Identification of immune genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major

The parasitic copepod Sinergasilus major is an important pathogen of grass carp Ctenopharyngodon idella. To understand the immune response of grass carp to the copepod infection, suppression subtractive hybridization method was employed to characterize genes up-regulation during the copepod infection in liver and gills of the fish. One hundred and twenty-two dot blot positive clones from infected subtracted library were sequenced. Searching available databases by using these nucleotide sequences revealed that 23 genes are immune-related, including known acute-phase reactants, and four novel genes encoding proteins such as source of immunodominant MHC-associated peptides (SIMP), TNF receptor-associated factor 2 binding protein (T2BP), poliovirus receptor-related protein 1 precursor, glycoprotein A repetitions predominant (GARP). The differential expression of seven immune genes, i.e. GARP, alpha-2-macroglobulin, MHC class I, C3, SIMP, T2BP, transferrin, as a result of infection was further confirmed by RT-PCR, with the up-regulation of alpha-2-macroglobulin, MHC class I, C3, SIMP and T2BP in the liver of infected fish, and down-regulation of SIMP in the gills of infected fish. The present study provides foundation for understanding grass carp immune response and candidate genes for further analysis.

Identification of two novel interferon-stimulated genes from cultured CAB cells induced by UV-inactivated grass carp hemorrhage virus

Interferon (IFN) exerts its antiviral effect by inducing the expression of a number of IFN-stimulated genes (ISGs) to establish a host antiviral state. Earlier studies identified some important fish IFN system genes from IFN-induced CAB cells (crucian carp Carassius auratus L. embryonic blastulae cells) after treatment with UV-inactivated GCHV (grass carp hemorrhage virus). Herein, the cloning of 2 novel IFN-stimulated genes, termed Gig1 and Gig2, is described for the same cell system. The complete cDNA sequences of Gig1 and Gig2 contain 1244 bp encoding for a 194-amino-acid protein and 693 bp for a 158-amino-acid protein, respectively. A search of public databases revealed that these are 2 novel IFN-stimulated genes, since neither significant homologous genes nor conserved motifs were identified. Active GCHV, UV-inactivated GCHV and CAB IFN-containing supernatant (ICS) induced transcription of these genes and distinct kinetics were observed. An analysis of differences in expression between the 2 genes and the IFN signal factors CaSTAT1 and CaIRF7 indicated that GCHV infection activated different signal pathways for their up-regulation. Upon virus infection, the transcription of Gig1 but not of Gig2 is strongly suppressed by cycloheximide (CHX). In contrast, following treatment with CAB IFN-containing supernatant, CHX does not inhibit either gene transcription. The results suggest that GCHV infection can induce expression of both Gig1 and Gig2 via newly synthesized CAB IFN, most probably through the JAK-STAT signal pathway, and can also directly activate Gig2 transcription without ongoing protein synthesis.

Complete genome sequence of lymphocystis disease virus isolated from China

Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1.

移动对象在空间网络数据库上的kNN查询

中国计算机学会

移动对象数据库模型、查询语言及实时交通流分析

提出一种移动对象数据库模型——Dynamic Transportation Network Based Moving Objects Database(简称DTNMOD),并给出了DTNMOD中基于移动对象时空轨迹的网络实时动态交通流分析方法.在DTNMOD中,交通网络被表示成动态的时空网络,可以描述交通状态、拓扑结构以及交通参数随时间的变化过程;网络受限的移动对象则用网络移动点表示.DTNMOD模型包含了完整的数据类型和查询操作的定义,因此可以在任何可扩充数据库(如PostgreSQL或SECONDO)中实现,从而得到完整的数据库模型和查询语言.为了对相关模型的性能进行比较与分析,基于PostgreSQL实现了一个原型系统并进行了一系列的实验.实验结果表明,DTNMOD提供了良好的区域查询及连接查询性能.

机密数据库泄漏源检测与量化评估方案

针对机密数据库泄漏源检测问题以及现有技术存在的不足,提出了一种基于复合型数据库水印的检测方法与量化评估方案.设计了复合型数据库水印的添加与检测算法,利用水印添加与数据分发协议,将机密数据与其所有者和用户的身份绑定,实现了泄漏源检测与追查过程中对参与双方的公平保护.使用概率分析的方法实现了检测结果的可靠性量化评估.最后通过实验分析了算法的计算性能,验证了方案的有效性和可行性.实验结果表明该方案具有较高的实用价值.

