A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac

With the availability of complete genome sequence for Drosophila melanogaster, one of the next strategic goals for fly researchers is a complete gene knockout collection. The P-element transposon, the workhorse of D. melanogaster molecular genetics, has a pronounced nonrandom insertion spectrum. It has been estimated that 87% saturation of the approximately 13,500-gene complement of D. melanogaster might require generating and analyzing up to 150,000 insertions. We describe specific improvements to the lepidopteran transposon piggyBac and the P element that enabled us to tag and disrupt genes in D. melanogaster more efficiently. We generated over 29,000 inserts resulting in 53% gene saturation and a more diverse collection of phenotypically stronger insertional alleles. We found that piggyBac has distinct global and local gene-tagging behavior from that of P elements. Notably, piggyBac excisions from the germ line are nearly always precise, piggyBac does not share chromosomal hotspots associated with P and piggyBac is more effective at gene disruption because it lacks the P bias for insertion in 5' regulatory sequences.     

Improved D-IDS using the federated peer-to-peer architecture

A number of IDSs have been proposed for a networked or distributed environment. A modified D-IDS using federated peer-to-peer architecture, MCR (Multicast Reflector) and modified shaker protocol were proposed. The suggested scheme can be implemented easily and performs the information sharing between low-level IDS agents. As all users within a group monitor each other's, the common control server can perform detect intrusions with less cost and support the detection of the inside intruders.     

Repetitive shuttling of a motor protein on DNA

Many helicases modulate recombination, an essential process that needs to be tightly controlled. Mutations in some human disease helicases cause increased recombination, genome instability and cancer. To elucidate the potential mode of action of these enzymes, here we developed a single-molecule fluorescence assay that can visualize DNA binding and translocation of Escherichia coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae Srs2. Individual Rep monomers were observed to move on single-stranded (ss)DNA in the 3' to 5' direction using ATP hydrolysis. Strikingly, on hitting a blockade, such as duplex DNA or streptavidin, the protein abruptly snapped back close to its initial position, followed by further cycles of translocation and snapback. This repetitive shuttling is likely to be caused by a blockade-induced protein conformational change that enhances DNA affinity for the protein's secondary DNA binding site, thereby resulting in a transient DNA loop. Repetitive shuttling was also observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment analogue, and the presence of Rep delayed formation of a filament of recombination protein RecA on ssDNA. Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates.     

Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation.

Enzyme-based chemical transformations typically proceed with high selectivity under mild conditions, and are becoming increasingly important in the pharmaceutical and chemical industries. Cytochrome P450 monooxygenases (P450s) constitute a large family of enzymes of particular interest in this regard. Their biological functions, such as detoxification of xenobiotics and steroidogenesis, are based on the ability to catalyse the insertion of oxygen into a wide variety of compounds. Such a catalytic transformation might find technological applications in areas ranging from gene therapy and environmental remediation to the selective synthesis of pharmaceuticals and chemicals. But relatively low turnover rates (particularly towards non-natural substrates), low stability and the need for electron-donating cofactors prohibit the practical use of P450s as isolated enzymes. Here we report the directed evolution of the P450 from Pseudomonas putida to create mutants that hydroxylate naphthalene in the absence of cofactors through the 'peroxide shunt' pathway with more than 20-fold higher activity than the native enzyme. We are able to screen efficiently for improved mutants by coexpressing them with horseradish peroxidase, which converts the products of the P450 reaction into fluorescent compounds amenable to digital imaging screening. This system should allow us to select and develop mono- and di-oxygenases into practically useful biocatalysts for the hydroxylation of a wide range of aromatic compounds.     

Improved D-IDS using the federated peer-to-peer architecture

A number of IDSs have been proposed for a networked or distributed environment. A modified D-IDS using federated peer-to-peer architecture, MCR （Multicast Reflector） and modified shaker protocol were proposed. The suggested scheme can be implemented easily and performs the information sharing between low-level IDS agents. As all users within a group monitor each other＇s, the common control server can perform detect intrusions with less cost and support the detection of the inside intruders.     

Ordered nanoporous arrays of carbon supporting high dispersions of platinum nanoparticles.

