A suite of parallel vectors for baculovirus expression

The expression of proteins using recombinant baculoviruses is a mature and widely used technology. However, some aspects of the technology continue to detract from high throughput use and the basis of the final observed expression level is poorly understood. Here, we describe the design and use of a set of vectors developed around a unified cloning strategy that allow parallel expression of target proteins in the baculovirus system as N-terminal or C-terminal fusions. Using several protein kinases as tests we found that amino-terminal fusion to maltose binding protein rescued expression of the poorly expressed human kinase Cot but had only a marginal effect on expression of a well-expressed kinase IKK-2. In addition, MBP fusion proteins were found to be secreted from the expressing cell. Use of a carboxyl-terminal GFP tagging vector showed that fluorescence measurement paralleled expression level and was a convenient readout in the context of insect cell expression, an observation that was further supported with additional non-kinase targets. The expression of the target proteins using the same vectors in vitro showed that differences in expression level were wholly dependent on the environment of the expressing cell and an investigation of the time course of expression showed it could affect substantially the observed expression level for poorly but not well-expressed proteins. Our vector suite approach shows that rapid expression survey can be achieved within the baculovirus system and in addition, goes some way to identifying the underlying basis of the expression level obtained. (c) 2006 Elsevier Inc. All rights reserved.

Rapid parallel expression in E-coli and insect cells: Analysis of five lef gene products of the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV)

A number of strategies are emerging for the high throughput (HTP) expression of recombinant proteins to enable structural and functional study. Here we describe a workable HTP strategy based on parallel protein expression in E. coli and insect cells. Using this system we provide comparative expression data for five proteins derived from the Autographa californica polyhedrosis virus genome that vary in amino acid composition and in molecular weight. Although the proteins are part of a set of factors known to be required for viral late gene expression, the precise function of three of the five, late expression factors (lefs) 6, 7 and 10, is unknown. Rapid expression and characterisation has allowed the determination of their ability to bind DNA and shown a cellular location consistent with their properties. Our data point to the utility of a parallel expression strategy to rapidly obtain workable protein expression levels from many open reading frames (ORFs).

Supramolecular parallel beta-sheet and amyloid-like fibril forming peptides using delta-aminovaleric acid residue

Four terminally blocked tripeptides containing delta-aminovaleric acid residue self-assemble to form supramolecular beta-sheet structures as are revealed from their FT-IR data. Single crystal X-ray diffraction studies of two representative peptides also show that they form parallel beta-sheet structures. Self-aggregation of these beta-sheet forming peptides leads to the formation of fibrillar structures, as is evident from scanning electron microscopic (SEM) and transmission electron microscopic (TEM) images. These peptide fibrils bind to a physiological dye, Congo red and exhibit a typical green-gold birefringence under polarized light, showing close resemblance to neurodegenerative disease causing amyloid fibrils. (c) 2005 Elsevier Ltd. All rights reserved.

Studies on the parallel synthesis and evaluation of new heterocyclic extractants for the partitioning of minor actinides

Multiple parallel synthesis and evaluation have been combined in order to identify new nitrogen heterocycles for the partitioning of minor actinides(III) such as americium(III) from lanthanides such as europium(Ill). An array of triazine-containing molecules was made using multiple parallel syntheses from diketones and amide hydrazides. An excess of each of the resulting purified reagents was dissolved in 1,1,2,2-tetrachloroethane containing 2-bromodecanoic acid, and equilibrated with an aqueous solution containing the radiotracers Eu-152 and Am-241 in nitric acid ([Eu] + [Am] < 400 nanomol dm(-3)). Gamma counting of the organic and aqueous phases led to the identification of several new reagents for the selective extraction of americium(III). In particular, 6-(2-pyridyl)-2-(5,6-dialkyl-1,2,4-triazaphenyl)pyridines were found to be effective reagents for the separation of americium(III) from europium(III), (SFAm/Eu was ca. 30 in [HNO3] = 0.013 mol/L).

2D parallel interpenetration of [M-2(bpp)(4)X-4] [M, Fe(II)/Co(II); bpp, 4,4 '-trimethylenedipyridine; X, SCN-SeCN- and N-3(-)] complexes: pseudohalide-dependent conformation of bpp

Three coordination complexes of Co(II)/Fe(II) with 4,4'-trimethylenedipyridine (bpp) and pseudohalides (SCN-, SeCN- and N-3(-)) have been synthesized. The complexes have been characterized by X-ray single crystal structure determination. They are isomorphous having 2D layers in which two independent wavy nets display parallel interwoven structures. Pseudohalide binds metal centers through N terminal and occupies the trans axial positions of the octahedral metal coordination environment. Pseudohalide remains pendant on both sides of the polymeric layer and help the stacking through hydrogen bonding. The conformation of bpp in the interpenetrated nets is observed to be dependent on the choice of pseudohalide. (C) 2008 Elsevier Inc. All rights reserved.

Giving up on modern foreign languages? Students' perceptions of learning French

This article reports on the findings of an investigation into the attitudes of English students aged 16 to 19 years towards French and how they view the reasons behind their level of achievement. Those students who attributed success to effort, high ability, and effective learning strategies had higher levels of achievement, and students intending to continue French after age 16 were more likely than noncontinuers to attribute success to these factors. Low ability and task difficulty were the main reasons cited for lack of achievement in French, whereas the possible role of learning strategies tended to be overlooked by students. It is argued that learners' self-concept and motivation might be enhanced through approaches that encourage learners to explore the causal links between the strategies they employ and their academic performance, thereby changing the attributions they make for success or failure.

The remote distractor effect in saccade programming: channel interactions and lateral inhibition

We explored the dependency of the saccadic remote distractor effect (RDE) on the spatial frequency content of target and distractor Gabor patches. A robust RDE was obtained with low-medium spatial frequency distractors, regardless of the spatial frequency of the tat-get. High spatial frequency distractors interfered to a similar extent when the target was of the same spatial frequency. We developed a quantitative model based on lateral inhibition within an oculomotor decision unit. This lateral inhibition mechanism cannot account for the interaction observed between target and distractor spatial frequency, pointing to the existence of channel interactions at an earlier level. (C) 2004 Elsevier Ltd. All rights reserved.

Parallel and distributed computing with Java

The Java language first came to public attention in 1995. Within a year, it was being speculated that Java may be a good language for parallel and distributed computing. Its core features, including being objected oriented and platform independence, as well as having built-in network support and threads, has encouraged this view. Today, Java is being used in almost every type of computer-based system, ranging from sensor networks to high performance computing platforms, and from enterprise applications through to complex research-based.simulations. In this paper the key features that make Java a good language for parallel and distributed computing are first discussed. Two Java-based middleware systems, namely MPJ Express, an MPI-like Java messaging system, and Tycho, a wide-area asynchronous messaging framework with an integrated virtual registry are then discussed. The paper concludes by highlighting the advantages of using Java as middleware to support distributed applications.

Hierarchical composite regular parallel architecture

The design space of emerging heterogenous multi-core architectures with re-configurability element makes it feasible to design mixed fine-grained and coarse-grained parallel architectures. This paper presents a hierarchical composite array design which extends the curret design space of regular array design by combining a sequence of transformations. This technique is applied to derive a new design of a pipelined parallel regular array with different dataflow between phases of computation.