人载脂蛋白APOA5的克隆表达纯化及相互作用蛋白研究

人类的载脂蛋白A5（apolipoprotein A5，APOA5）是一个新近发现的载脂蛋白家族成员。它在血浆中的含量比其他载脂蛋白低1-2个数量级，但能显著影响血浆三酰甘油水平，对血脂代谢具有重要意义，可以作为降血脂药物治疗中一个强有力的潜在靶标。 由于APOA5在血浆中含量低，直接从血浆中分离纯化很困难，国内一直没有报道简易可靠的纯化方法。为进一步研究APOA5的生物学特性，探讨其与TG代谢中的其它关键成分之间的相互关系，揭示其在脂类代谢相关疾病中的重要地位，必须有大量的蛋白和抗体用于基础研究。因此本研究首先利用基因工程技术，诱导表达纯化APOA5蛋白，免疫动物制备多克隆抗体，为进一步研究人肝脏细胞中APOA5的相互作用蛋白，研究APOA5蛋白在肝脏细胞中的功能奠定基础。 为了深入研究APOA5在肝脏中如何行使功能，我们采用细菌双杂交技术寻找与APOA5相互作用的蛋白因子。并采用Pull-down技术，免疫荧光及免疫共沉淀技术进一步确证其在体外和体内的相互作用关系，为进一步阐明APOA5在体内的生理功能提供了新的线索。 第一部分 APOA5基因的克隆、原核表达、纯化及其多克隆抗体的制备 本研究首先应用基因克隆技术，从人肝癌细胞系SMMC-7721的cDNA中扩增出1.1 kb的ApoA5基因全长序列。然后将其克隆至表达载体pThioHisD，构建原核表达载体pTH-APOA5。该重组质粒转化至大肠杆菌 BL21（DE3），成功实现人APOA5融合蛋白在大肠杆菌中的表达。经发酵得到高效表达的融合蛋白。 融合蛋白在 IPGT 诱导下以包涵体的形式大量表达。利用融合蛋白上的一段组氨酸序列，用镍离子亲和柱进行纯化和复性后，获得较高纯度的人APOA5融合蛋白。利用该融合蛋白免疫新西兰大耳白兔，获得了高效价的兔抗人APOA5多克隆抗体，Western Blot结果显示此多克隆抗体与APOA5特异性结合。 第二部分 细菌双杂交筛选与APOA5相互作用的蛋白 本实验首先构建了pBT-APOA5重组质粒，经双酶切、PCR和测序鉴定证明重组诱饵质粒构建成功，并进行了表达、自激活鉴定。Western Blot鉴定证实报告菌株中表达了分子量为 68 kD左右的重组融合蛋白，与预测的分子量APOA5（41 kD）/lamda cI （27 kD）一致。自激活实验证明诱饵蛋白不能单独激活报告基因，可用于筛选人肝脏cDNA文库。经过双重抗性筛选和回复筛选，分离出10个阳性克隆。对结果进行生物信息学分析，得到7个与APOA5相互作用的蛋白，其中BI1为细胞凋亡调节因子；ATP6、CYTB、ND2、COX-1为线粒体表达蛋白； ALB、TTR为血清蛋白。 第三部分 APOA5与BI1相互作用的确证 首先构建了BI1的原核表达载体pGEX-5X-3-BI1，利用Pull-down实验检测了APOA5与BI1在体外具有相互作用。然后构建了BI1的真核表达载体pCDNA3.1-HA-BI1和APOA5的真核表达载体pCDNA3.1-APOA5，并验证其表达。通过免疫荧光细胞内共定位研究发现，靶蛋白APOA5主要分布于胞浆，与BI1在HEK293细胞有共定位，即APOA5与BI1存在相互作用的可能。最后利用免疫共沉淀手段，在HEK293细胞中确证了靶蛋白APOA5与BI1在体内的相互作用。 上述研究结果，为深入研究APOA5在体内的生物学功能提供了新的思路。 Apolipoprotein A5 (APOA5) is a newly discovered protein belongs to apolipoprotein family. APOA5’s concentration is 1-2 orders of magnitude lower than other apolipoproteins in the circulation. APOA5 significantly affected plasma triglyceride levels, which is important on lipid metabolism. APOA5 has strong potential to be used as a hypolipidemic drug target. Large amount of APOA5 protein and antibodies are needed in basic research, such as biological characteristics study of the APOA5, its relationship with other key components in TG metabolism, its role played in Lipid metabolism-related diseases. Due to its low concentration in plasma, separation and purification of APOA5 from the plasma is very difficult. Until now no report on simple and reliable method for purification has been published in China. In this study, we firstly got APOA5 recombinant protein using genetic engineering technology. The purified recombinant protein was used to immunize rabbits to get antiserum. It is important for further study of the APOA5 protein-interacting protein. And it lays the foundation for studing APOA5 function in liver. In order to study APOA5 function in liver, we used bacterial two-hybrid technology to find the APOA5 protein interactor. Pull-down, immunofluorescence