Targeted disruption of the Pak5 and Pak6 genes in mice leads to deficits in learning and locomotion

PAK6 is a member of the group B family of PAK serine/threonine kinases, and is highly expressed in the brain. The group B PAKs, including PAK4, PAK5, and PAK6, were first identified as effector proteins for the Rho GTPase Cdc42. They have important roles in filopodia formation, the extension of neurons, and cell survival. Pak4 knockout mice die in utero, and the embryos have several abnormalities, including a defect in the development of motor neurons. In contrast, Pak5 knockout mice do not have any noticeable abnormalities. So far nothing is known about the biological function of Pak6. To address this, we have deleted the Pak6 gene in mice. Since Pak6 and Pak5 are both expressed in the brain, we also generated Pak5/Pak6 double knockout mice. These mice were viable and fertile, but had several locomotor and behavioral deficits. Our results indicate that Pak5 and Pak6 together are not required for viability, but are required for a normal level of locomotion and activity as well as for learning and memory. This is consistent with a role for the group B PAKs in the nervous system.     

Electroporation as an efficient method of gene transfer

Gene transfer by electroporation is an indispensable method for the study of developmental biology, especially for the study using chick embryos. Here we briefly review the principles of the method, and its application to chick embryos. Methods of transient misexpression and long-term misexpression by retrovirus vector or transposon system, and knockdown by small interference RNA are reviewed.     

An ensemble model for predicting Rhopalosiphum padi abundance

A novel modeling method is proposed to predict the abundance of the main vector of barley yellow dwarf virus in autumn sown cereal crops. An ensemble model based on artificial neural networks (ANN) was developed to predict the number of Rhopalosiphum padi (L.) (Homoptera: Aphididae) caught in traps during the autumn flight period at Lincoln, Canterbury, New Zealand, over the period 1982–2003. Artificial neural networks were trained using historical weather data and aphid data collected from a suction trap. Model results were compared with those obtained using multiple regression (MR) models using the same independent variables. Both ANN and MR models were validated by leave‐one‐out validation, in other words, by sequentially jackknifing each year out of the data set, fitting a model to the remaining data, then using that model to predict the number of aphids for each jackknifed year. A linear ensemble of ANN models further improved the predictions and represented the trends in the number of aphids over the 22‐year period very well. The r² between the predicted and observed numbers of aphids for the ANN models changed from 0.68 to 0.83 using the linear ensemble model, but the ensemble approach did not change the prediction for the MR models. The absolute mean error (ABSME) of prediction was much lower for the ANN ensemble predictions compared to that for the MR models. The ABMSE for the ANN models dropped from 109 to 86 aphids compared to an ABMSE reduction from 245 to 220 aphids for the MR models. We discuss the potential for ensemble models for predicting insect abundance when long‐term historical data are available.     

Microbial control of Agrilus planipennis (Coleoptera: Buprestidae) with Beauveria bassiana strain GHA: field applications

The effects of Beauveria bassiana strain GHA, applied as BotaniGard ES, on newly colonised and well-established populations of emerald ash borer, Agrilus planipennis (Coleoptera: Buprestidae) were evaluated in the field using foliar and trunk sprays in Michigan in 2004–2005. Results from field trials at a newly colonised white ash site showed a 41% reduction in A. planipennis population in fungal-treated trees compared with that of untreated controls. In addition, fungal infection was also found in 20% of the larval population within 14 days of incubation under laboratory conditions. At a site with a well-established Agrilus planipennis population in green ash trees, larval density was reduced by 47% for trees treated with the fungus compared with that of the controls; 21% of larvae from the current generation were found infected after 14 days of laboratory incubation. Fungal-treated green ash trees also produced fewer adults emerging in the next generation, with a 63% reduction in adult density observed in treated trees compared to that of controls. As a result, fungal-treated trees sustained 42% less crown dieback than did controls. A. planipennis larval density was negatively correlated with trunk height above the ground, and positively correlated with log diameter. Results of laboratory leaf bioassays on A. planipennis adults showed that fungal conidia persisted well under field conditions, with mortality of 78–100% at 7 – days post – exposure for leaves collected between 2 and 264 h after application. Potential strategies for using B. bassiana strain GHA for managing A. planipennis are discussed.     

