Phylogenetic studies of three sinipercid fishes (Perciformes: Sinipercidae) based on complete mitochondrial DNA sequences

The sinipercids are a group of 12 species of freshwater percoid fish endemic to East Asia and their phylogenetic placements have perplexed generations of taxonomists. We cloned and sequenced the complete mitochondrial DNA (mtDNA) of three sinipercid fishes (Siniperca chuatsi, S. kneri, and S. scherzeri) to characterize and compare their mitochondrial genomes. The mitochondrial genomes of S. chuatsi, S. kneri, and S. scherzeri were 16,496, 17,002, and 16,585?bp in length, respectively. The organization of the three mitochondrial genomes is similar to those reported from other fish mitochondrial genomes, which contains 37 genes (13 protein-coding genes, 2 ribosomal RNAs, and 22 transfer RNAs) and a major non-coding control region. Among the 13 protein-coding genes of all the three sinipercid fishes, three reading-frame overlaps were found on the same strand. There is an 81-bp tandem repeat cluster at the end of CSB-3 in the S. scherzeri control region. The complete mitochondrial genomes of the three sinipercids should be useful for the evolutionary studies of sinipercids and other vertebrate species.     

Profiling cellular protein complexes by proximity ligation with dual tag microarray readout

Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.     

Chimeric piggyBac transposases for genomic targeting in human cells

Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy.     

贵州喀斯特石漠化类型及程度评价

由于碳酸盐岩岩性的不同,发育的喀斯特在生态环境因子的组合和质量上有较大的差异,形成了不同的石漠化类型。在大量典型样地调查的基础上,以岩性、小生境种数、小生境组合、裸露石面面积、石砾含量和土壤总量6个因子为指标,通过聚类分析和主成分分析相结合的方法,将喀斯特石漠化分为2大类型,即显性石漠化和隐性石漠化,分析比较了它们的主要特征。同时,分别石漠化类型选择了程度评价指标,用坐标综合评定法和欧氏距离公式建立了石漠化数量评价模型,即石漠化程度指数模型,用石漠化程度指数对样地的石漠化现状进行了评价,将6 4个样地划分为未石漠化,轻度、中度、强度和极强度5个等级。     

Effectiveness of the cleaning performance of the bacterial strain <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. SG06-02 in the petroleum hydrocarbon-polluted intestine of clams

Summary A petroleum-degrading bacterial strain Acinetobacter sp. SG06-02 in the oil-polluted intestine of shellfish clam was previously isolated in our laboratory. In this study, the effectiveness of using its cleaning performance on petroleum pollution was investigated. The accumulation of petroleum in the intestine of Ruditapes philippinarum was determined by a UV spectrophotometer. The survival and growth of the SG06-02 strain in the intestine of Ruditapes philippinarum after oil pollution were examined. The concentration of oil accumulated in the Ruditapes philippinarum intestine quickly rises to 254.97 mg/kg within 12 h after placing the clam in the petroleum-polluted seawater tank. After decontaminating using Acinetaobacter we found that the concentration of petroleum inside the Ruditapes philippinarum declined significantly. The results of the cleaning experiments showed that the degradation activity by using bioremediation method of the strain SG06-02 increased 12.8∼30.2% compared to using clean seawater in 4 days. This research indicated that the petroleum degrading bacteria could survive and was effective in cleaning oily pollutants in the seashell.     

A dual-tag microarray platform for high-performance nucleic acid and protein analyses

DNA microarrays serve to monitor a wide range of molecular events, but emerging applications like measurements of weakly expressed genes or of proteins and their interaction patterns will require enhanced performance to improve specificity of detection and dynamic range. To further extend the utility of DNA microarray-based approaches we present a high-performance tag microarray procedure that enables probe-based analysis of as little as 100 target cDNA molecules, and with a linear dynamic range close to 10⁵. Furthermore, the protocol radically decreases the risk of cross-hybridization on microarrays compared to current approaches, and it also allows for quantification by single-molecule analysis and real-time on-chip monitoring of rolling-circle amplification. We provide proof of concept for microarray-based measurement of both mRNA molecules and of proteins, converted to tag DNA sequences by padlock and proximity probe ligation, respectively.     

Transgenic breeding of anti-Bombyx mori L. nuclear polyhedrosis virus silkworm Bombyx mori

Silkworm strains resistant to Bombyx mori L. nuclear polyhedrosis virus were obtained through transgenic experiments. piggyBac transposon with an A3 promoter were randomly inserted into the silkworm, driving the enhanced green fluorescent protein (EGFP) reporter gene into the silkworm genome. Polymerase chain reaction results verified the insertion of the extraneous EGFP gene, and fluorescence microscopy showed that the EGFP was expressed in the midgut tissue. The morbidity ratio of the nuclear polyhedrosis decreased from 90% in the original silkworm strain to 66.7% in the transgenic silkworm strain. Compared with the resistance to the Bombyx mori L. nuclear polyhedrosis virus in the Qiufeng strain, which is commonly used in the production, there was an increase of 33 centesimal points in the transgenic silkworms. The antivirotic character in the Chunhua x Qiuyue strain, which was bred from a different transgenic family, was about 10 centesimal points higher than that in the Qiufeng x Baiyu, another crossbreed used in production. Our results indicated a good application value of the transposon-inserted mutation in the breeding of anti-BmNPV silkworm strain.     

<Emphasis Type="Italic">In planta</Emphasis> production and characterization of a hyperthermostable GH10 xylanase in transgenic sugarcane

Sugarcane (Saccharum sp. hybrids) is one of the most efficient and sustainable feedstocks for commercial production of fuel ethanol. Recent efforts focus on the integration of first and second generation bioethanol conversion technologies for sugarcane to increase biofuel yields. This integrated process will utilize both the cell wall bound sugars of the abundant lignocellulosic sugarcane residues in addition to the sucrose from stem internodes. Enzymatic hydrolysis of lignocellulosic biomass into its component sugars requires significant amounts of cell wall degrading enzymes. In planta production of xylanases has the potential to reduce costs associated with enzymatic hydrolysis but has been reported to compromise plant growth and development. To address this problem, we expressed a hyperthermostable GH10 xylanase, xyl10B in transgenic sugarcane which displays optimal catalytic activity at 105?°C and only residual catalytic activity at temperatures below 70?°C. Transgene integration and expression in sugarcane were confirmed by Southern blot, RT-PCR, ELISA and western blot following biolistic co-transfer of minimal expression cassettes of xyl10B and the selectable neomycin phosphotransferase II. Xylanase activity was detected in 17 transgenic lines with a fluorogenic xylanase activity assay. Up to 1.2% of the total soluble protein fraction of vegetative progenies with integration of chloroplast targeted expression represented the recombinant Xyl10B protein. Xyl10B activity was stable in vegetative progenies. Tissues retained 75% of the xylanase activity after drying of leaves at 35?°C and a 2 month storage period. Transgenic sugarcane plants producing Xyl10B did not differ from non-transgenic sugarcane in growth and development under greenhouse conditions. Sugarcane xylan and bagasse were used as substrate for enzymatic hydrolysis with the in planta produced Xyl10B. TLC and HPLC analysis of hydrolysis products confirmed the superior catalytic activity and stability of the in planta produced Xyl10B with xylobiose as a prominent degradation product. These findings will contribute to advancing consolidated processing of lignocellulosic sugarcane biomass.     

Synthesis and biological evaluation of a new series of cinnamic acid amide derivatives as potent haemostatic agents containing a 2-aminothiazole substructure

Ten new cinnamic acid derivatives containing a 2-aminothiazole substructure were designed and synthesized. This series of compounds exhibited good thermostabilities as demonstrated by thermogravimetric analysis. In coagulation assays (prothrombin time, activated partial thromboplastin time and thrombin time) in vitro, most compounds demonstrated excellent activities to promote blood coagulation. Among the studied series, compounds N1, N4, N5 and W5 exhibited a significant coagulation activity. Further studies indicated that compound N5 (IC₅₀ = 1.87 μmol/L) displayed the most suitable efficacy of promoting platelet aggregation than the clinically used haemostatic drug etamsylate (IC₅₀ = 46.22 μmol/L). Furthermore, the relationship between the functional groups of the compounds and the corresponding blood coagulant activity was explored in this study.