and immunoprecipitation techniques were used to further confirm the interaction between APOA5 with its interactor in vitro and in vivo. All of these stdudies provided new clues on its physiological functions in vivo. Part I: Cloning, prokaryotic expression, purification and polyclonal antibody preparation of APOA5 First of all, we amplified APOA5 CDS sequence from the human hepatoma cell line SMMC-7721, and subcloned into Expression vector pThioHisD, and got the recombinants named pTH-APOA5. The plasmid was transformed to BL21 (DE3). E. coli BL21(DE3) cells bearing the pTH-APOA5 plasmid were cultured and APOA5 protein synthesis was induced by the addition of IPTG. Recombinant protein was expression in the form of inclusion. Inclusion bodies were dissolved in phosphate-buffered saline containing 8 M urea and 40 mM imidazole, then applied to a Ni2+ affinity column, and were eluted in a buffer containing 4 M urea and 200 mM imidazole. Fractions containing the APOA5 protein were pooled and dialyzed against buffer containing phosphate-buffered saline. Antiserum to recombinant human APOA5 was generated by immuning rabbit. Western Blot showed that this antiserum specific binding with APOA5. Part II Two-hybrid system screening protein interactions with the APOA5 The coding sequence of human APOA5 was amplified using synthetic oligonucleotide primers from pTH-APOA5 vector and was subcloned into the pBT plasmidc to yield pBT-APOA5 vector. DNA sequencing was performed to verify that no unwanted mutations occurred during the process of plasmid vector construction. We verified recombinant protein expression and tested self-activation by pBT-APOA5 prior to screening. Western Blot verified inducing a 68 kD band, consistent with the predicted molecular weight (APOA5 41 kD, lamda cI 27 kD). pBT-APOA5 can be used for screening human liver cDNA library because it can not self-activation. Totally 10 positive clones were isolated. The nucleotide sequence of the positive clones were determined and compared to NCBI nucleotide sequence databases. We got 7 protein which interact with APOA5, included BI1(Apoptosis regulator); ATP6, CYTB, ND2, COX-1(Mitochondrial protein) and ALB, TTR(Serum protein). Part III Confirming of interaction between APOA5 with BI1 pGEX-5X-3-BI1 vector was subcloned at first. Pull-down experiments were used to detect the interaction between APOA5 with BI1 in vitro. Later, pCDNA3.1-HA-BI1 and pCDNA3.1-APOA5 were subcloned. Through immunofluorescence co-localization study, we found APOA5 mainly distributed in the cytoplasm. APOA5 is co-localization with BI1 in HEK293 cells. Finally, we verified interaction between APOA5 with BI1 in vivo through immunoprecipitation.

