Browsing by Author "Ryan, Jennifer"

Now showing 1 - 4 of 4
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    Gene targets of mouse miR-709: regulation of distinct pools
    (Nature, 2016-01) Surendran, Sneha; Jideonwo, Victoria N.; Merchun, Chris; Ahn, Miwon; Murray, John; Ryan, Jennifer; Dunn, Kenneth W.; Kota, Janaiah; Morral, Núria; Department of Medical & Molecular Genetics, IU School of Medicine
    MicroRNA (miRNA) are short non-coding RNA molecules that regulate multiple cellular processes, including development, cell differentiation, proliferation and death. Nevertheless, little is known on whether miRNA control the same gene networks in different tissues. miR-709 is an abundant miRNA expressed ubiquitously. Through transcriptome analysis, we have identified targets of miR-709 in hepatocytes. miR-709 represses genes implicated in cytoskeleton organization, extracellular matrix attachment, and fatty acid metabolism. Remarkably, none of the previously identified targets in non-hepatic tissues are silenced by miR-709 in hepatocytes, even though several of these genes are abundantly expressed in liver. In addition, miR-709 is upregulated in hepatocellular carcinoma, suggesting it participates in the genetic reprogramming that takes place during cell division, when cytoskeleton remodeling requires substantial changes in gene expression. In summary, the present study shows that miR-709 does not repress the same pool of genes in separate cell types. These results underscore the need for validating gene targets in every tissue a miRNA is expressed.
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    Intravital Multiphoton Microscopy with Fluorescent Bile Salts in Rats as an In Vivo Biomarker for Hepatobiliary Transport Inhibition
    (ASPET, 2018-05) Ryan, Jennifer; Morgan, Ryan E.; Chen, Yuan; Volak, Laurie P.; Dunn, Robert T.; Dunn, Kenneth W.; Medicine, School of Medicine
    The bile salt export pump (BSEP) is expressed at the canalicular domain of hepatocytes, where it mediates the elimination of monovalent bile salts into the bile. Inhibition of BSEP is considered a susceptibility factor for drug-induced liver injury that often goes undetected during nonclinical testing. Although in vitro assays exist for screening BSEP inhibition, a reliable and specific method for confirming Bsep inhibition in vivo would be a valuable follow up to a BSEP screening strategy, helping to put a translatable context around in vitro inhibition data, incorporating processes such as metabolism, protein binding, and other exposure properties that are lacking in most in vitro BSEP models. Here, we describe studies in which methods of quantitative intravital microscopy were used to identify dose-dependent effects of two known BSEP/Bsep inhibitors, 2-[4-[4-(butylcarbamoyl)-2-[(2,4-dichlorophenyl)sulfonylamino]phenoxy]-3-methoxyphenyl]acetic acid (AMG-009) and bosentan, on hepatocellular transport of the fluorescent bile salts cholylglycyl amidofluorescein and cholyl-lysyl-fluorescein in rats. Results of these studies demonstrate that the intravital microscopy approach is capable of detecting Bsep inhibition at drug doses well below those found to increase serum bile acid levels, and also indicate that basolateral efflux transporters play a significant role in preventing cytosolic accumulation of bile acids under conditions of Bsep inhibition in rats. Studies of this kind can both improve our understanding of exposures needed to inhibit Bsep in vivo and provide unique insights into drug effects in ways that can improve our ability interpret animal studies for the prediction of human drug hepatotoxicity.
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    A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy
    (American Psychological Society, 2015-12-01) Tao, Wen; Rubart, Michael; Ryan, Jennifer; Xiao, Xiao; Qiao, Chunping; Hato, Takashi; Davidson, Michael W.; Dunn, Kenneth W.; Day, Richard N.; Department of Medicine, IU School of Medicine
    The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes.
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    Quantitative Intravital Microscopy of Liver Transport
    (Office of the Vice Chancellor for Research, 2013-04-05) Ryan, Jennifer; Ghabril, Marwan; Decker, Brian; Dunn, Kenneth W.
    Because if its unique ability to collect fluorescence images from deep in biological tissues, intravital multiphoton microscopy has become a valuable tool in several areas of biological research, including neurobiology, cancer biology and immunology. Here we describe methods of quantitative intravital microscopy that we have developed to characterize cholestatic liver injury. Special methods of tissue immobilization, multiphoton microscopy and digital image analysis were developed to support dynamic measurements of the kinetics of transport from the sinusoids into the cytosol and from the cytosol into the bile canaliculi in individual hepatocytes in vivo. Using a combination of different fluorescent probes, we have combined transport assays with measures of microvascular function, inflammation and cell viability to provide integrated measures of liver injury. The sensitivity of this approach is demonstrated in quantitative analyses of the acute effects of cholestatic drugs and the effects of chronic kidney disease.