Utilization of microalgae has been hampered by limited tools for creating loss-of-function mutants. Furthermore, modified strains for deployment into the field must be free of antibiotic resistance genes and face fewer regulatory hurdles if they are transgene free. The oleaginous microalga, Nannochloropsis oceanica CCMP1779, is an emerging model for microalgal lipid metabolism. We present a one-vector episomal CRISPR/Cas9 system for N. oceanica that enables the generation of marker-free mutant lines. The CEN/ARS6 region from Saccharomyces cerevisiae was included in the vector to facilitate its maintenance as circular extrachromosal DNA. The vector utilizes a bidirectional promoter to produce both Cas9 and a ribozyme flanked sgRNA. This system efficiently generates targeted mutations, and allows the loss of episomal DNA after the removal of selection pressure, resulting in marker-free non-transgenic engineered lines. To test this system, we disrupted the nitrate reductase gene (NR) and subsequently removed the CRISPR episome to generate non-transgenic marker-free nitrate reductase knockout lines (NR-KO).
To evaluate local procedures for and barriers to testing Medicaid patients for lead toxicity, a retrospective review of 675 charts of Medicaid patients' age 12 to 36 months form the 7 practices who serve the vast majority of Medicaid children in Kalamazoo, Michigan was undertaken. We identified and tested a model for barriers to patients and physicians obtaining blood lead levels. Only 27.6% had blood lead levels substantially lower than the universal testing mandated by the Centers for Medicare and Medicaid Services. Physicians overestimated the number of children having blood lead tests, even with adjusting for patient non-compliance. Practices varied sharply in their approach to screening. Of 489 patients without findable lead test results, 139 (28.4%) were attributable to previous screening, 98 (20.0%) to patient failure, 181 (37.0%) to physician failure, 52 (10.6%) to patient + physician failure and 19 (3.9%) were indeterminate. There is need for improvement, standardization and unification of lead screening guidelines. Understanding barriers to blood lead testing is also necessary to develop credible data to promote shifts in public policy regarding lead abatement. Further studies should be done, such as interviews and questionnaires to determine why physicians fail to order blood lead tests, and patients fail to appear for a test that has been ordered.
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