Soil microbial community analysis using terminal restriction fragment length polymorphisms

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a polymerase chain reaction (PCR)-fingerprinting method that is commonly used for comparative microbial community analysis. The method can be used to analyze communities of bacteria, archaea, fungi, other phylogenetic groups or subgroups, as well as functional genes. The method is rapid, highly reproducible, and often yields a higher number of operational taxonomic units than other, commonly used PCR-fingerprinting methods. Sizing of terminal restriction fragments (T-RFs) can now be done using capillary sequencing technology allowing samples contained in 96- or 384-well plates to be sized in an overnight run. Many multivariate statistical approaches have been used to interpret and compare T-RFLP fingerprints derived from different communities. Detrended correspondence analysis and the additive main effects with multiplicative interaction model are particularly useful for revealing trends in T-RFLP data. Due to biases inherent in the method, linking the size of T-RFs derived from complex communities to existing sequence databases to infer their taxonomic position is not very robust. This approach has been used successfully, however, to identify and follow the dynamics of members within very simple or model communities. The T-RFLP approach has been used successfully to analyze the composition of microbial communities in soil, water, marine, and lacustrine sediments, biofilms, feces, in and on plant tissues, and in the digestive tracts of insects and mammals. The T-RFLP method is a user-friendly molecular approach to microbial community analysis that is adding significant information to studies of microbial populations in many environments.

Research on storing raster metadata in spatial database

Storage of raster metadata is a key topic in spatial database. Although there are a few of abstract standards on raster metadata, there is not implement standard about it. This paper concludes three storage models implemented in current spatial databases and discusses their advantages and disadvantages. After that analyzing, the paper proposes a mixed storage method which is used the relational table to store structured metadata and used XML to store non-structured metadata, and gives its implementation solution. © 2010 IEEE.

Semisupervised Dimensionality Reduction and Classification Through Virtual Label Regression

Semisupervised dimensionality reduction has been attracting much attention as it not only utilizes both labeled and unlabeled data simultaneously, but also works well in the situation of out-of-sample. This paper proposes an effective approach of semisupervised dimensionality reduction through label propagation and label regression. Different from previous efforts, the new approach propagates the label information from labeled to unlabeled data with a well-designed mechanism of random walks, in which outliers are effectively detected and the obtained virtual labels of unlabeled data can be well encoded in a weighted regression model. These virtual labels are thereafter regressed with a linear model to calculate the projection matrix for dimensionality reduction. By this means, when the manifold or the clustering assumption of data is satisfied, the labels of labeled data can be correctly propagated to the unlabeled data; and thus, the proposed approach utilizes the labeled and the unlabeled data more effectively than previous work. Experimental results are carried out upon several databases, and the advantage of the new approach is well demonstrated.

Fast Haar Transform Based Feature Extraction for Face Representation and Recognition

Subspace learning is the process of finding a proper feature subspace and then projecting high-dimensional data onto the learned low-dimensional subspace. The projection operation requires many floating-point multiplications and additions, which makes the projection process computationally expensive. To tackle this problem, this paper proposes two simple-but-effective fast subspace learning and image projection methods, fast Haar transform (FHT) based principal component analysis and FHT based spectral regression discriminant analysis. The advantages of these two methods result from employing both the FHT for subspace learning and the integral vector for feature extraction. Experimental results on three face databases demonstrated their effectiveness and efficiency.

CIAC COMPREHENSIVE INFORMATION-SYSTEM OF RARE-EARTHS

The CIAC (Changchun Institute of Applied Chemistry) Comprehensive information System of Rare Earths is composed of three subsystems, namely, extraction data, physicochemical properties, and reference data. This paper describes the databases pertaining to the extraction of rare earths and their physicochemical properties and discusses the relationships between data retrieval and optimization and between the structures of the extractants and the efficiency with which they are extracted. Expert systems for rare earth extraction and calculation of thermodynamic parameters are described, and an application of pattern recognition to the problems of classification of compounds of the rare earths and prediction of their properties is reported.

The construction of a cDNA library enriched for immune genes and the analysis of 7535 ESTs from Chinese mitten crab Eriocheir sinensis

Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture crustacean species in China. A cDNA library was constructed from hemocytes of E. sinensis challenged with the mixture of Listonella anguillarum and Staphylococcus aureus, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. Single-pass 5' sequencing of 10368 clones yielded 7535 high quality ESTs (Expressed Sequence Tags) and these ESTs were assembled into 2943 unigenes. BLAST analysis revealed that 1706 unigenes (58.0% of the total) or 4593 ESTs (61.0% of the total) were novel genes that had no significant matches to any protein sequences in the public databases. The rest 1237 unigenes; (42.0% of the total) were closely matched to the known genes or sequences deposited in public databases, which could be classed into 20 or 23 classifications according to "molecular function" or "biological process" respectively based on the Gene Ontology (GO). And 221 unigenes (7.5% of all 2943 unigenes, 17.9% of matched unigenes) or 969 ESTs (12.9% of all 7535 ESTs, 32.9% of matched ESTs) were identified to be immune genes. The relative higher proportion of immune-related genes in the present cDNA library than that in the normal library of E. sinensis and other crustaceans libraries, and the differences and changes in percentage and quantity of some key immune-related genes especially the immune inducible genes between two E. sinensis cDNA libraries may derive from the bacteria challenge to the Chinese mitten crab. The results provided a well-characterized EST resource for the genomics community, gene discovery especially for the identification of host-defense genes and pathways in crabs as well as other crustaceans. (C) 2009 Elsevier Ltd. All rights reserved.

