Mark Gosink

Identification of tubular injury microRNA biomarkers in urine: comparison of next-generation sequencing and qPCR-based profiling platforms

BackgroundMicroRNAs (miRNAs) are small, non-coding RNAs that regulate protein levels post-transcriptionally. miRNAs play important regulatory roles in many cellular processes and have been implicated in several diseases. Recent studies have reported significant levels of miRNAs in a variety of body fluids, raising the possibility that miRNAs could serve as useful biomarkers. Next-generation sequencing (NGS) is increasingly employed in biomedical investigations. Although concordance between this platform and qRT-PCR based assays has been reported in high quality specimens, information is lacking on comparisons in biofluids especially urine. Here we describe the changes in miRNA expression patterns in a rodent model of renal tubular injury (gentamicin). Our aim is to compare RNA sequencing and qPCR based miRNA profiling in urine specimen from control and rats with confirmed tubular injury.ResultsOur preliminary examination of the concordance between miRNA-seq and qRT-PCR in urine specimen suggests minimal agreement between platforms probably due to the differences in sensitivity. Our results suggest that although miRNA-seq has superior specificity, it may not detect low abundant miRNAs in urine samples. Specifically, miRNA-seq did not detect some sequences which were identified by qRT-PCR. On the other hand, the qRT-PCR analysis was not able to detect the miRNA isoforms, which made up the majority of miRNA changes detected by NGS.ConclusionsTo our knowledge, this is the first time that miRNA profiling platforms including NGS have been compared in urine specimen. miRNAs identified by both platforms, let-7d, miR-203, and miR-320, may potentially serve as promising novel urinary biomarkers for drug induced renal tubular epithelial injury.

The Role of Hypoxia in 2-Butoxyethanol–Induced Hemangiosarcoma

To understand the molecular mechanisms underlying compound-induced hemangiosarcomas in mice, and therefore, their human relevance, a systems biology approach was undertaken using transcriptomics and Causal Network Modeling from mice treated with 2-butoxyethanol (2-BE). 2-BE is a hemolytic agent that induces hemangiosarcomas in mice. We hypothesized that the hemolysis induced by 2-BE would result in local tissue hypoxia, a well-documented trigger for endothelial cell proliferation leading to hemangiosarcoma. Gene expression data from bone marrow (BM), liver, and spleen of mice exposed to a single dose (4 h) or seven daily doses of 2-BE were used to develop a mechanistic model of hemangiosarcoma. The resulting mechanistic model confirms previous work proposing that 2-BE induces macrophage activation and inflammation in the liver. In addition, the model supports local tissue hypoxia in the liver and spleen, coupled with increased erythropoeitin signaling and erythropoiesis in the spleen and BM, and suppression of mechanisms that contribute to genomic stability, events that could be contributing factors to hemangiosarcoma formation. Finally, an immunohistochemistry method (Hypoxyprobe) demonstrated that tissue hypoxia was present in the spleen and BM. Together, the results of this study identify molecular mechanisms that initiate hemangiosarcoma, a key step in understanding safety concerns that can impact drug decision processes, and identified hypoxia as a possible contributing factor for 2-BE–induced hemangiosarcoma in mice.

SAG/ROC2/Rbx2/Hrt2, a Component of SCF E3 Ubiquitin Ligase: Genomic Structure, a Splicing Variant, and Two Family Pseudogenes

We have recently cloned and characterized an evolutionarily conserved gene, Sensitive to Apoptosis Gene (SAG), which encodes a redox-sensitive antioxidant protein that protects cells from apoptosis induced by redox agents. The SAG protein was later found to be the second family member of ROC/Rbx/Hrt, a component of the Skp1-cullin-F box protein (SCF) E3 ubiquitin ligase, being required for yeast growth and capable of promoting cell growth during serum starvation. Here, we report the genomic structure of the SAG gene that consists of four exons and three introns. We also report the characterization of a SAG splicing variant (SAG-v), that contains an additional exon (exon 2; 264 bp) not present in wildtype SAG. The inclusion of exon 2 disrupts the SAG ORF and gives rise to a protein of 108 amino acids that contains the first 59 amino acids identical to SAG and a 49-amino acid novel sequence at the C terminus. The entire RING-finger domain of SAG was not translated because of several inframe stop codons within the exon 2. The SAG-v protein was expressed in multiple human tissues as well as cell lines, but at a much lower level than wildtype SAG. Unlike SAG, SAG-v was not able to rescue yeast cells from lethality in a ySAG knockout, nor did it bind to cullin-1 or have ligase activity, probably because of the lack of the RING-finger domain. Finally, we report the identification of two SAG family pseudogenes, SAGP1 and SAGP2, that share 36% or 47% sequence identity with ROC1/Rbx1/Hrt1 and 30% or 88% with SAG, respectively. Both genes are intronless with two inframe stop codons.

