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.

VEGF gene silencing by cytomegalovirus promoter driven ShRNA expression vector results in vascular development defects in zebrafish

Zebrafish has been generally considered as an excellent model in case of drug screening, disease model establishment, and vertebrate embryonic development study. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against VEGF gene in zebrafish was tested, and its effect on vascular development was assed, too. Using RT-qPCR, blood vessel staining, and in situ hybridization, we confirmed certain transcriptional activity and down regulation of gene expression by the vector. In situ hybridization analysis indicated selective inhibition of NRP1 expression in the VEGF gene loss of function model, which might imply in turn that VEGF could not only activate endothelial cells directly but also could contribute to stimulating angiogenesis in vivo by a mechanism that involved up-regulation of its cognate receptor expression in zebrafish. This contributed to a better understanding of molecular mechanisms of cardiovascular development. The system improved the success rate in making inducible knockdown and widened the possibilities for better therapeutic targets in zebrafish.

Construction and characterization of a bacterial artificial chromosome library of marine macroalga Porphyra yezoensis (Rhodophyta)

A large-DNA-fragment library is necessary for research into the Porphyra genome. In this study, a bacterial artificial chromosome (BAC) library of Porphyra yezoensis was constructed and characterized. The library contains 54,144 BAC clones with an average insert size of about 65 kb and fewer than 0.7% of clones without large inserts. Therefore, its capacity is more than 6.6 P. yezoensis genome equivalents, and the probability of recovering any nuclear DNA sequence from the library is higher than 99%. The library shows good fidelity and stability. A putative trehalose-6-phosphate synthase (TPS) gene was successfully screened out from the library. The above results show that the library is useful for gene cloning and genomic research in P. yezoensis.

Size and time of day affect the response of postlarvae and early juvenile grooved tiger prawns Penaeus semisulcatus De Haan (Decapoda:Penaeidae) to natural and artificial seagrass in the laboratory

Juvenile tiger prawns (Penaeus semisulcatus De Haan and P. esculentus Haswell) show a strong association with vegetated habitats and are rarely caught on non-vegetated areas. This pattern of distribution may be caused by postlarvae selecting vegetation when they settle, or to differences in post-settlement mortality in different habitats. In this study, we examined whether the postlarvae and early juvenile stages of P. semisulcatus would distinguish between seagrass (Zostera capricorni Aschers) without epiphytes, artificial seagrass and bare substratum in the laboratory. The responses of prawns reared from the egg to different stages of postlarval and juvenile development were tested to determine whether, and when, each size class showed a response to a particular habitat. Five size classes of postlarvae (average carapace lengths [CL] of 1.2, 1.4, 1.6, 1.7 and 2.0 mm) were offered a choice between Z. capricorni and bare sand. Small size classes of postlarvae either did not respond to Z. capricorni (1.2 and 1.6 mm CL), or were more abundant on bare substratum than Z. capricorni. In contrast, the largest size classes of postlarvae (1.7 and 2.0 mm CL) were more abundant on Z. capricorni during the day but not at night. The behaviour of postlarvae changed markedly at a size of 1.7 mm CL (22 days from the first nauplius): smaller postlarvae frequently swam in the water column; 1.7 and 2.0 mm CL postlarvae spent much more. time resting on the substrate and perched on seagrass leaves. This size at which postlarvae first respond to seagrass during the day, and show mainly benthic behaviour, is similar to the size at which they are found on shallow seagrass beds in northern Australia. Large postlarvae (2.7 mm CL) and juveniles (4.1 mm CL) both were more abundant on artificial seagrass than bare sand during the day but not at night, indicating that they respond to structured habitats. When large postlarvae (2.4 mm CL) and juveniles (3.5 mm CL) were offered a choice between Z. capricorni without epiphytes and artificial seagrass, they were more abundant on the Z. capricorni, which suggests that chemical cues from seagrass may explain some of the responses of P. semisulcatus to seagrass. (C) 1997 Elsevier Science B.V.

Construction and characterization of two bacterial artificial chromosome libraries of Zhikong scallop, Chlamys farreri Jones et Preston, and identification of BAC clones containing the genes involved in its innate immune system

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.

Construction of bacterial artificial chromosome libraries for Zhikong Scallop Chlamys farreri

Two Large-insert genomic bacterial artificial chromosome (BAC) libraries of Zhikong scallop Chlamys farreri were constructed to promote our genetic and genomic research. High-quality megabase-sized DNA was isolated from the adductor muscle of the scallop and partially digested by BamH I and Mbo I, respectively. The BamH I library consisted of 53 760 clones while the Mbo I library consisted of 7 680clones. Approximately 96 % of the clones in BamH I library contained nuclear DNA inserts in average size of 100 kb, providing a coverage of 5.3 haploid genome equivalents. Similarly, the Mbo I library with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1 haploid genome equivalents.

Optimal Fuzzy PID Controller with Incomplete Derivation and Its Simulation Research on Application of Intelligent Artificial Legs

提出了一种新的最优模糊PID控制器，它由两部分组成，即在线模糊推理机构和带有不完全微分的常规PID控制器，在模糊推理机构中，引入了三个可调节因子xp,xi和xd，其作用是进一步修改和优化模糊推理的结果，以使控制器对一个给定对象具有最优的控制效果，可调节因子的最优值采用ITAE准则及Nelder和Mead提出的柔性多面体最优搜索算法加以确定，这种PID控制器被用来控制由作者设计的智能人工腿中的一个直流电机，仿真结果表明该控制器的设计是非常有效的，它可被用于控制各种不同的对象和过程。

Direct observation of subsurface oxygen on the defects of Pd(100)

Oxygen adsorption and desorption on a Pd(100) surface with a mesoscopic defect were studied by photoemission electron microscopy (PEEM). The defect surface, with an area of approximately 200 x 60 mu m(2), behaved differently from the perfect Pd(100) surface towards the adsorption of oxygen. When saturated, both surface oxygen and subsurface oxygen coexisted on the defect surface, whereas only surface oxygen was present on the Pd(100) surface. Upon heating, subsurface oxygen diffused back to the surface and desorbed with surface oxygen at the same time. The difference in oxygen adsorption ability between the defect surface and the perfect Pd(100) surface can be attributed to different structures of these two surfaces. (C) 1999 Elsevier Science B.V. All rights reserved.