Horizontal transfer of genes between species is an important mechanism for bacterial genome evolution. In Escherichia coli, conjugation is the transfer from a donor (F(+)) to a recipient (F(-)) cell through cell-to-cell contact. We demonstrate what we believe to be a novel qPCR method for quantifying the transfer kinetics of the F plasmid in a population by enumerating the relative abundance of genetic loci unique to the plasmid and the chromosome. This approach allows us to query the plasmid transfer rate without the need for selective culturing with unprecedented single locus resolution. We fit the results to a mass action model where the rate of plasmid growth includes the lag time of newly formed F(+) transconjugants and the recovery time between successive conjugation events of the F(+) donors. By assaying defined mixtures of genotypically identical donor and recipient cells at constant inoculation densities, we extract an F plasmid transfer rate of 5 × 10(-10) (cells/mL · min)(-1). We confirm a plasmid/chromosome ratio of 1:1 in homogenous F(+) populations throughout batch growth. Surprisingly, in some mixture experiments we observe an excess of F plasmid in the early saturation phase that equilibrates to a final ratio of one plasmid per chromosome.
Cancer growth and proliferation rely on intracellular iron availability. We studied the effects of Deferiprone (DFP), a chelator of intracellular iron, on three prostate cancer cell lines: murine, metastatic TRAMP-C2; murine, non-metastatic Myc-CaP; and human, non-metastatic 22rv1. The effects of DFP were evaluated at different cellular levels: cell culture proliferation and migration; metabolism of live cells (time-course multi-nuclear magnetic resonance spectroscopy cell perfusion studies, with 1-13C-glucose; and Seahorse extracellular flux analysis); and expression (Western Blot) and activity of mitochondrial aconitase (m-Acon), an iron-dependent enzyme. The half and 90% inhibitory concentration (IC50 and IC90, respectively) of DFP for the three cell lines after 48 h incubation ranged within 51–67 μM and 81–186 μM, respectively. Exposure to 100 μM DFP led to: (i) Significant inhibition of cell migration after different exposure times, ranging from 12 h (TRAMP-C2) to 48 h (22rv1), in agreement with the respective cell doubling times; (ii) Significantly decreased glucose consumption and glucose-driven TCA cycle activity in metastatic TRAMP-C2 cells, during the first 10 h of exposure, and impaired cellular bioenergetics and membrane phospholipid turnover after 23 h exposure, consistent with a cytostatic effect of DFP. At this time point, all cell lines studied showed (iii) significant decreases in mitochondrial functional parameters associated with oxygen consumption rate, and (iv) both significantly lower m-Acon expression and activity. Our results indicate the potential of DFP to inhibit prostate cancer proliferation at clinically relevant doses and plasma concentrations.
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