Microevolutionary dynamics of eccDNA in Chinese hamster ovary cells grown in fed-batch cultures under control and lactate-stressed conditions.

Chinese hamster ovary (CHO) cell lines are widely used to manufacture biopharmaceuticals. However, CHO cells are not an optimal expression host due to the intrinsic plasticity of the CHO genome. Genome plasticity can lead to chromosomal rearrangements, transgene exclusion, and phenotypic drift. A poorly understood genomic element of CHO cell line instability is extrachromosomal circular DNA (eccDNA) in gene expression and regulation. EccDNA can facilitate ultra-high gene expression and are found within many eukaryotes including humans, yeast, and plants. EccDNA confers genetic heterogeneity, providing selective advantages to individual cells in response to dynamic environments. In CHO cell cultures, maintaining genetic homogeneity is critical to ensuring consistent productivity and product quality. Understanding eccDNA structure, function, and microevolutionary dynamics under various culture conditions could reveal potential engineering targets for cell line optimization. In this study, eccDNA sequences were investigated at the beginning and end of two-week fed-batch cultures in an ambr®250 bioreactor under control and lactate-stressed conditions. This work characterized structure and function of eccDNA in a CHO-K1 clone. Gene annotation identified 1551 unique eccDNA genes including cancer driver genes and genes involved in protein production. Furthermore, RNA-seq data is integrated to identify transcriptionally active eccDNA genes.

Thiazide Diuretic Utilization Within the VA.

Background: The 2017 American College of Cardiology/American Heart Association blood pressure guideline recommends chlorthalidone as the preferred thiazide diuretic. We aimed to better understand thiazide prescribing patterns within the US Department of Veterans Affairs (VA). Methods: A retrospective analysis was conducted of patients with a prescription for hydrochlorothiazide (HCTZ), chlorthalidone, indapamide, or any combination products containing these from January 1, 2016, to January 21, 2022. The primary objective was to determine the utilization rates of each thiazide in the active cohort, assessed via χ2 test with Bonferroni correction. Secondary objectives included concomitant potassium or magnesium supplementation, blood pressure rates and control, and thiazide use from January 1, 2016, to December 31, 2021. Results: Of 628,994 active thiazide prescriptions, utilization rates differed significantly between thiazide groups (P < .001). Rates for HCTZ, chlorthalidone, and indapamide were 84.6%, 14.9%, and 0.5%, respectively. HCTZ use decreased from 90.2% to 83.5% (P < .001) and chlorthalidone use increased from 9.3% to 16.0% (P < .001). Between thiazide groups, rates of blood pressure control were not significantly different (P = .58). Potassium or magnesium supplementation was significantly different between groups (P < .001). The highest concomitant supplementation was with indapamide followed by chlorthalidone and HCTZ with rates of 27.1%, 22.6%, and 12.4%, respectively. Conclusions: Despite guideline recommendations for chlorthalidone, HCTZ is the most prescribed thiazide diuretic within the VA. However, there was a significant trend toward increased chlorthalidone prescribing from 2016 to 2021. Application of these data may guide further research to increase guideline-recommended therapy.