasma atomic emission spectrometry

Target transformation factor analysis was used to correct spectral interference in inductively coupled plasma atomic emission spectrometry (ICP-BES) for the determination of rare earth impurities in high purity thulium oxide. Data matrix was constructed with pure and mixture vectors and background vector. A method based on an error evaluation function was proposed to optimize the peak position, so the influence of the peak position shift in spectral scans on the determination was eliminated or reduced. Satisfactory results were obtained using factor analysis and the proposed peak position optimization method.

Probabilistic distribution of one-phase structure seminvariants for an isomorphous pair of structures: Theoretical basis and initial applications

Given a special type of triplet of reciprocal-lattice vectors in the monoclinic and orthorhombic systems, there exist eight three-phase structure seminvariants (3PSSs) for a pair of isomorphous structures. The first neighborhood of each of these 3PSSs is defined by the six magnitudes and the joint probability distribution of the corresponding six structure factors is derived according to Hauptman's neighborhood principle. This distribution leads to the conditional probability distribution of each of the 3PSSs, assuming as known the six magnitudes in its first neighborhood. The conditional probability distributions can be directly used to yield the reliable estimates (0 or pi) of the one-phase structure seminvariants (1PSSs) in the favorable case that the variances of the distributions happen to be small [Hauptman (1975). Acta Cryst. A31, 680-687]. The relevant parameters in the formulas for the monoclinic and orthorhombic systems are given in a tabular form. The applications suggest that the method is efficient for estimating the 1PSSs with values of 0 or pi.

Suppression of zebrafish VEGF gene by cytomegalovirus promoter-driven short hairpin constructs induces vascular development defects and down regulation NRP1 expression

The usage of RNA interference for gene knockdown in zebrafish through expression of the small interfering RNA mediators from DNA vectors has created a lot of excitement in the research community. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against vascular endothelial growth factor (VEGF) gene in zebrafish was tested, and its effects on VEGF-mediated vasculogenesis and angiogenesis were evaluated. Altogether four vectors targeting various locations of VEGF gene were constructed, and pSI-V4 was proven to be the most effective one. Microinjection of pSI-V4 into the zebrafish embryos resulted in defective vascular formation and down regulation of VEGF expression. In situ hybridization analysis indicated that silencing VEGF gene expression by pSI-V4 resulted in down regulation of neuropilin-1 (NRP1), a potent VEGF receptor. Knockdown of VEGF expression by morpholino gave the same result. This provided evidence that the VEGF-mediated angiogenesis in zebrafish was in part dependent on NRP1 expression. The results contributed to a better understanding of molecular mechanisms of cardiovascular development and provided a potential promoter for making inducible knockdown in zebrafish.

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coil has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sg PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with alpha-(32)p labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.

A new wind vector algorithm for C-band SAR

Ocean wind speed and wind direction are estimated simultaneously using the normalized radar cross sections or' corresponding to two neighboring (25-km) blocks, within a given synthetic aperture radar (SAR) image, having slightly different incidence angles. This method is motivated by the methodology used for scatterometer data. The wind direction ambiguity is removed by using the direction closest to that given by a buoy or some other source of information. We demonstrate this method with 11 EN-VISAT Advanced SAR sensor images of the Gulf of Mexico and coastal waters of the North Atlantic. Estimated wind vectors are compared with wind measurements from buoys and scatterometer data. We show that this method can surpass other methods in some cases, even those with insufficient visible wind-induced streaks in the SAR images, to extract wind vectors.

Wind-vector estimation for RADARSAT-1 SAR images: Validation of wind-direction estimates based upon geometry diversity

In this letter, a new wind-vector algorithm is presented that uses radar backscatter sigma(0) measurements at two adjacent subscenes of RADARSAT-1 synthetic aperture radar (SAR) images, with each subscene having slightly different geometry. Resultant wind vectors are validated using in situ buoy measurements and compared with wind vectors determined from a hybrid wind-retrieval model using wind directions determined by spectral analysis of wind-induced image streaks and observed by colocated QuikSCAT measurements. The hybrid wind-retrieval model consists of CMOD-IFR2 [applicable to C-band vertical-vertical (W) polarization] and a C-band copolarization ratio according to Kirchhoff scattering. The new algorithm displays improved skill in wind-vector estimation for RADARSAT-1 SAR data when compared to conventional wind-retrieval methodology. In addition, unlike conventional methods, the present method is applicable to RADARSAT-1 images both with and without visible streaks. However, this method requires ancillary data such as buoy measurements to resolve the ambiguity in retrieved wind direction.