Serum microRNA signatures as "liquid biopsies" for interrogating hepatotoxic mechanisms and liver pathogenesis in human

MicroRNAs (miRNAs) released into the peripheral circulation upon cellular injury have shown a promise as a new class of tissue-specific biomarkers. We were first to demonstrate that next-generation sequencing analysis of serum from human subjects with acetaminophen-induced liver injury revealed a specific signature of circulating miRNAs. We consequently hypothesized that different types of hepatic liver impairments might feature distinct signatures of circulating miRNAs and that this approach might be useful as minimally invasive diagnostic “liquid biopsies” enabling the interrogation of underlying molecular mechanisms of injury in distant tissues. Therefore we examined serum circulating miRNAs in a total of 72 serum samples from a group of 53 subjects that included patients with accidental acetaminophen overdose, hepatitis B infection, liver cirrhosis and type 2 diabetes as well as gender- and age-matched healthy subjects with no evidence of liver disease. The miRNA signatures were identified using next-generation sequencing that provided analysis for the whole miRNome, including miRNA isoforms. Compared to the healthy subjects, a total of 179 miRNAs showed altered serum levels across the diseased subjects. Although many subjects have elevated alanine aminotransferase suggesting liver impairments, we identified distinct miRNA signatures for different impairments with minimum overlap. Furthermore, the bioinformatics analysis of miRNA signatures revealed relevant molecular pathways associated with the mechanisms of toxicity and or pathogenesis of disease. Interestingly, the high proportion of miRNA isoforms present in the respective signatures indicated a new level of complexity in cellular response to stress or disease. Our study demonstrates for the first time that signatures of circulating miRNAs show specificity for liver injury phenotypes and, once validated, might become useful for diagnosis of organ pathologies as “liquid biopsies”.

GenSensor Suite: A Web-Based Tool for the Analysis of Gene and Protein Interactions, Pathways, and Regulation

The GenSensor Suite consists of four web tools for elucidating relationships among genes and proteins. GenPath results show which biochemical, regulatory, or other gene set categories are over- or under-represented in an input list compared to a background list. All common gene sets are available for searching in GenPath, plus some specialized sets. Users can add custom background lists. GenInteract builds an interaction gene list from a single gene input and then analyzes this in GenPath. GenPubMed uses a PubMed query to identify a list of PubMed IDs, from which a gene list is extracted and queried in GenPath. GenViewer allows the user to query one gene set against another in GenPath. GenPath results are presented with relevant P- and q-values in an uncluttered, fully linked, and integrated table. Users can easily copy this table and paste it directly into a spreadsheet or document.

The genomic sequence of lymphocryptovirus from cynomolgus macaque.

Lymphocryptoviruses such as Epstein-Barr virus (EBV) cause persistent infections in human and non-human primates, and suppression of the immune system can increase the risk of lymphocryptovirus (LCV)-associated tumor development in both human and non-human primates. To enable LCV infection as a non-clinical model to study effects of therapeutics on EBV immunity, we determined the genomic DNA sequence of the LCV from cynomolgus macaque, a species commonly used for non-clinical testing. Comparison to rhesus macaque LCV and human EBV sequences indicates that LCV from the cynomolgus macaque has the same genomic arrangement and a high degree of similarity in most genes, especially with rhesus macaque LCV. Genes showing lower similarity were those encoding proteins involved in latency and/or tumor promotion or immune evasion. The genomic sequence of LCV from cynomolgus macaque should aid the development of non-clinical tools for identifying therapeutics that impact LCV immunity and carry potential lymphoma risk.

ToxEvaluator: an integrated computational platform to aid the interpretation of toxicology study-related findings

Attempts are frequently made to investigate adverse findings from preclinical toxicology studies in order to better understand underlying toxicity mechanisms. These efforts often begin with limited information, including a description of the adverse finding, knowledge of the structure of the chemical associated with its cause and the intended pharmacological target. ToxEvaluator was developed jointly by Pfizer and the Comparative Toxicogenomics Database (http://ctdbase.org) team at North Carolina State University as an in silico platform to facilitate interpretation of toxicity findings in light of prior knowledge. Through the integration of a diverse set of in silico tools that leverage a number of public and proprietary databases, ToxEvaluator streamlines the process of aggregating and interrogating diverse sources of information. The user enters compound and target identifiers, and selects adverse event descriptors from a safety lexicon and mapped MeSH disease terms. ToxEvaluator provides a summary report with multiple distinct areas organized according to what target or structural aspects have been linked to the adverse finding, including primary pharmacology, structurally similar proprietary compounds, structurally similar public domain compounds, predicted secondary (i.e. off-target) pharmacology and known secondary pharmacology. Similar proprietary compounds and their associated in vivo toxicity findings are reported, along with a link to relevant supporting documents. For similar public domain compounds and interacting targets, ToxEvaluator integrates relationships curated in Comparative Toxicogenomics Database, returning all direct and inferred linkages between them. As an example of its utility, we demonstrate how ToxEvaluator rapidly identified direct (primary pharmacology) and indirect (secondary pharmacology) linkages between cerivastatin and myopathy.

Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples

Archival formalin-fixed paraffin-embedded (FFPE) tissue samples offer a vast but largely untapped resource for genomic research. The primary technical issues limiting use of FFPE samples are RNA yield and quality. In this study, we evaluated methods to demodify RNA highly fragmented and crosslinked by formalin fixation. Primary endpoints were RNA recovery, RNA-sequencing quality metrics, and transcriptional responses to a reference chemical (phenobarbital, PB). Frozen mouse liver samples from control and PB groups (n = 6/group) were divided and preserved for 3 months as follows: frozen (FR); 70% ethanol (OH); 10% buffered formalin for 18 h followed by ethanol (18F); or 10% buffered formalin (3F). Samples from OH, 18F, and 3F groups were processed to FFPE blocks and sectioned for RNA isolation. Additional sections from 3F received the following demodification protocols to mitigate RNA damage: short heated incubation with Tris-Acetate-EDTA buffer; overnight heated incubation with an organocatalyst using 2 different isolation kits; or overnight heated incubation without organocatalyst. Ribo-depleted, stranded, total RNA libraries were built and sequenced using the Illumina HiSeq 2500 platform. Overnight incubation (± organocatalyst) increased RNA yield >3-fold and RNA integrity numbers and fragment analysis values by > 1.5- and >3.0-fold, respectively, versus 3F. Postsequencing metrics also showed reduced bias in gene coverage and deletion rates for overnight incubation groups. All demodification groups had increased overlap for differentially expressed genes (77%-84%) and enriched pathways (91%-97%) with FR, with the highest overlap in the organocatalyst groups. These results demonstrate simple changes in RNA isolation methods that can enhance genomic analyses of FFPE samples.

Use of Rat Primary Mesenteric Cells for the Prediction of PDE4 Inhibitor Drug-Induced Vascular Injury

Drug-induced vascular injury (DIVI) in preclinical studies can delay, if not terminate, a drug development program. Clinical detection of DIVI can be very difficult as there are no definitive biomarkers known to reliably detect this disorder in all instances. The preclinical identification of DIVI requires detailed microscopic examination of a wide range of tissues although one of the most commonly affected areas in rats is the mesenteric vasculature. The reason for this predisposition of mesenteric arteries in rats as well as the exact mechanism and cell types involved in the initial development of these lesions have not been fully elucidated. We hypothesized that by using a mixed culture of cells from rat mesenteric tissue, we would be able to identify an RNA expression signature that could predict the invivo development of DIVI. Five compounds designed to inhibit Phosphodiesterase 4 activity (PDE4i) were chosen as positive controls. PDE4is are well known to induce DIVI in the mesenteric vasculature of rats and there is microscopic evidence that this is associated, at least in part, with a proinflammatory mechanism. We surveyed, by qRT-PCR, the expression of 96 genes known to be involved in inflammation and using a Random-Forest model, identified 12 genes predictive of invivo DIVI outcomes in rats. Using these genes, we were able to cross-validate the ability of the Random-Forest modeling to predict the concentration at which PDE4i caused DIVI invivo.

Induction of endogenous retroelements as a potential mechanism for mouse-specific drug-induced carcinogenicity

A number of chemical compounds have been shown to induce liver tumors in mice but not in other species. While several mechanisms for this species-specific tumorigenicity have been proposed, no definitive mechanism has been established. We examined the effects of the nongenotoxic rodent hepatic carcinogen, WY-14,643, in male mice from a high liver tumor susceptible strain (C3H/HeJ), and from a low tumor susceptible strain (C57BL/6). WY-14,643, a PPARα activator induced widespread increases in the expression of some endogenous retroelements, namely members of LTR and LINE elements in both strains. The expression of a number of known retroviral defense genes was also elevated. We also demonstrated that basal immune-mediated viral defense was elevated in C57BL/6 mice (the resistant strain) and that WY-14,643 further activated those immuno-defense processes. We propose that the previously reported >100X activity of retroelements in mice drives mouse-specific tumorigenicity. We also propose that C57BL/6’s competent immune to retroviral activation allows it to remove cells before the activation of these elements can result in significant chromosomal insertions and mutation. Finally, we showed that WY-14,643 treatment induced gene signatures of DNA recombination in the sensitive C3H/HeJ strain.