Expressed sequence tags from the zhikong scallop (Chlamys farreri): Discovery and annotation of host-defense genes

A high-quality cDNA library was constructed from whole body tissues of the zhikong scallop, Chlamys farreri, challenged by Listonella anguillarum. A total of 5720 clones were sequenced, yielding 5123 expressed sequence tags (ESTs). Among the 3326 unique genes identified, 2289 (69%) genes had no significant (E-value < 1e-5) matches to known sequences in public databases and 194 (6%) matched proteins of unknown functions. The remaining 843 (25%) genes that exhibited homology with genes of known functions, showed broad involvement in metabolic processes (31%), cell structure and motility (20%), gene and protein expression (12%), cell signaling and cell communication (8%), cell division (4%), and notably, 25% of those genes were related to immune function. They included stress response genes, complement-like genes, proteinase and proteinase inhibitors, immune recognition receptors and immune effectors. The EST collection obtained in this study provides a useful resource for gene discovery and especially for the identification of host-defense genes and systems in scallops and other molluscs. (C) 2009 Elsevier Ltd. All rights reserved.

Isolation and mapping of telomeric pentanucleotide (TAACC)(n) repeats of the pacific whiteleg shrimp, Penaeus vannamei, using fluorescence in situ hybridization

To develop genetic and physical maps for shrimp, accurate information on the actual number of chromosomes and a large number of genetic markers is needed. Previous reports have shown two different chromosome numbers for the Pacific whiteleg shrimp, Penaeus vannamei, the most important penaeid shrimp species cultured in the Western hemisphere. Preliminary results obtained by direct sequencing of clones from a Sau3A-digested genomic library of P. vannamei ovary identified a large number of (TAACC/GGTTA)-containing SSRs. The objectives of this study were to (1) examine the frequency of (TAACC)(n) repeats in 662 P. vannamei genomic clones that were directly sequenced, and perform homology searches of these clones, (2) confirm the number of chromosomes in testis of P. vannamei, and (3) localize the TAACC repeats in P. vannamei chromosome spreads using fluorescence in situ hybridization (FISH). Results for objective I showed that 395 out of the 662 clones sequenced contained single or multiple SSRs with three or more repeat motifs, 199 of which contained variable tandem repeats of the pentanucleotide (TAACC/GGTTA),, with 3 to 14 copies per sequence. The frequency of (TAACC)n repeats in P. vannamei is 4.68 kb for SSRs with five or more repeat motifs. Sequence comparisons using the BLASTN nonredundant and expressed sequence tag (EST) databases indicated that most of the TAACC-containing clones were similar to either the core pentanucleotide repeat in PVPENTREP locus (GenBank accession no. X82619) or portions of 28S rRNA. Transposable elements (transposase for Tn1000 and reverse transcriptase family members), hypothetical or unnamed protein products, and genes of known function such as 18S and 28S rRNAs, heat shock protein 70, and thrombospondin were identified in non-TAACC-containing clones. For objective 2, the meiotic chromosome number of P. vannamei was confirmed as N = 44. For objective 3, four FISH probes (P1 to P4) containing different numbers of TAACC repeats produced positive signals on telomeres of P. vannamei chromosomes. A few chromosomes had positive signals interstitially. Probe signal strength and chromosome coverage differed in the general order of P1 > P2 > P3 > P4, which correlated with the length of TAACC repeats within the probes: 83, 66, 35, and 30 bp, respectively, suggesting that the TAACC repeats, and not the flanking sequences, produced the TAACC signals at chromosome ends and TAACC is likely the telomere sequence for P. vannamei.