Fluorescence-based assays provide sensitive and adaptable methods for point of care testing, environmental monitoring, studies of protein abundance and activity, and a wide variety of additional ...applications. Currently, their utility in remote and low-resource environments is limited by the need for technically complicated or expensive instruments to read out fluorescence signal. Here we describe the Genes in Space Fluorescence Viewer (GiS Viewer), a portable, durable viewer for rapid molecular assay readout that can be used to visualize fluorescence in the red and green ranges. The GiS Viewer can be used to visualize any assay run in standard PCR tubes and contains a heating element. Results are visible by eye or can be imaged with a smartphone or tablet for downstream quantification. We demonstrate the capabilities of the GiS Viewer using two case studies-detection of SARS-CoV-2 RNA using RT-LAMP and quantification of drug-induced changes in gene expression via qRT-PCR on Earth and aboard the International Space Station. We show that the GiS Viewer provides a reliable method to visualize fluorescence in space without the need to return samples to Earth and can further be used to assess the results of RT-LAMP and qRT-PCR assays on Earth.
Hepatocyte nuclear factor 4 alpha (HNF4α), the master regulator of hepatocyte differentiation, has been recently shown to inhibit hepatocyte proliferation by way of unknown mechanisms. We ...investigated the mechanisms of HNF4α‐induced inhibition of hepatocyte proliferation using a novel tamoxifen (TAM)‐inducible, hepatocyte‐specific HNF4α knockdown mouse model. Hepatocyte‐specific deletion of HNF4α in adult mice resulted in increased hepatocyte proliferation, with a significant increase in liver‐to‐body‐weight ratio. We determined global gene expression changes using Illumina HiSeq‐based RNA sequencing, which revealed that a significant number of up‐regulated genes following deletion of HNF4α were associated with cancer pathogenesis, cell cycle control, and cell proliferation. The pathway analysis further revealed that c‐Myc‐regulated gene expression network was highly activated following HNF4α deletion. To determine whether deletion of HNF4α affects cancer pathogenesis, HNF4α knockdown was induced in mice treated with the known hepatic carcinogen diethylnitrosamine (DEN). Deletion of HNF4α significantly increased the number and size of DEN‐induced hepatic tumors. Pathological analysis revealed that tumors in HNF4α‐deleted mice were well‐differentiated hepatocellular carcinoma (HCC) and mixed HCC‐cholangiocarcinoma. Analysis of tumors and surrounding normal liver tissue in DEN‐treated HNF4α knockout mice showed significant induction in c‐Myc expression. Taken together, deletion of HNF4α in adult hepatocytes results in increased hepatocyte proliferation and promotion of DEN‐induced hepatic tumors secondary to aberrant c‐Myc activation. (HEPATOLOGY 2013;57:2480–2490)
Hepatocellular carcinoma (HCC) is the most common hepatic malignancy and the third leading cause of cancer related deaths. Previous studies have implicated bile acids in pathogenesis of HCC, but the ...mechanisms are not known. We investigated the mechanisms of HCC tumor promotion by bile acids the diethylnitrosamine (DEN)-initiation-cholic acid (CA)-induced tumor promotion protocol in mice. The data show that 0.2% CA treatment resulted in threefold increase in number and size of DEN-induced liver tumors. All tumors observed in DEN-treated mice were well-differentiated HCCs. The HCCs observed in DEN-treated CA-fed mice exhibited extensive CD3-, CD20-, and CD45-positive inflammatory cell aggregates. Microarray-based global gene expression studies combined with Ingenuity Pathway Analysis revealed significant activation of NF-κB and Nanog in the DEN-treated 0.2% CA-fed livers. Further studies showed significantly higher TNF-α and IL-1β mRNA, a marked increase in total and phosphorylated-p65 and phosphorylated IκBα (degradation form) in livers of DEN-treated 0.2% CA-fed mice. Treatment of primary mouse hepatocytes with various bile acids showed significant induction of stemness genes including Nanog, KLF4, Sox2, and Oct4. Quantification of total and 20 specific bile acids in liver, and serum revealed a tumor-associated bile acid signature. Finally, quantification of total serum bile acids in normal, cirrhotic, and HCC human samples revealed increased bile acids in serum of cirrhotic and HCC patients. Taken together, these data indicate that bile acids are mechanistically involved pathogenesis of HCC and may promote HCC formation via activation of inflammatory signaling.
