Cre-mediated autoexcision of selectable marker genes in soybean, cotton, canola and maize transgenic plants.

KEY MESSAGE: Efficient selectable marker gene autoexcision in transgenic plants of soybean, cotton, canola, and maize is achieved by effective Cre recombinase expression. Selectable marker genes are often required for efficient generation of transgenic plants in plant transformation but are not desired once the transgenic events are obtained. We have developed Cre/loxP autoexcision systems to remove selectable marker genes in soybean, cotton, canola and maize. We tested a set of vectors with diverse promoters and identified promising promoters to drive cre expression for each of the four crops. We evaluated both the efficiency of generating primary transgenic events with low transgene copy numbers, and the frequency of marker-free progeny in the next generation. The best performing vectors gave no obvious decrease in the transformation frequency in each crop and generated homozygous marker-free progeny in the next generation. We found that effective expression of Cre recombinase for marker gene autoexcision can be species dependent. Among the vectors tested, the best autoexcision frequency (41%) in soybean transformation came from using the soybean RSP1 promoter for cre expression. The cre gene expressed by soybean RSP1 promoter with an Arabidopsis AtpE intron delivered the best autoexcision frequency (69%) in cotton transformation. The cre gene expressed by the embryo-specific eUSP88 promoter from Vicia faba conferred the best marker excision frequency (32%) in canola transformation. Finally, the cre gene expressed by the rice CDC45-1 promoter resulted in 44% autoexcision in maize transformation. The Cre/loxP recombinase system enables the generation of selectable marker-free transgenic plants for commercial product development in four agriculturally important crops and provides further improvement opportunities for more specific and better marker excision efficiency.

A Novel Approach to ICU Survivor Care: A Population Health Quality Improvement Project.

OBJECTIVES: Deliver a novel interdisciplinary care process for ICU survivor care and their primary family caregivers, and assess mortality, readmission rates, and economic impact compared with usual care. DESIGN: Population health quality improvement comparative study with retrospective data analysis. SETTING: A single tertiary care rural hospital with medical/surgical, neuroscience, trauma, and cardiac ICUs. PATIENTS: ICU survivors. INTERVENTIONS: Reorganization of existing post discharge health care delivery resources to form an ICU survivor clinic care process and compare this new process to post discharge usual care process. MEASUREMENTS AND MAIN RESULTS: Demographic data, Acute Physiology and Chronic Health Evaluation IV scores, and Charlson Comorbidity Index scores were extracted from the electronic health record. Additional data was extracted from the care manager database. Economic data were extracted from the Geisinger Health Plan database and analyzed by a health economist. During 13-month period analyzed, patients in the ICU survivor care had reduced mortality compared with usual care, as determined by the Kaplan-Meier method (ICU survivor care 0.89 vs usual care 0.71; log-rank p = 0.0108) and risk-adjusted stabilized inverse probability of treatment weighting (hazard ratio, 0.157; 95% CI, 0.058-0.427). Readmission for ICU survivor care versus usual care: at 30 days (10.4% vs 26.3%; stabilized inverse probability of treatment weighting hazard ratio, 0.539; 95% CI, 0.224-1.297) and at 60 days (16.7% vs 34.7%; stabilized inverse probability of treatment weighting hazard ratio, 0.525; 95% CI, 0.240-1.145). Financial data analysis indicates estimated annual cost savings to Geisinger Health Plan ranges from $247,052 to $424,846 during the time period analyzed. CONCLUSIONS: Our ICU survivor care process results in decreased mortality and a net annual cost savings to the insurer compared with usual care processes. There was no statistically significant difference in readmission rates.

Vacuolar glyphosate-sequestration correlates with glyphosate resistance in ryegrass (Lolium spp.) from Australia, South America, and Europe: a 31P NMR investigation.

Lolium spp., ryegrass, variants from Australia, Brazil, Chile, and Italy showing differing levels of glyphosate resistance were examined by (31)P NMR. Extents of glyphosate (i) resistance (LD(50)), (ii) inhibition of 5-enopyruvyl-shikimate-3-phosphate synthase (EPSPS) activity (IC(50)), and (iii) translocation were quantified for glyphosate-resistant (GR) and glyphosate-sensitive (GS) Lolium multiflorum Lam. variants from Chile and Brazil. For comparison, LD(50) and IC(50) data for Lolium rigidum Gaudin variants from Italy were also analyzed. All variants showed similar cellular uptake of glyphosate by (31)P NMR. All GR variants showed glyphosate sequestration within the cell vacuole, whereas there was minimal or no vacuole sequestration in the GS variants. The extent of vacuole sequestration correlated qualitatively with the level of resistance. Previous (31)P NMR studies of horseweed ( Conyza canadensis (L.) Cronquist) revealed that glyphosate sequestration imparted glyphosate resistance. Data presented herein suggest that glyphosate vacuolar sequestration is strongly contributing, if not the major contributing, resistance mechanism in ryegrass as well.