The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems.

The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications).

Acute pediatric stroke: contributors to institutional cost.

BACKGROUND AND PURPOSE: Recent studies examined the overall cost of pediatric stroke, but there are little data regarding the sources of these costs. We examined an administrative database that collected charges from 24 US children's hospitals to determine the sources of costs for acute hospital care of stroke. METHODS: We used International Classification of Diseases, 9th Revision codes to search the Pediatric Health Information System. From 2003 to 2009 there were 1667 patients who had a primary diagnosis of stroke, 703 of which were hemorrhagic and 964 were ischemic. Individual costs, excluding physician charges, were gathered under 7 categories that were ranked to determine which contributed the most to total cost. Individual costs were ranked within their categories. We analyzed costs based on stroke type. Total costs were adjusted using the US Consumer Price Index to compare increases with the rate of inflation. RESULTS: Median total cost for any stroke was $19,548 (interquartile range, $10,764-$40,721). The category "other/nursing" contributed the most to hospital costs followed by imaging, laboratory, and pharmacy. Brain MRI and CT contributed the most to imaging costs. Hemorrhagic strokes (median $24,843) were more expensive than ischemic strokes (median $16,954). Total cost increased from 2003 to 2009, but no overall annual trend emerged after controlling for gender, age, race, and hospital. CONCLUSIONS: This is the first in-depth analysis of cost for pediatric stroke care. The highest cost categories are potential targets for cost containment but are also crucial for effective diagnosis and treatment. Necessary yet prudent use of imaging technologies and inpatient stays may be strategies for cost containment.

Strategy of the use of 28S rRNA as a housekeeping gene in real-time quantitative PCR analysis of gene transcription in insect cells infected by viruses.

Quantitative real-time reverse transcription-PCR (qRT-PCR) has been used widely to measure gene transcription regulation in cells. qRT-PCR must include one or more internal housekeeping genes to normalize data collection. A strategy to use the host cell 28S rRNA as a housekeeping gene in qRT-PCR analysis of gene transcription of insect cells infected by baculovirus and ascovirus was developed. It has been found that the 28S rRNA reverse primer can be incorporated in the oligo-dT-primed cDNA synthesis reaction. In such a way, amplification of 28S cDNA showed lower and less variable cycle thresholds in cells infected by viruses than by using only oligo-dT and other published housekeeping genes such as the TATA box binding protein (TBP) gene, the peptidyl prolyl isomerase A (PPI) gene and the ribosomal protein 13 (L13) gene. Incorporation of the 28S reverse primer in oligo-dT-primed cDNA synthesis also does not interfere with the detection of other polymerase II transcribed genes.

Characterization of a virion occlusion-defective Autographa californica multiple nucleopolyhedrovirus mutant lacking the p26, p10 and p74 genes.

Nucleopolyhedroviruses (NPVs), family Baculoviridae, are insect-specific viruses with the potential to control insect pests in agriculture and forestry. NPVs are occluded in polyhedral occlusion bodies. Polyhedra protect virions from inactivation in the environment as well as assisting virions in horizontal transmission in the insect population. The process of virion occlusion in the polyhedra is undefined and the genes that regulate the virion occlusion process have not been well investigated yet. An Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutant (AcDef) that has a 2136 bp DNA deletion, including p26, p10 and p74 genes, has been isolated. No virions were detected in the polyhedra of AcDef. Restoration of all the missing sequences into AcDef led to proper virion occlusion. Individual gene deletion of either p10 or p26 could not abolish virion occlusion in the polyhedra of AcMNPV, but p10 deletion reduced virion occlusion efficiency more than threefold compared with the wild-type AcMNPV. Previous studies by other research groups on deletion of AcMNPV gene p74 suggested that p74 is a per os infectivity factor, and deletion of the p74 gene did not eliminate virion occlusion. Collectively, the three genes (p26, p10 and p74) may act in concert to regulate the virion occlusion process. Therefore, p26, p10 and p74 are all required for proper virion occlusion in the polyhedra of AcMNPV.