Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

Trichosanthin suppresses the elevation of p38 MAPK, and Bcl-2 induced by HSV-1 infection in Vero cells

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) effective against HIV-1 and HSV-1 replication. The mechanism of its antiviral activity is not clear. Many believe that it is related to ribosome inactivation. Some RIPs and viral infectio

In vitro anti-HIV and -HSV activity and safety of sodium rutin sulfate as a microbicide candidate

Sodium rutin sulfate (SRS) is a sulfated rutin modified from the natural flavonol glycoside rutin. Here, we investigated its in vitro anti-HIV and -HSV activities and its cytotoxic profile. Fifty percent inhibitory concentration (IC50) values of SRS against HIV-1 X4 virus IIIB, HIV-1 R5 isolates Ada-M and Ba-L were 2.3 +/- 0.2, 4.5 +/- 2.0 and 8.5 +/- 3.8 mu M with a selectivity index (SI) of 563, 575 and 329, respectively. Its IC50 against primary R5 HIV-1 isolate from Yunnan province in China was 13.1 +/- 5.5 mu M, with a Sl of 197. In contrast, unsulfated rutin had no activity against any of the HIV-1 isolates tested. Further study indicated that SRS blocked viral entry and virus-cell fusion likely through interacting with the HIV- I envelope glycoprotein. SRS also demonstrated some activity against human herpes simplex virus (HSV) with an IC50 of 88.3 +/- 0.1 mu M and a Sl of 30. The 50% cytotoxicity concentration (CC50) of SRS was >3.0 mM, as determined in human genital ME 180, HeLa and primary human foreskin fibroblast cells. Minimum inhibitory concentration of SRS for vaginal lactobacilli was >3.0 mM. These results collectively indicate that SRS represents a novel candidate for anti-HIV-1/HSV microbicide development. (C) 2007 Elsevier B.V. All rights reserved.

Development of a positive-negative selection procedure for gene targeting in fish cells

The gene targeting technique is a powerful tool for analyzing functions of cloned genes and for generating transgenic animals with site-directed integration of foreign genes. In order to develop this technique in fish, positive-negative selection (PNS) and homologous recombination vectors were constructed, and their expression was examined in fish cells. A vector (pNK) for PNS consists of the neomycin resistance gene (neo) as a positive selectable marker gene and the herpes simplex virus (HSV) thymidine kinase (tk) gene as a negative selectable marker gene. Positive selection with geneticin (G418) of epithelioma papulosum of carp (EPC) cells transfected with linearized pNK vector yielded 350 colonies, while double selection of transfected EPC cells with G418 and gancyclovir (Gc) resulted in nearly complete cell death, demonstrating that the PNS procedure is effective in fish cells. Homologous recombination vectors consist of the Xiphophorus melanoma receptor kinase (X mrk(Y)) gene as homologous sequence in addition to the neo and tk genes. Conditions for homologous recombination vector transfection and drug selection were established. After verification of the feasibility of expression of homologous recombination vectors in EPC cells, the first gene targeting experiments were attempted in the Xiphophorus melanoma cell line, PSM. Positive-negative selection of the targeting vector-transfectants led to a low enrichment in this particular cell line. The reasons for the low enrichment in PSM cells were discussed. (C) 2002 Elsevier Science B.V. All rights reserved.

一种遥感图像融合增强的实现方法

本发明涉及一种遥感图像融合增强的实现方法，包括以下步骤：将选取的多光谱遥感影像与全色波段影像进行配准；将多光谱遥感影像的波段进行LBV数据变换，得到处理后的L、B、V图像；将处理后的L、B、V图像作为三个分量，将代表普通辐射水平的L当作I分量和V分量，选择进行IHS变换融合、IHS变换同小波变换相结合的融合以及HSV变换同小波变换相结合的融合；通过肉眼观察最后的图像融合结果是否满意；采用多种评价标准对结果图像进行评价。本发明方法采用了利用多个光谱波段进行数据变换的LBV数据变换方法，使图像光谱信息更加丰富，突破了以往传统的图像融合方法只利用3个光谱波段与高分辨率图像进行融合的局限，是对传统融合方法的一大突破。

The dazzling array of basal branches in the mtDNA macrohaplogroup M from India as inferred from complete genomes

Many efforts based on complete mitochondrial DNA (mtDNA) genomes have been made to depict the global mtDNA landscape, but the phylogeny of Indian macrohaplogroup M has not yet been resolved in detail. To fill this lacuna, we took the same strategy as in o

Phylogeny of mitochondrial DNA macrohaplogroup N in India, based on complete sequencing: Implications for the peopling of South Asia

To resolve the phylogeny of the autochthonous mitochondrial DNA (mtDNA) haplogroups of India and determine the relationship between the Indian and western Eurasian mtDNA pools more precisely, a diverse subset of 75 macrohaplogroup N lineages was chosen fo

Enhancement of the anti-herpetic effect of trichosanthin by acyclovir and interferon

Trichosanthin (TCS) is a type I ribosome-inactivating protein that has a wide range of pharmacological activities. The present study investigated the effectiveness of TCS on herpes simplex virus (HSV-1). The anti-viral activity and toxicity of TCS on Vero

