scholarly journals Experimental Evolution as a High-Throughput Screen for Genetic Adaptations

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Vaughn S. Cooper
Keyword(s):  
High Throughput ◽  
Experimental Evolution ◽  
High Frequency ◽  
Population Genetic ◽  
Evolutionary Process ◽  
High Throughput Screen ◽  
Adaptive Traits ◽  
Controlled Conditions ◽  
Large Populations ◽  
Selective Environment

ABSTRACT Experimental evolution is a method in which populations of organisms, often microbes, are founded by one or more ancestors of known genotype and then propagated under controlled conditions to study the evolutionary process. These evolving populations are influenced by all population genetic forces, including selection, mutation, drift, and recombination, and the relative contributions of these forces may be seen as mysterious. Here, I describe why the outcomes of experimental evolution should be viewed with greater certainty because the force of selection typically dominates. Importantly, any mutant rising rapidly to high frequency in large populations must have acquired adaptive traits in the selective environment. Sequencing the genomes of these mutants can identify genes or pathways that contribute to an adaptation. I review the logic and simple mathematics why this evolve-and-resequence approach is a powerful way to find the mutations or mutation combinations that best increase fitness in any new environment.

scholarly journals High Throughput Screen Identifies the DNMT1 (DNA Methyltransferase-1) Inhibitor, 5-Azacytidine, as a Potent Inducer of PTEN (Phosphatase and Tensin Homolog)

2020 ◽  
Vol 40 (8) ◽  
pp. 1854-1869
Author(s):  
Keith A. Strand ◽  
Sizhao Lu ◽  
Marie F. Mutryn ◽  
Linfeng Li ◽  
Qiong Zhou ◽  
...  
Keyword(s):  
Protein Expression ◽  
High Throughput ◽  
Knockout Mice ◽  
Vascular Remodeling ◽  
Dna Methyltransferase ◽  
Dna Methyltransferase 1 ◽  
Protective Effects ◽  
High Throughput Screen ◽  
Phosphatase And Tensin Homolog ◽  
Tensin Homolog

Objective: Our recent work demonstrates that PTEN (phosphatase and tensin homolog) is an important regulator of smooth muscle cell (SMC) phenotype. SMC-specific PTEN deletion promotes spontaneous vascular remodeling and PTEN loss correlates with increased atherosclerotic lesion severity in human coronary arteries. In mice, PTEN overexpression reduces plaque area and preserves SMC contractile protein expression in atherosclerosis and blunts Ang II (angiotensin II)-induced pathological vascular remodeling, suggesting that pharmacological PTEN upregulation could be a novel therapeutic approach to treat vascular disease. Approach and Results: To identify novel PTEN activators, we conducted a high-throughput screen using a fluorescence based PTEN promoter-reporter assay. After screening ≈3400 compounds, 11 hit compounds were chosen based on level of activity and mechanism of action. Following in vitro confirmation, we focused on 5-azacytidine, a DNMT1 (DNA methyltransferase-1) inhibitor, for further analysis. In addition to PTEN upregulation, 5-azacytidine treatment increased expression of genes associated with a differentiated SMC phenotype. 5-Azacytidine treatment also maintained contractile gene expression and reduced inflammatory cytokine expression after PDGF (platelet-derived growth factor) stimulation, suggesting 5-azacytidine blocks PDGF-induced SMC de-differentiation. However, these protective effects were lost in PTEN-deficient SMCs. These findings were confirmed in vivo using carotid ligation in SMC-specific PTEN knockout mice treated with 5-azacytidine. In wild type controls, 5-azacytidine reduced neointimal formation and inflammation while maintaining contractile protein expression. In contrast, 5-azacytidine was ineffective in PTEN knockout mice, indicating that the protective effects of 5-azacytidine are mediated through SMC PTEN upregulation. Conclusions: Our data indicates 5-azacytidine upregulates PTEN expression in SMCs, promoting maintenance of SMC differentiation and reducing pathological vascular remodeling in a PTEN-dependent manner.

scholarly journals Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

2011 ◽  
Vol 16 (8) ◽  
pp. 869-877 ◽  
Author(s):  
Duncan I. Mackie ◽  
David L. Roman
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interaction ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Screening Method ◽  
Fold Increase ◽  
Rgs Proteins ◽  
High Throughput Screen ◽  
Protein Protein Interaction

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 µM in dose–response experiments. Four exhibited IC50 values <6 µM while inhibiting the Gαo–RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

scholarly journals Long-Term Experimental Evolution in Escherichia coli. VI. Environmental Constraints on Adaptation and Divergence

Genetics ◽  
1997 ◽  
Vol 146 (2) ◽  
pp. 471-479 ◽  
Author(s):  
Michael Travisano
Keyword(s):  
Escherichia Coli ◽  
Genetic Variation ◽  
Experimental Evolution ◽  
Population Genetic ◽  
Environmental Constraints ◽  
Asymmetric Pattern ◽  
Nutrient Environment ◽  
Mean Fitness ◽  
Population Genetic Variation

The effect of environment on adaptation and divergence was examined in two sets of populations of Escherichia coli selected for 1000 generations in either maltose- or glucose-limited media. Twelve replicate populations selected in maltose-limited medium improved in fitness in the selected environment, by an average of 22.5%. Statistically significant among-population genetic variation for fitness was observed during the course of the propagation, but this variation was small relative to the fitness improvement. Mean fitness in a novel nutrient environment, glucose-limited medium, improved to the same extent as in the selected environment, with no statistically significant among-population genetic variation. In contrast, 12 replicate populations previously selected for 1000 generations in glucose-limited medium showed no improvement, as a group, in fitness in maltose-limited medium and substantial genetic variation. This asymmetric pattern of correlated responses suggests that small changes in the environment can have profound effects on adaptation and divergence.

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