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Miller, E. J.

Publications and source records attributed to Miller, E. J..

2 recordsLinked to original sources

High Throughput Viability Assay for Microbiology

Counting viable cells is a universal practice in microbiology. The colony forming unit (CFU) assay has remained the gold standard to measure viability across disciplines; however, it is time-intensive and resource-consuming. Herein, we describe the Geometric Viability Assay (GVA) that replicates CFU measurements over 6-orders of magnitude while reducing over 10-fold the time and consumables. GVA computes a samples viable cell count based on the distribution of embedded colonies growing inside a pipette tip. GVA is compatible with gram-positive and -negative planktonic bacteria, biofilms, and yeast. Laborious CFU experiments such as checkerboard assays, treatment time-courses, and drug screens against slow-growing cells are simplified by GVA. We therefore screened a drug library against exponential and stationary phase E. coli leading to the discovery of the ROS-mediated, bactericidal mechanism of diphenyliodonium. The ease and low cost of GVA evinces it can accelerate existing viability assays and enable measurements at previously impractical scales.

microbiology↗

Extracellular Superoxide Dismutase (EC-SOD) Regulates Gene Methylation and Cardiac Fibrosis During Chronic Hypoxic Stress.

BackgroundChronic hypoxic stress induces epigenetic modifications in cardiac fibroblasts, such as inactivation of tumor suppressor genes (RASSF1A), and activation of kinases (ERK1/2). The effects of the antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), on these epigenetic changes has not been fully explored. ObjectivesTo define the effect of EC-SOD overexpression on cardiac fibrosis induced by chronic hypoxia. MethodsWild type C57B6 male mice (WT) and transgenic males with an extra copy of human hEC-SOD (TG) were housed in hypoxia (10% O2) for 21 days. Right ventricular tissue was studied for cardiac fibrosis markers using RT-PCR and Western Blot analyses. Downstream effects were studied, for both RASSF-1 expression and methylation and its relation to ERK1/2, using in-vivo & in-vitro models ResultsThere were significant increases in markers of cardiac fibrosis : Collagen 1, Alpha Smooth Muscle Actin (ASMA) and SNAIL, in the WT hypoxic animals as compared to the TG hypoxic group (p< 0.05). Expression of DNA methylation enzymes (DNMT 1,2) was significantly increased in the WT hypoxic mice as compared to the hypoxic TG mice (p<0.001). RASSF1A expression was significantly lower and ERK1/2 was significantly higher in hypoxia WT compared to the hypoxic TG group (p<0.05). Use of SiRNA to block RASSF1A gene expression in murine cardiac fibroblast tissue culture led to increased fibroblast proliferation (p<0.05). Methylation of RASSF1A promoter region showed a significant reduction in the TG hypoxic group compared to the WT hypoxic group (0.59 vs 0.75 respectively). ConclusionsEC-SOD significantly attenuates RASSF1A gene methylation, and plays a pivotal role cardiac fibrosis induced by hypoxia.

developmental biology↗