Nanostructured carbon materials are potentially of great technological interest for the development of electronic, catalytic and hydrogen-storage systems. Here we describe a general strategy for the synthesis of highly ordered, rigid arrays of nanoporous carbon having uniform but tunable diameters (typically 6 nanometres inside and 9 nanometres outside). These structures are formed by using ordered mesoporous silicas as templates, the removal of which leaves a partially ordered graphitic framework. The resulting material supports a high dispersion of platinum nanoparticles, exceeding that of other common microporous carbon materials (such as carbon black, charcoal and activated carbon fibres). The platinum cluster diameter can be controlled to below 3 nanometres, and the high dispersion of these metal clusters gives rise to promising electrocatalytic activity for oxygen reduction, which could prove to be practically relevant for fuel-cell technologies. These nanomaterials can also be prepared in the form of free-standing films by using ordered silica films as the templates.     

集成电路电磁适应性的时钟信号调制

电磁干扰(EMI)是瞬时功能故障的主要来源之一,原因是电源供电线上的噪声注入引起了VDD和GND的额定值的波动.介绍了一种新的方法来增强片上系统(SoC)关于电源和接地电压瞬变时的信号完整性,并且这种方法完全符合IEC 61000-4-29 标准.其基本思想是设计和IEC 61000-4-29兼容的集成电路,通过局部和动态地将时钟占空比去适应传播延迟的变化和扰动的逻辑路径.当无法满足时,该方法导致暂时把集成电路的工作频率减小到满足该标准的最小值.根据异常电网活动,时钟占空比调制(CDCM)是通过使用正和/或负边沿时钟展宽逻辑(CSL)块来实现的.基于这个概念,在尽可能保持时钟高速频率的同时,数字电路对于电源供电线上波动的耐受性将会更强.该方法可被视作一种在线提供动态自适应同步的监视技术.通过SPICE模拟,实验结果表明此方法的有效性.     

TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity

Retinoic-acid-inducible gene-I (RIG-I; also called DDX58) is a cytosolic viral RNA receptor that interacts with MAVS (also called VISA, IPS-1 or Cardif) to induce type I interferon-mediated host protective innate immunity against viral infection. Furthermore, members of the tripartite motif (TRIM) protein family, which contain a cluster of a RING-finger domain, a B box/coiled-coil domain and a SPRY domain, are involved in various cellular processes, including cell proliferation and antiviral activity. Here we report that the amino-terminal caspase recruitment domains (CARDs) of RIG-I undergo robust ubiquitination induced by TRIM25 in mammalian cells. The carboxy-terminal SPRY domain of TRIM25 interacts with the N-terminal CARDs of RIG-I; this interaction effectively delivers the Lys 63-linked ubiquitin moiety to the N-terminal CARDs of RIG-I, resulting in a marked increase in RIG-I downstream signalling activity. The Lys 172 residue of RIG-I is critical for efficient TRIM25-mediated ubiquitination and for MAVS binding, as well as the ability of RIG-I to induce antiviral signal transduction. Furthermore, gene targeting demonstrates that TRIM25 is essential not only for RIG-I ubiquitination but also for RIG-I-mediated interferon- production and antiviral activity in response to RNA virus infection. Thus, we demonstrate that TRIM25 E3 ubiquitin ligase induces the Lys 63-linked ubiquitination of RIG-I, which is crucial for the cytosolic RIG-I signalling pathway to elicit host antiviral innate immunity.     

State space trend-cycle decomposition of the ARIMA(1,1,1) process

It has been common practice to decompose an integrated time series into a random walk trend and a stationary cycle using the state space model. Application of state space trend-cycle decomposition, however, often results in a misleading interpretation of the model, especially when the observability of the state space model and the redundant relationships among the model parameters are not properly considered. In this study, it is shown that spurious trend-cycle decomposition, discussed by Nelson (1988), results from an unobservable state space model, and the usual assumption of independent noise processes in the model results in parameter redundancy. Equivalence relationships for the ARIMA(1,1,1) process and the state space model consisting of a random walk trend and an AR(1) cycle, where the noise processes of the trend and of the cycle are generally correlated, are also derived. © 1997 John Wiley & Sons, Ltd.