Evaluation of the parasitoid Copidosoma koehleri for biological control of the potato tuber moth,Phthorimaea operculella,in Israeli potato fields

The potato tuber moth Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) is a major agricultural pest of solaneceous crops in warm countries worldwide. The encyrtid polyembryonic parasitoid Copidosoma koehleri (Blanchard) has been successfully introduced for biological control of the moth in potato fields in South Africa and Australia; however, augmentative releases of the parasitoid in trial plots and in commercial potato fields in Israel did not reduce pest populations or infestation levels more than chemical treatment. Copidosoma koehleri accounted for 4–5% of parasitism on tuber moth caterpillars, while most parasitism was due to local species of larval parasitoids. The abundance and composition of local parasitoids did not differ between C. koehleri release plots and conventionally treated control plots. These findings can be interpreted as failure of the introduced parasitoids to survive and locate their hosts, or as mortality of C. koehleri within hosts in the field. Sentinel hosts, placed in trial plots and collected after 24 h, were rarely parasitised by C. koehleri, supporting the first interpretation. To test the second hypothesis, hosts parasitised by C. koehleri were placed in field plots for a week, collected, and reared out in the laboratory. The emergence rates of C. koehleri from these hosts resembled those of lab-reared controls, suggesting that mortality within hosts in the field is not a major cause of C. koehleri's poor biocontrol performance.     

Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large-scale processes

We set out to investigate if E. coli genotype plays a significant role in host strain selection for optimal processing of plasmid DNA based on both quality and quantity of supercoiling. Firstly 17 E. coli commercial and non-commercial strains were selected and their available genetic backgrounds were researched in the open literature. Growth characteristics of all the strains were considered and made impartial by using a common medium and growth condition platform. By keeping the growth conditions constant for each strain/plasmid combination, we are only looking at one variable which is the host strain. The second step was to attempt to correlate the findings with common genotype characteristics (e.g. mutations such as endA or recA). We found that one can screen the number of strains which are likely to give good productivity early on, before any further optimisation and verification is performed, both for small and large plasmids. Also, it is worth noting that complex plasmid interactions with each strain prevent the use of genotype alone in making an intelligent choice for supercoiling optimisation. This leads to a third optimisation step selecting a few of the potentially high performing strains based on high DNA yield and supercoiling, with a view to identify the factors which need improvement in strain design and bioreactor optimisation. We found that high specific growth rates of some strains did not affect the level of DNA supercoiling but did influence the total plasmid yield, potentially an important aspect in the design of fermentation strategy. Interestingly, a few host/plasmid combinations result in what appears to be runaway plasmid replication.     

Production of recombinant proteins in clonal root cultures using episomal expression vectors

We have developed a fully contained system for expressing recombinant proteins that is based on clonal root cultures and episomal expression vectors. Clonal root lines expressing green fluorescent protein (GFP) or human growth hormone were generated from Nicotiana benthamiana leaves infected with the tobacco mosaic virus-based vector 30B after exposure to Agrobacterium rhizogenes. These lines accumulated GFP at over 50 mg per kg fresh tissue, a level that is comparable with other plant production systems in early stage development. Accumulation of both hGH and GFP in the clonal root lines was sustained over a 3-year period, and in the absence of antibiotic selection. This technology shows promise for commercial production of vaccine antigens and therapeutic proteins in contained facilities.     

基于支持向量机方法的蛋白可溶性预测

按照蛋白质序列中残基的相对可溶性,将其分为两类(表面／内部)和三类(表面／中间／内部)进行预测。选择不同窗宽和参数对数据进行训练和预测,以确保得到最好的分类效果,并同其他已有方法进行比较。对同一数据集不同分类阈值的预测结果显示,支持向量机方法对蛋白质可溶性的整体预测效果优于神经网络和信息论的方法。其中,对两类数据的最优分类结果达到79．0％,对三类数据的最优分类结果达到67．5％,表明支持向量机是蛋白质残基可溶性预测的一种有效方法。