乳头瘤病毒及协同因子与宫颈癌的关系

本研究旨在探讨宫颈癌前病变和宫颈癌的发生发展与人乳头瘤病毒及协同因子 (HSV ,CMV)的关系。对81例不同宫颈病变组织进行HPV16 / 18和HPV6 / 11原位杂交 ,同时对 10 3例不同宫颈病变组织用DNA扩增法检测HPV、HSV和CMV。结果表明病毒DNA原位杂交信号的分布与HE染色中挖空细胞的分布一致。HPV16 / 18与不同宫颈病变组织原位杂交阳性率平均为 5 1% ,HPV6 / 11的则为 6 4%。经PCR检测 ,HPV16 / 18、HPV6 / 11、HSV、CMV在不同宫

Rana grylio virus thymidine kinase gene: an early gene of iridovirus encoding for a cytoplasmic protein

The presence of thymidine kinase (TK) is a feature of many large DNA viruses. Here, a TK gene homologue was cloned and characterized from Rana grylio virus (RGV), a member of family Iridoviridae. RGV TK encodes a protein of 195 aa with a predicted molecular mass of 22.1 kDa. Homologues of the protein were present in all the currently sequenced iridoviruses, and phylogenetic analysis showed that it was much close to cellular TK type 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Subsequently, Western blotting revealed TK expression increased with time from 6 h post-infection in RGV-infected cells. Using drug inhibition analysis by protein synthesis inhibitor (cycloheximide) and DNA replication inhibitor (cytosine arabinofuranoside), RGV TK was classified as the early expression gene during in vitro infection. Subcellular localization by TK-GFP fusion protein expression and immunofluorescence staining showed RGV TK was an exclusively cytoplasmic protein in fish cells. Collectively, current data indicate that RGV TK was an early gene of iridovirus which encoded a cytoplasmic protein in fish cells.

一种基于亮度分析的图像分割方法

图像分割是数字图像处理领域的重要研究内容。随着数字图像处理技术的发展和相关学科的进步，图像分割在图像编辑、计算机视觉、医疗影像、遥感图像等方面都取得了良好的应用，而且具有巨大的应用潜力。 图像分割是一个既很基础又很复杂的问题。经过多年的发展，研究者提出了很多优秀的算法，取得了令人鼓舞的成就。但由于图像分割本身的不确定性和复杂性，还没有一种算法能够解决好所有的分割问题。为了使图像分割的结果更加符合用户需求，交互式的分割方法逐渐成为了图像分割算法的主流。交互方式的简化也就成为了研究者努力的目标。 另一方面，图切分技术(Graph-Cut)是应用于图像分割算法的一种重要工具。Lazy Snapping方法利用人工交互操作来提取图像信息，然后使用图切分来分割图像，取得了较好的分割效果。而且该方法的交互界面简单易用，对模糊边界有较快的响应速度，引起了研究者的重视，是一种结合人工交互与图切分技术的重要分割方法。 鉴于Lazy Snapping方法在处理色彩丰富、对比强烈的图像时出现的交互操作过多、分割容易出错的问题，本文提出了一种基于亮度分析的图像分割方法。该方法在HSV空间下，以亮度为依据来提取前景和背景的特征，取代或简化了Lazy Snapping中的人工操作步骤，然后用图切分技术来进行分割。这种方法能够提取到较准确的图像特征，有效地减轻了用户操作量，明显地改善了分割结果。

Polarization patterns control based on photoinduced anisotropy of photochromic fulgide

We investigate the photoinduced anisotropy of a photochromic material of pyrrylfulgide/PMMA films. It is proven that when the film is illuminated with a linear polarization light, an optical axis that has the same polarization as the excitation light could be induced in the film. A matrix of light spots with different polarizations is recorded on the pyrrylfulgide/PMMA film. When reading out with non-polarization light, the matrix of light spots shows no information of patterns. However, when reading out with different linear polarization lights, different patterns could be observed. The experiment confirms that the pyrrylfulgide/PMMA film could be used to record two different polarization patterns in a matrix of spots. This property may be applied in camouflage technology.

Synthesis and application on parallel image storage of bisfurylfulgide

A bisfurylfulgide, E, E-3,4-bis[1-(2,5-dimethyl-3-furyl)ethylidene]-3,4-dihydrofuran-2,5-dione, is synthesized by Stobbe condensation reaction. The molecular structure of target compound is confirmed by single crystal X-ray crystallography analysis. It shows that the distances between two possible reaction sites of molecule are 0.3394 and 0.3406 nm respectively, which is favorable to photocyclization. The photochromic properties of this compound in different solvents are investigated, and the result shows that the compound exhibits excellent photochromic behavior. The primary result of applied research on parallel image storage is also presented.

Optical image processing using the photoinduced anisotropy of pyrrylfulgide

A synthesized photochromic compound-pyrrylfulgide-is prepared as a thin film doped in a polymethylmethacrylate (PMMA) matrix. Under irradiation by UV light, the film converts from the bleached state into a colored state that has a maximum absorption at 635 nm and is thermally stable at room temperature. When the colored state is irradiated by a linearly polarized 650 nm laser, the film returns to the bleached state; photoinduced anisotropy is produced during this process. Application of optical image processing methods using the photoinduced anisotropy of the pyrrylfulgide/PMMA film is described. Examples in non-Fourier optical image processing, such as contrast reversal and image subtraction and summation, as well as in Fourier optical image processing, such as low-pass filtering and edge enhancement, are presented. (c) 2006 Optical Society of America.