Enhanced gene expression from retroviral vectors

<p>Abstract</p> <p>Background</p> <p>Retroviruses are widely used to transfer genes to mammalian cells efficiently and stably. However, genetic elements required for high-level gene expression are incompatible with standard systems. The retroviral RNA genome is produced by cellular transcription and post-transcriptional processing within packaging cells: Introns present in the retroviral genomic transcript are removed by splicing, while polyadenylation signals lead to the production of ineffective truncated genomes. Furthermore strong enhancer/promoters within the retroviral payload lead to detrimental competition with the retroviral enhancer/promoter.</p> <p>Results</p> <p>By exploiting a new method of producing the retroviral genome <it>in vitro </it>it is possible to produce infectious retroviral particles carrying a high-level expression cassette that completely prohibits production of infectious retroviral particles by conventional methods.</p> <p>We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron, the GFP open reading frame and a strong polyadenylation signal. This cassette was cloned into both a conventional MMLV retroviral vector and a vector designed to allow <it>in vitro </it>transcription of the retroviral genome by T7 RNA polymerase.</p> <p>When the conventional retroviral vector was transfected into packaging cells, the expression cassette drove strong GFP expression, but no infectious retrovirus was produced. Introduction of the <it>in vitro </it>produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression. However, infectious retrovirus was easily recovered, and when used to infect target primary human cells led to very high GFP expression – up to 3.5 times greater than conventional retroviral LTR-driven expression.</p> <p>Conclusion</p> <p>Retroviral vectors carrying an optimized high-level expression cassette do not produce infectious virions when introduced into packaging cells by transfection of DNA. Infectious retrovirus carrying the same cassette is readily produced when packaging cells are transfected with <it>in vitro </it>transcribed retroviral genomic RNA. The applications of this technique are not limited to producing the higher levels of transgene expression demonstrated here. For example, novel reporters with alternatively spliced exon-intron configurations could readily be transduced into virtually any cell. Furthermore, because the <it>in vitro </it>transcripts are not translated within the packaging cells, retroviruses carrying genes lethal to the packaging cells can also be produced.</p

Do Hospitals Cross-Subsidize?

Despite its salience as a regulatory tool to ensure the delivery of unprofitable medical services, cross-subsidization of services within hospital systems has been notoriously difficult to detect and quantify. We use repeated shocks to a profitable service in the market for hospital-based medical care to test for cross-subsidization of unprofitable services. Using patient-level data from general short-term hospitals in Arizona and Colorado before and after entry by cardiac specialty hospitals, we study how incumbent hospitals adjusted their provision of three uncontested services that are widely considered to be unprofitable. We estimate that the hospitals most exposed to entry reduced their provision of psychiatric, substance-abuse, and trauma care services at a rate of about one uncontested-service admission for every four cardiac admissions they stood to lose. Although entry by single-specialty hospitals may adversely affect the provision of unprofitable uncontested services, these findings warrant further evaluation of service-line cross-subsidization as a means to finance them

Statistical Communication Theory

Contains reports on four research projects

Do Hospitals Cross Subsidize?

Cross-subsidies are often considered the principal mechanism through which hospitals provide unprofitable care. Yet, hospitals’ reliance on and extent of cross-subsidization are difficult to establish. We exploit entry by cardiac specialty hospitals as an exogenous shock to incumbent hospitals’ profitability and in turn to their ability to cross-subsidize unprofitable services. Using patient-level data from general short-term hospitals in Arizona and Colorado before and after entry, we find that the hospitals most exposed to entry reduced their provision of services considered to be unprofitable (psychiatric, substance- abuse, and trauma care) and expanded their admissions for neurosurgery, a highly profitable service.

Stochasticity and evolutionary stability

In stochastic dynamical systems, different concepts of stability can be obtained in different limits. A particularly interesting example is evolutionary game theory, which is traditionally based on infinite populations, where strict Nash equilibria correspond to stable fixed points that are always evolutionarily stable. However, in finite populations stochastic effects can drive the system away from strict Nash equilibria, which gives rise to a new concept for evolutionary stability. The conventional and the new stability concepts may apparently contradict each other leading to conflicting predictions in large yet finite populations. We show that the two concepts can be derived from the frequency dependent Moran process in different limits. Our results help to determine the appropriate stability concept in large finite populations. The general validity of our findings is demonstrated showing that the same results are valid employing vastly different co-evolutionary processes

Statistical Communication Theory

Contains research objectives and reports on eight research projects

Statistical Communication Theory

Contains reports on six research projects

Statistical Communication Theory

Contains reports on nine research projects

Revisiting diagenesis on the Ontong Java Plateau: Evidence for authigenic crust precipitation in Globorotalia tumida

The calcite tests of foraminifera lie in marine sediments for thousands to millions of years, before being analysed to generate trace element and isotope palaeoproxy records. These sediments constitute a distinct physio-chemical environment from the conditions in which the tests formed. Storage in sediments can modify the trace element and isotopic content of foraminiferal calcite through diagenetic alteration, which has the potential to confound their palaeoceanographic interpretation. A previous study of G. tumida from the Ontong Java Plateau, western equatorial Pacific, found that preferential dissolution of higher-Mg chamber calcite, and the preservation of a low-Mg crust on the tests significantly reduced whole-test Mg/Ca and Sr/Ca [Brown and Elderfield, 1996]. Here, we revisit these specimens with a combination of synchrotron X-ray computed tomography (sXCT) and electron probe micro-analyses (EPMA) to re-evaluate the nature of their diagenetic alteration. The dissolution of higher-Mg calcite with depth was directly observed in the sXCT data, confirming the inference of the previous study. The sXCT data further reveal a thickening of the chemically and structurally distinct calcite crust with depth. We propose that these crusts have a diagenetic origin, driven by the simultaneous dissolution of high-Mg chamber calcite and precipitation of low-Mg crust from the resulting modified pore-water solution. While the breadth of the study is limited by the nature of the techniques, the observation of both dissolution and re-precipitation of foraminiferal calcite serves to demonstrate the action of two simultaneous diagenetic alteration processes, with significant impacts on the resulting palaeoproxy signals.The authors would like to acknowledge Aleksey Sadekov, Gerald Langer, India Weidle, Alberto de Fanis, Andrew Bodey, Joan Vila-Comamala and Ulrich Wagner for their help with the project. The work was funded by the Diamond Light Source and by the ERC (2010-NEWLOG ADG-267931 grant to HE).This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/2014PA00275

Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation

Background: Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics. Methods and Findings: To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF. Conclusions: These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation