Use of a dual reporter plasmid to demonstrate bactofection with an attenuated aroa- derivative of Pasteurella multocida b:2

A reporter plasmid pSRG has been developed which expresses red fluorescent protein (RFP) from a constitutive prokaryotic promoter within Pasteurella multocida B:2 and green fluorescent protein (GFP) from a constitutive eukaryotic promoter within mammalian cells. This construct has been used to determine the location and viability of the bacteria when moving from the extracellular environment into the intracellular compartment of mammalian cells. Invasion assays with embryonic bovine lung (EBL) cells and an attenuated AroA- derivative of Pasteurella multocida B:2 (strain JRMT12), harbouring the plasmid pSRG, showed that RFP-expressing bacteria could be detected intracellularly at 3 h post-invasion. At this stage, some EBL cells harbouring RFP-expressing bacteria were observed to express GFP simultaneously, indicating release of the plasmid into the intracellular environment. At 5 h post-invasion, more EBL cells were expressing GFP, while still harbouring RFP-expressing bacteria. Concurrently, some EBL cells were shown to express only GFP, indicating loss of viable bacteria within these cells. These experiments proved the functionality of the pSRG dual reporter system and the potential of P. multocida B:2 JRMT12 for bactofection and delivery of a DNA vaccine

Transcriptome Alteration in the Diabetic Heart by Rosiglitazone: Implications for Cardiovascular Mortality

BACKGROUND: Recently, the type 2 diabetes medication, rosiglitazone, has come under scrutiny for possibly increasing the risk of cardiac disease and death. To investigate the effects of rosiglitazone on the diabetic heart, we performed cardiac transcriptional profiling and imaging studies of a murine model of type 2 diabetes, the C57BL/KLS-lepr(db)/lepr(db) (db/db) mouse. METHODS AND FINDINGS: We compared cardiac gene expression profiles from three groups: untreated db/db mice, db/db mice after rosiglitazone treatment, and non-diabetic db/+ mice. Prior to sacrifice, we also performed cardiac magnetic resonance (CMR) and echocardiography. As expected, overall the db/db gene expression signature was markedly different from control, but to our surprise was not significantly reversed with rosiglitazone. In particular, we have uncovered a number of rosiglitazone modulated genes and pathways that may play a role in the pathophysiology of the increase in cardiac mortality as seen in several recent meta-analyses. Specifically, the cumulative upregulation of (1) a matrix metalloproteinase gene that has previously been implicated in plaque rupture, (2) potassium channel genes involved in membrane potential maintenance and action potential generation, and (3) sphingolipid and ceramide metabolism-related genes, together give cause for concern over rosiglitazone's safety. Lastly, in vivo imaging studies revealed minimal differences between rosiglitazone-treated and untreated db/db mouse hearts, indicating that rosiglitazone's effects on gene expression in the heart do not immediately turn into detectable gross functional changes. CONCLUSIONS: This study maps the genomic expression patterns in the hearts of the db/db murine model of diabetes and illustrates the impact of rosiglitazone on these patterns. The db/db gene expression signature was markedly different from control, and was not reversed with rosiglitazone. A smaller number of unique and interesting changes in gene expression were noted with rosiglitazone treatment. Further study of these genes and molecular pathways will provide important insights into the cardiac decompensation associated with both diabetes and rosiglitazone treatment

Food safety advantages of combining advanced decontamination processes

No abstract available

Food safety advantages of combining advanced decontamination processes

No abstract available

Bioluminescence as a Reporter of Intracellular Survival of <i>Bordetella bronchiseptica</i> in Murine Phagocytes

ABSTRACT The uptake and persistence of Bordetella bronchiseptica was characterized in murine phagocytes by using a novel bioluminescence-based reporter system. A mini-Tn 5 promoter probe carrying the intact lux operon from the terrestrial bacterium Photorhabdus luminescens which allowed measurement of light output without the addition of exogenous substrate was constructed. It was used to create a pool of bioluminescent fusion strains of B. bronchiseptica . The internalization and persistence in murine macrophages of a constitutive bioluminescent strain of B. bronchiseptica was monitored by luminometry and by fluorescence and electron microscopy. The number of bacteria internalized, in a microfilament-dependent process, by a mouse macrophage-like cell line after 2 h was approximately 1% of the inoculum for several different multiplicities of infection (MOI). At an MOI of &lt;500:1 (bacteria to macrophages), viable numbers of intracellular bacteria declined over a 4-day period. However, at an MOI of ≥500:1, long-term survival was enhanced, with viable bacteria recovered up to 4 days postinfection with little decline in numbers, indicating that a critical population size may have been essential for intracellular persistence. No evidence of macrophage killing by intracellular bacteria was detected over the 4-day period. Intracellular bioluminescent B. bronchiseptica organisms in mouse peritoneal cells were detected at 24 and 48 h after intraperitoneal injection of mice. Bioluminescence is shown to act as a convenient real-time technique for monitoring of intracellular survival of B. bronchiseptica in vitro and may provide a suitable means for examining the role of long-term intracellular survival of the bacterium in the host. </jats:p