A Fully Automated Process Using a Magnetic Particle Based Kit for Removal of Dye Terminators from Sequencing Reactions

Prolinx,® Inc. of Bothell, WA has developed the RapXtract™ 384 Dye Terminator Removal Kit for full automation of DNA sequencing reaction purification. The RapXtract product line is based upon proprietary superparamagnetic particle technology that eliminates the need for centrifugation, vacuum filtration, or modified primers to achieve purification of sequencing reactions. The kit described here is pre-dispensed in a 384-well microtiter plate and run on the TECAN GENESIS Workstation 150 (Tecan U.S. Inc., Research Triangle Park, NC). This system enables rapid purification of up to 384 sequencing reactions in a single run.As the completion of the Human Genome Project nears, it is imperative for biotechnology and pharmaceutical companies to increase throughput of DNA sequencing in order to be competitive in the drug discovery and validation process. The “race to market” requires a shift from standard DNA sequencing processes-including DNA sequencing reaction purification-towards complete walk-away automation for all steps.Existing sequencing reaction purification methods (Table 1) require considerable resources including: plastic and other laboratory consumables; specialized equipment, such as high-speed centrifuges or vacuum filtration apparatus; and labor-intensive protocols requiring large amounts of technician time. As a result, walk-away automation of standard purification methods is difficult and expensive.     

Characterization of silica-based monoliths with bimodal pore size distribution

Band dispersion was studied and the retention thermodynamics addressed for insulin and angiotensin II on C18 silica monoliths with a bimodal pore size distribution, covering linear mobile-phase velocities up to 1 cm/s and different temperatures. These data suggest that the influence of average column pressure on retention (between 0 and 10 MPa) is not negligible. Plate height curves were interpreted with the van Deemter equation by assuming an independent contribution from mechanical and non-mechanical dispersion mechanisms. This analysis revealed diffusion-limited mass transfer in the mesoporous silica skeleton which, in turn, allowed us to calculate an equivalent dispersion particle diameter (d(disp) = 3 microm) using the C-term parameter of the van Deemter equation. The resulting superposition of reduced plate height curves for monolithic and particulate beds confirmed that this view presents an adequate analogy. The macroporous interskeleton network responsible for the hydraulic permeability of a monolith was translated to the interparticle pore space of particulate beds, and an equivalent permeability particle diameter (d(perm) = 15 microm) was obtained by scaling based on the Kozeny-Carman equation.     

Prediction of chromatographic retention and protein identification in liquid chromatography/mass spectrometry

Liquid chromatography coupled on- or off-line with mass spectrometry is rapidly advancing as a tool in proteomics capable of dealing with the inherent complexity in biology and complementing conventional approaches based on two-dimensional gel electrophoresis. Proteins can be identified by proteolytic digestion and peptide mass fingerprinting or by searching databases using short-sequence tags generated by tandem mass spectrometry. This paper shows that information on the chromatographic behavior of peptides can assist protein identification by peptide mass fingerprinting in liquid chromatography/mass spectrometry. This additional information is significant and already available at no extra experimental cost.     

The SARS Crisis: Was Anybody Responsible?

As scholars in fields such as media studies, crisis studies and public policy studies have argued, there exists a fundamental link between crises and the media. Once an event has been interpreted as a crisis, questions of accountability inevitably appear on the media agenda, and the struggle to attribute blame and responsibility to a specific entity or entities—the blame game—thus becomes an inexorable part of the crisis process. Focusing on three liberal Western newspapers with an international, primarily Western elite readership and a reputation for in‐depth analysis of global events, The Economist, the Financial Times and the International Herald Tribune, this article employs Iyengar's and Valkenburg's notions of responsibility frames to examine whether initial coverage of the 2003 SARS crisis in these accounts held any particular entity accountable for the crisis, looks at three key themes used to communicate to the reader a particular way of thinking about responsibility for SARS and examines some of the consequences of the kind of responsibility frame constructed around the SARS crisis in these accounts. As our findings show, there is an entity that the early news accounts studied consistently held responsible for the 2003 SARS crisis, the Chinese system, and the corresponding responsibility frame at operation in these accounts is thematic rather than episodic in nature, since it consistently places the SARS crisis within a broader context (a product of “China” itself and/or of societal‐governmental forces in China) rather than in relation to a specific episode or as the result of the particular actions of individuals. The SARS crisis narrative therefore presented in these accounts tells the story of an anachronistic Chinese system faced with a contemporary health threat that, by its very nature, it is incapable of assessing accurately or managing responsibly. By way of conclusion, we argue that, while the use of such a thematic frame to explain China's role in the 2003 SARS crisis may be accurate in certain respects, this frame falls short in other respects and proves particularly inadequate to the challenge of capturing the economic complexities of China's role during the crisis.     

DEVAN: a tool for detailed video analysis of user test data

A tool was developed for structured and detailed analysis of video data from user tests of interactive systems. It makes use of a table format for representing an interaction at multiple levels of abstraction. Interactions are segmented based on threshold times for pauses between actions. Usability problems are found using a list of observable indications for the occurrence of problems. The tool was evaluated by having two analysts apply it to three data sets from user tests on two different products. The segmentation technique proved to yield meaningful segments that helped in understanding the interaction. The interaction table was explicit enough to discuss in detail what had caused the differences in the analysts' lists of usability problems. The results suggested that the majority of differences were caused by unavoidable differences in interpretations of subjects' behaviour and that only minor improvements should be expected by refining the tool.     

Cocaine-conditioned place preference in young precocial domestic fowl.

Experiment 1 established conditioned place preference (CPP) control procedures for young precocial domestic fowl (test day age?=?13–25 days). Chicks displayed a preference (greater choice time) for white stimulus cards on first exposure to white versus red cards and equivalent preference for these stimulus cards after 6 exposures. Chicks in Experiment 2 displayed a cocaine CPP at each dose (0.5, 1.0, and 2.0 mg/kg intraperitoneally) after 6 cocaine-red card and 6 saline-white card pairings. This CPP decreased and was no longer significant over 2 additional nonreinforced test days. Experiment 3 results replicated the cocaine CPP (2.0 mg/kg) and demonstrated that the CPP did not develop when cocaine was administered several hours after card exposure. To our knowledge, these results are the first to demonstrate drug CPP in a nonmammalian model and support the notion that drug reinforcement processes are highly conserved. This precocial avian model may prove a useful addition to existing altricial models for the study of determinants of human drug use and abuse. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Gas chromatographic separation of linoleic acid hydroperoxides as trimethylsilyl ethers of methyl hydroxystearates

The primary products, 9- and 13-hydroperoxy-octadecadienoic acids, from lipoxygenase catalyzed oxidation of linoleic acid, were converted into the trimethylsilyl ethers of methyl 9- and 13-hydroxystearates which were completely separated by gas chromatography on an OV-17 methyl silicone (50% phenyl groups), capillary column.     

Tocotrienol-rich fraction from palm oil affects gene expression in tumors resulting from MCF-7 cell inoculation in athymic mice

It has recently been shown that tocotrienols are the components of vitamin E responsible for inhibiting the growth of human breast cancer cells in vitro, through an estrogen-independent mechanism. Although tocotrienols act on cell proliferation in a dose-dependent manner and can induce programmed cell death, no specific gene regulation has yet been identified. To investigate the molecular basis of the effect of tocotrienols, we injected MCF-7 breast cancer cells into athymic nude mice. Mice were fed orally with 1 mg/d of tocotrienol-rich fraction (TRF) for 20 wk. At end of the 20 wk, there was a significant delay in the onset, incidence, and size of the tumors in nude mice supplemented with TRF compared with the controls. At autopsy, the tumor tissue was excised and analyzed for gene expression by means of a cDNA array technique. Thirty out of 1176 genes were significantly affected. Ten genes were down-regulated and 20 genes up-regulated with respect to untreated animals, and some genes in particular were involved in regulating the immune system and its function. The expression of the interferon-inducible transmembrane protein-1 gene was significantly up-regulated in tumors excised from TRF-treated animals compared with control mice. Within the group of genes related to the immune system, we also found that the CD59 glycoprotein precursor gene was up-regulated. Among the functional class of intracellular transducers/effectors/modulators, the c-myc gene was significantly down-regulated in tumors by TRF treatment. Our observations indicate that TRF supplementation significantly and specifically affects MCF-7 cell response after tumor formation in vivo and therefore the host immune function. The observed effect on gene expression is possibly exerted independently from the antioxidant activity typical of this family of molecules.