靶向斑马鱼VEGF的shRNA表达载体的构建及其对斑马鱼胚胎血管发育的影响

血管内皮生长因子(vascular endothelial growth factor, VEGF)是一种多功能的细胞因子，其主要作用是促进血管内皮细胞增殖和增加血管通透性，是肿瘤及正常组织血管生成的中心调控因素，以VEGF为靶点的肿瘤血管靶向性治疗成为近几年肿瘤治疗的新途径。RNAi是近年来新发展的一项反向遗传学技术，是一种研究基因功能的有力工具。斑马鱼作为一种重要的模式生物，被广泛用于胚胎的分子发育机制、疾病模型的构建以及药物筛选等研究中。然而在斑马鱼中运用RNAi技术进行基因功能研究是一个相对较新的领域，研究资料较少，并且目前进行的斑马鱼RNAi实验中，siRNA大都是通过化学方法或体外转录合成的。体外合成的siRNA在进入体内后会被降解而无法达到持久阻抑基因表达的目的。因此本研究旨在探讨VEGF特异性siRNA表达载体对斑马鱼VEGF基因的沉默作用，通过分析表型及相关细胞因子的变化，阐明VEGF对斑马鱼胚胎血管生成的影响及作用机制。 研究通过计算机辅助设计软件，针对斑马鱼VEGF mRNA不同位点设计合成了4段含siRNA特异序列的DNA单链，经退火，克隆入pSilencer 4.1-CMV neo载体CMV启动子下游，构建了重组质粒pS1-VEGF、pS2-VEGF、pS3-VEGF及pS4-VEGF。 通过显微注射的方法将载体导入1-2细胞期斑马鱼体内，于胚胎发育的48 h采用RT-PCR的方法检测VEGF基因的表达量，研究不同干扰序列对VEGF基因表达的干涉作用。结果显示，针对不同位点的表达载体对VEGF基因表达的抑制效率有显著差异。它们对VEGF mRNA的抑制率分别为80.5%，42.8%，12.5%，40.7%。通过筛选我们得到了一条具有高效抑制作用的载体pS1-VEGF，该载体的相应序列靶向斑马鱼两个主要异构体VEGF165和VEGF121的共有外显子序列。 形态学检测结果显示，注射了pS1-VEGF的胚胎出现了心包膜水肿、血流速度减慢、循环红细胞堆积等症状。定量碱性磷酸酶染色显示，注射pS1-VEGF能够抑制斑马鱼胚胎新生血管的形成，当注射剂量为0.4 ng时，血管生成的抑制率为31.8%。NBT/BCIP血管染色显示，注射该载体后72 h，50%的斑马鱼肠下静脉、节间血管以及其它血管的发育受到不同程度的抑制。随着注射剂量的加大，血管发育受抑制的情况也随之加重，当注射剂量为1 ng时，只有心脏、头部及卵黄有血液循环。对干扰效果的特异性进行了研究，结果表明pS1-VEGF对斑马鱼内源基因胸苷酸合成酶（thymidylate synthase, TS）基因的表达没有明显的抑制作用。针对TS基因的shRNA表达载体及与斑马鱼没有同源性的对照载体对VEGF基因表达也没有明显的抑制作用。浓度梯度实验表明在0-1.2 ng的范围内干扰效果具有剂量依赖性。 以胚胎整体原位杂交的方法检测质粒对VEGF基因受体NRP1基因表达的影响，发现VEGF特异性shRNA表达载体能够引起NRP1基因表达的降低，说明斑马鱼中VEGF所介导的血管生成作用至少在部分上是依赖于NRP通路所调节的。 本研究工作为进一步研究斑马鱼基因功能、VEGF调控网络提供了一个快速、有效的手段，为阐明斑马鱼的血管生成机制提供了新的资料，为采用RNAi技术，以VEGF为靶点，以斑马鱼为模型对肿瘤进行基因治疗研究奠定了基础。

基于模糊集理论的二维线性鉴别分析新方法

二维线性鉴别分析（2DLDA）是一种直接基于矩阵的特征提取方法，跳过传统的基于Fisher鉴别准则的线性鉴别分析方法中必须先将二维矩阵转化成一维矢量的过程，有效地提高了特征提取速度且避免了小样本问题，其识别率优于传统的Fisherface方法。结合模糊集理论，提出了一种新的2DLDA算法——模糊2DLDA（FIDLDA）算法。首先采用FKNN算法得到相应的样本分布信息，并按其对最后得到的特征向量所作的贡献融入到特征抽取过程中，得到有效的样本特征向量集。实验表明，F2DLDA算法的性能优于传统的2DLDA算法和Fisherface方法。

改进的统计不相关最优鉴别矢量集

该文对统计不相关最优鉴别矢量集算法进行研究,在分析统计不相关最优鉴别矢量集算法的基础上提出了一种改进的方法。该方法在类内散布矩阵的特征空间中求解统计不相关最优鉴别矢量集。为了加快特征抽取速度,利用基于图像鉴别分析的维数压缩方法,对图像数据进行了压缩。在ORL和Yale人脸数据库的数值实验,验证本文所提出的方法的有效性。