Doxycycline alters metabolism and proliferation of human cell lines.

The tetracycline antibiotics are widely used in biomedical research as mediators of inducible gene expression systems. Despite many known effects of tetracyclines on mammalian cells-including inhibition of the mitochondrial ribosome-there have been few reports on potential off-target effects at concentrations commonly used in inducible systems. Here, we report that in human cell lines, commonly used concentrations of doxycycline change gene expression patterns and concomitantly shift metabolism towards a more glycolytic phenotype, evidenced by increased lactate secretion and reduced oxygen consumption. We also show that these concentrations are sufficient to slow proliferation. These findings suggest that researchers using doxycycline in inducible expression systems should design appropriate controls to account for potential confounding effects of the drug on cellular metabolism

High-throughput mutagenesis identifies regulatory interactions and drug resistance mechanisms in Src kinase

Thesis (Ph.D.)--University of Washington, 2019The majority of the human proteome is phosphorylated by the eukaryotic protein kinase (EPK) enzyme family. EPKs are subject to multiple layers of regulation that modulate both the magnitude and specificity of their activity and this regulation is crucial, as loss of control over activity often results in disease. Commonly, this regulation is governed by interactions between the catalytic kinase domain and other regions of the protein. A classic example of intramolecular EPK regulation was delineated in the tyrosine kinase Src, whose regulatory SH2 and SH3 domains interface with its catalytic domain to constrain activity. Although this mechanism of Src intramolecular regulation is broadly well established, our knowledge of how the individual catalytic domain residues contribute to regulation is less well understood. To investigate this, we use saturation mutagenesis to functionally interrogate thousands of single missense mutants of the catalytic domain of Src kinase. We identify 27 residues located throughout the catalytic domain that, when mutated, result in increased Src activity. As expected, several of these residues hyper-activate Src by disrupting known mechanisms of SH2 or SH3 domain regulation. However, we also identify a patch of residues on Src’s C-lobe which strongly affects its activity but whose mechanism cannot be explained by our current understanding of Src regulation. We investigate this patch and surprisingly find that mutations here affect activity in a manner dependent on Src’s membrane-binding SH4 domain. Moreover, these mutations cause Src to associate more with membranes both in vitro and in cells, implying these mutations alter SH4 domain conformation. Our results suggest a novel functional interaction between Src’s SH4 and catalytic domain that couples localization and activity and thus our study adds an additional layer to the canonical mechanisms of Src kinase regulation. In a follow-up project, we use this same system to systematically identify mutations that confer resistance to three different conformation-selective inhibitors of Src. We identify dozens of resistance mutations, including some that are specific to a given inhibitor. These “unique” resistance mutations are of particular interest, because these residues may contribute to the adoption of specific conformations of Src’s catalytic domain. In summary, this work uncovers a previously unidentified regulatory role for Src’s SH4 domain. Moreover, it lays the groundwork for future investigations into the nature of these newly identified resistance mutants for conformation-selective inhibitors

Californians' Beliefs About Income Inequality

Rising income inequality and wealth in America have attracted substantial public attention in recent years. As evidence has mounted that the gap betwen the affluent and the poor has been increasing, elected officials, scholars, journalists, and even figures from popular culture have discussed the causes and effects of this phenomenon. The focus of the debate has often been the economic trends rather than the views of a broad sampling of Americans.  But public opinion can condition policy responses, and it is important to measure public opinion on economic inequality, its causes, and potential remedies. This IGS Research Brief seeks to understand public opinion in California about this issue. It describes and analyzes the results of an IGS Poll on income inequality, its perceived causes, and the views of Californians regarding possible responses

Californians' Beliefs About Income Inequality

Rising income inequality and wealth in America have attracted substantial public attention in recent years. As evidence has mounted that the gap betwen the affluent and the poor has been increasing, elected officials, scholars, journalists, and even figures from popular culture have discussed the causes and effects of this phenomenon. The focus of the debate has often been the economic trends rather than the views of a broad sampling of Americans.  But public opinion can condition policy responses, and it is important to measure public opinion on economic inequality, its causes, and potential remedies. This IGS Research Brief seeks to understand public opinion in California about this issue. It describes and analyzes the results of an IGS Poll on income inequality, its perceived causes, and the views of Californians regarding possible responses

Californians' Beliefs About Income Inequality

Rising income inequality and wealth in America have attracted substantial public attention in recent years. As evidence has mounted that the gap betwen the affluent and the poor has been increasing, elected officials, scholars, journalists, and even figures from popular culture have discussed the causes and effects of this phenomenon. The focus of the debate has often been the economic trends rather than the views of a broad sampling of Americans.  But public opinion can condition policy responses, and it is important to measure public opinion on economic inequality, its causes, and potential remedies. This IGS Research Brief seeks to understand public opinion in California about this issue. It describes and analyzes the results of an IGS Poll on income inequality, its perceived causes, and the views of Californians regarding possible responses

Doxycycline alters metabolism and proliferation of human cell lines.

The tetracycline antibiotics are widely used in biomedical research as mediators of inducible gene expression systems. Despite many known effects of tetracyclines on mammalian cells-including inhibition of the mitochondrial ribosome-there have been few reports on potential off-target effects at concentrations commonly used in inducible systems. Here, we report that in human cell lines, commonly used concentrations of doxycycline change gene expression patterns and concomitantly shift metabolism towards a more glycolytic phenotype, evidenced by increased lactate secretion and reduced oxygen consumption. We also show that these concentrations are sufficient to slow proliferation. These findings suggest that researchers using doxycycline in inducible expression systems should design appropriate controls to account for potential confounding effects of the drug on cellular metabolism