Hepatocyte nuclear factor 4α (HNF4α) is an orphan nuclear receptor commonly known as the master regulator of hepatic differentiation, owing to the large number of hepatocyte-specific genes it ...regulates. Whereas the role of HNF4α in hepatocyte differentiation is well
recognized and extensively studied, its role in regulation of cell proliferation is relatively less known. Recent studies have revealed that HNF4α inhibits proliferation not only of hepatocytes but also cells in colon and kidney. Further, a growing number of studies have demonstrated
that inhibition or loss of HNF4α promotes tumorigenesis in the liver and colon, and reexpression of HNF4α results in decreased cancer growth. Studies using tissue-specific conditional knockout mice, knock-in studies, and combinatorial bioinformatics of RNA/ChIP-sequencing data
indicate that the mechanisms of HNF4α-mediated inhibition of cell proliferation are multifold, involving epigenetic repression of promitogenic genes, significant cross talk with other cell cycle regulators including c-Myc and cyclin D1, and regulation of miRNAs. Furthermore, studies
indicate that posttranslational modifications of HNF4α may change its activity and may be at the core of its dual role as a differentiation factor and repressor of proliferation. This review summarizes recent findings on the role of HNF4α in cell proliferation and highlights the
newly understood function of this old receptor.
Hepatocyte nuclear factor 4 alpha (HNF4α) is known as the master regulator of hepatic differentiation, which regulates over 60% of the hepatocyte specific genes. Recent studies including this ...(Walesky et al. Am J Physiol Gastrointest Liver Physiol. 304:G26-37, 2013) demonstrated that HNF4α also inhibits hepatocyte proliferation via repression of pro-mitogenic genes. In this study hepatocyte specific HNF4α knockout mice were generated using 2–3month old HNF4α-floxed mice treated with Cre recombinase under Major Urinary Protein promoter delivered in AAV8 vector (MUP-iCre-AAV8). Control mice were treated with MUP-EGFP-AAV8. Livers were isolated from control and KO mice one week after AAV8 administration and used for gene array analysis. These data revealed several new negative target genes of HNF4α, majority of which are pro-mitogeneic genes inhibited by HNF4α in adult hepatocytes.
Background and Aims
Hepatocyte nuclear factor 4 alpha (HNF4α) is indispensable for hepatocyte differentiation and critical for maintaining liver health. Here, we demonstrate that loss of HNF4α ...activity is a crucial step in the pathogenesis of chronic liver diseases (CLDs) that lead to development of HCC.
Approach and Results
We developed an HNF4α target gene signature, which can accurately determine HNF4α activity, and performed an exhaustive in silico analysis using hierarchical and K‐means clustering, survival, and rank‐order analysis of 30 independent data sets containing over 3500 individual samples. The association of changes in HNF4α activity to CLD progression of various etiologies, including HCV‐ and HBV‐induced liver cirrhosis (LC), NAFLD/NASH, and HCC, was determined. Results revealed a step‐wise reduction in HNF4α activity with each progressive stage of pathogenesis. Cluster analysis of LC gene expression data sets using the HNF4α signature showed that loss of HNF4α activity was associated with progression of Child‐Pugh class, faster decompensation, incidence of HCC, and lower survival with and without HCC. A moderate decrease in HNF4α activity was observed in NAFLD from normal liver, but a further significant decline was observed in patients from NAFLD to NASH. In HCC, loss of HNF4α activity was associated with advanced disease, increased inflammatory changes, portal vein thrombosis, and substantially lower survival.
