Human IL-12 p40 as a reporter gene for high-throughput screening of engineered mouse embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>Establishing a suitable level of exogenous gene expression in mammalian cells in general, and embryonic stem (ES) cells in particular, is an important aspect of understanding pathways of cell differentiation, signal transduction and cell physiology. Despite its importance, this process remains challenging because of the poor correlation between the presence of introduced exogenous DNA and its transcription. Consequently, many transfected cells must be screened to identify those with an appropriate level of expression. To improve the screening process, we investigated the utility of the human interleukin 12 (IL-12) p40 cDNA as a reporter gene for studies of mammalian gene expression and for high-throughput screening of engineered mouse embryonic stem cells.</p> <p>Results</p> <p>A series of expression plasmids were used to study the utility of IL-12 p40 as an accurate reporter of gene activity. These studies included a characterization of the IL-12 p40 expression system in terms of: (i) a time course of IL-12 p40 accumulation in the medium of transfected cells; (ii) the dose-response relationship between the input DNA and IL-12 p40 mRNA levels and IL-12 p40 protein secretion; (iii) the utility of IL-12 p40 as a reporter gene for analyzing the activity of <it>cis</it>-acting genetic elements; (iv) expression of the IL-12 p40 reporter protein driven by an IRES element in a bicistronic mRNA; (v) utility of IL-12 p40 as a reporter gene in a high-throughput screening strategy to identify successful transformed mouse embryonic stem cells; (vi) demonstration of pluripotency of IL-12 p40 expressing ES cells <it>in vitro </it>and <it>in vivo</it>; and (vii) germline transmission of the IL-12 p40 reporter gene.</p> <p>Conclusion</p> <p>IL-12 p40 showed several advantages as a reporter gene in terms of sensitivity and ease of the detection procedure. The IL-12 p40 assay was rapid and simple, in as much as the reporter protein secreted from the transfected cells was accurately measured by ELISA using a small aliquot of the culture medium. Remarkably, expression of Il-12 p40 does not affect the pluripotency of mouse ES cells. To our knowledge, human IL-12 p40 is the first secreted reporter protein suitable for high-throughput screening of mouse ES cells. In comparison to other secreted reporters, such as the widely used alkaline phosphatase (SEAP) reporter, the IL-12 p40 reporter system offers other real advantages.</p

Evaluating trade-offs of a large, infrequent sediment diversion for restoration of a forested wetland in the Mississippi delta

© 2018 Elsevier Ltd Flood control levees cut off the supply of sediment to Mississippi delta coastal wetlands, and contribute to putting much of the delta on a trajectory for continued submergence in the 21st century. River sediment diversions have been proposed as a method to provide a sustainable supply of sediment to the delta, but the frequency and magnitude of these diversions needs further assessment. Previous studies suggested operating river sediment diversions based on the size and frequency of natural crevasse events, which were large (\u3e5000 m3/s) and infrequent (active \u3c once a year) in the last naturally active delta. This study builds on these previous works by quantitatively assessing tradeoffs for a large, infrequent diversion into the forested wetlands of the Maurepas swamp. Land building was estimated for several diversion sizes and years inactive using a delta progradation model. A benefit-cost analysis (BCA) combined model land building results with an ecosystem service valuation and estimated costs. Results demonstrated that land building is proportional to diversion size and inversely proportional to years inactive. Because benefits were assumed to scale linearly with land gain, and costs increase with diversion size, there are disadvantages to operating large diversions less often, compared to smaller diversions more often for the immediate project area. Literature suggests that infrequent operation would provide additional gains (through increased benefits and reduced ecosystem service costs) to the broader Lake Maurepas-Pontchartrain-Borgne ecosystem. Future research should incorporate these additional effects into this type of BCA, to see if this changes the outcome for large, infrequent diversions

Tracking Cardiac Rehabilitation Participation and Completion Among Medicare Beneficiaries to Inform the Efforts of a National Initiative

BACKGROUND: Despite cardiac rehabilitation (CR) being shown to improve health outcomes among patients with heart disease, its use has been suboptimal. In response, the Million Hearts Cardiac Rehabilitation Collaborative developed a road map to improve CR use, including increasing participation rates to ≥70% by 2022. This observational study provides current estimates to measure progress and identifies the populations and regions most at risk for CR service underutilization. METHODS AND RESULTS: We identified Medicare fee-for-service beneficiaries who were CR eligible in 2016, and assessed CR participation (≥1 CR session attended), timely initiation (participation within 21 days of event), and completion (≥36 sessions attended) through 2017. Measures were assessed overall, by beneficiary characteristics and geography, and by primary CR-qualifying event type (acute myocardial infarction hospitalization; coronary artery bypass surgery; heart valve repair/replacement; percutaneous coronary intervention; or heart/heart-lung transplant). Among 366 103 CR-eligible beneficiaries, 89 327 (24.4%) participated in CR, of whom 24.3% initiated within 21 days and 26.9% completed CR. Eligibility was highest in the East South Central Census Division (14.8 per 1000). Participation decreased with increasing age, was lower among women (18.9%) compared with men (28.6%; adjusted prevalence ratio: 0.91 [95% CI, 0.90-0.93]) was lower among Hispanics (13.2%) and non-Hispanic blacks (13.6%) compared with non-Hispanic whites (25.8%; adjusted prevalence ratio: 0.63 [0.61-0.66] and 0.70 [0.67-0.72], respectively), and varied by hospital referral region and Census Division (range: 18.6% [East South Central] to 39.1% [West North Central]) and by qualifying event type (range: 7.1% [acute myocardial infarction without procedure] to 55.3% [coronary artery bypass surgery only]). Timely initiation varied by geography and qualifying event type; completion varied by geography. CONCLUSIONS: Only 1 in 4 CR-eligible Medicare beneficiaries participated in CR and marked disparities were observed. Reinforcement of current effective strategies and development of new strategies will be critical to address the noted disparities and achieve the 70% participation goal