Conclusions
In conclusion, these data indicate that loss of HNF4α function is a common event in the pathogenesis of CLDs leading to HCC and is important from both diagnostic and therapeutic standpoints.
Abstract only
Cholangiocarcinoma (CC) is among the deadliest cancers worldwide, with a five‐year survival rate of ~15% in intrahepatic cholangiocarcinoma (ICC) and ~30% in extrahepatic ...cholangiocarcinoma (ECC). It is a rare cancer, affecting ~8,500 patients in the U.S. each year. The incidence of CC has increased, and there are few effective strategies for prevention, diagnosis, or treatment, indicating a significant unmet medical need. Often, altered developmental signaling can shed light on pathways that may also be altered during cancer pathogenesis. Therefore, our strategy is to understand mechanisms that are important in hepatobiliary development and examine these pathways in the context of CC pathogenesis in hopes of discovering novel therapeutic targets.
We have recently shown that loss of hepatocyte nuclear factor 4 alpha (HNF4α) contributes to the pathogenesis of CC. Further, Wnt/β‐catenin has recently been shown to be upregulated in patients with CC. Therefore, we hypothesized that an HNF4α/β‐catenin interaction may be important in the context of CC pathogenesis.
Hepatocytes and cholangiocytes are thought to originate from a bipotent progenitor cell population called hepatoblasts (Prox1‐positive). In the current study we have demonstrated the importance of proper HNF4α/β‐catenin signaling in the development of both hepatocytes and cholangiocytes within the zebrafish liver. HNF4α knockdown results in a loss of the hepatoblast marker Prox1. Interestingly, zebrafish maintain development of cholangiocytes following a loss of the Prox1‐positive hepatoblast population. Further, genetic or chemical increase in β‐catenin (APC‐/‐ ; Wnt agonist) results in a loss of HNF4α, Prox1, and a maintenance of cholangiocyte differentiation. Therefore, we hypothesize that over‐activation of β‐catenin results in a loss of HNF4α, causing hepatoblasts to preferentially differentiate into cholangiocytes.
These studies demonstrate that HNF4α/β‐catenin signaling are directly linked to regulate hepatobiliary development. Further investigation into the mechanisms by which these pathways interact can provide valuable knowledge in understanding hepatobiliary development and how alterations in these pathways may contribute to cholangiocarcinoma pathogenesis.
Support or Funding Information
Supported by a research fellowship from the Cholangiocarcinoma Foundation
Acetaminophen (APAP) overdose results in acute liver failure and has limited treatment options. Previous studies show that stimulating liver regeneration is critical for survival after APAP overdose, ...but the mechanisms remain unclear. In this study, we identified major signaling pathways involved in liver regeneration after APAP-induced acute liver injury using a novel incremental dose model. Liver injury and regeneration were studied in C57BL/6 mice treated with either 300 mg/kg (APAP300) or 600 mg/kg (APAP600) APAP. Mice treated with APAP300 developed extensive liver injury and robust liver regeneration. In contrast, APAP600-treated mice exhibited significant liver injury but substantial inhibition of liver regeneration, resulting in sustained injury and decreased survival. The inhibition of liver regeneration in the APAP600 group was associated with cell cycle arrest and decreased cyclin D1 expression. Several known regenerative pathways, including the IL-6/STAT-3 and epidermal growth factor receptor/c-Met/mitogen-activated protein kinase pathways, were activated, even at APAP600, where regeneration was inhibited. However, canonical Wnt/β-catenin and NF-κB pathways were activated only in APAP300-treated mice, where liver regeneration was stimulated. Furthermore, overexpression of a stable form of β-catenin, where serine 45 is mutated to aspartic acid, in mice resulted in improved liver regeneration after APAP overdose. Taken together, our incremental dose model has identified a differential role of several signaling pathways in liver regeneration after APAP overdose and highlighted canonical Wnt signaling as a potential target for regenerative therapies for APAP-induced acute liver failure.
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