Julie A. Hicks

MicroRNA Expression in Chicken Embryos

MicroRNA (miRNA) are small single-stranded noncoding RNA that posttranscriptionally regulate gene expression. A major role of miRNA is the regulation of gene expression in developmental processes. In this study, we constructed a small RNA library from 11-d-old chick embryos and used this library to examine the miRNA expression profile of the embryos. This small RNA library was sequenced by using 454 Life Sciences pyrosequencing technology. A total of 10,466 sequences were obtained and annotated as either known chicken miRNA, miRNA that shared homology with other species, or novel miRNA. We identified the expression of 110 known chicken miRNA, 36 homologous chicken miRNA (previously unannotated in the chicken but conserved with miRNA from other species), and 14 novel chicken-specific miRNA not identified in any other species. We also demonstrated that some of the identified chicken embryonic miRNA are differentially expressed among the developing spleen, liver, or bursa. The current study demonstrates that a very diverse and dynamic set of miRNA is expressed in the embryonic chick at 11 d of incubation. The identification of miRNA present in the embryonic chicken will further aid in understanding the complexity of gene regulation during vertebrate development.

Discovery of chicken microRNAs associated with lipogenesis and cell proliferation

The primary function of microRNA (miRNA, a class of small regulatory RNA) is to regulate gene expression. Studies of miRNA in mammals suggest that many liver-associated miRNAs are expressed, with a wide range of functions. To characterize miRNA expressed in the avian liver, we created two small RNA libraries from embryonic chick livers at embryonic day (E)15 and E20, a time at which the embryo begins to grow rapidly and so its energy demands increase. It is of interest to examine miRNAs expressed at these developmental stages because miRNAs involved in regulating metabolic pathways and cell proliferation are likely to be identified. The small RNA libraries were sequenced with 454 Life Sciences deep sequencing. Of the 49,937 sequences obtained, 29,390 represented known chicken miRNAs and 1,233 reads represented homologous miRNAs that have not been previously identified in chickens. Additionally, 1,032 reads represented 17 potential novel miRNAs not previously identified in any species. To further investigate the possible functions of avian liver miRNAs we identified the potential targets of two differentially expressed novel miRNAs, nc-miR-5 and nc-miR-33. These two miRNAs were predicted to target metabolic genes, including the lipid metabolism-associated gene fatty acid synthase (FAS), and genes involved in the control of cell proliferation, such as peroxisome proliferator-activated binding protein (Pparbp) and bone morphogenetic protein 4 (BMP4). Our findings demonstrate that a diverse group of miRNAs are expressed in developing avian livers. In addition, some of the identified miRNAs have been suggested to play a key role(s) in regulating metabolic pathways.

Characterization of the microRNAome in porcine reproductive and respiratory syndrome virus infected macrophages.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a member of the arterivirus family, is the causative agent of Porcine Reproductive and Respiratory Syndrome (PRRS). PRRS is characterized by late term abortions and respiratory disease, particularly in young pigs. Small regulatory RNAs termed microRNA (miRNA) are associated with gene regulation at the post-transcriptional level. MiRNAs are known to play many diverse and complex roles in viral infections. To discover the impact of PRRSV infections on the cellular miRNAome, Illumina deep sequencing was used to construct small RNA expression profiles from in vitro cultured PRRSV-infected porcine alveolar macrophages (PAMs). A total of forty cellular miRNAs were significantly differentially expressed within the first 48 hours post infection (hpi). The expression of six miRNAs, miR-30a-3p, miR-132, miR-27b*, miR-29b, miR-146a and miR-9-2, were altered at more than one time point. Target gene identification suggests that these miRNAs are involved in regulating immune signaling pathways, cytokine, and transcription factor production. The most highly repressed miRNA at 24 hpi was miR-147. A miR-147 mimic was utilized to maintain miR-147 levels in PRRSV-infected PAMs. PRRSV replication was negatively impacted by high levels of miR-147. Whether down-regulation of miR-147 is directly induced by PRRSV or if it is part of the cellular response and PRRSV indirectly benefits remains to be determined. No evidence could be found of PRRSV-encoded miRNAs. Overall, the present study has revealed that a large and diverse group of miRNAs are expressed in swine alveolar macrophages and that the expression of a subset of these miRNAs is altered in PRRSV infected macrophages.

Current knowledge of microRNA characterization in agricultural animals.

MicroRNA (miRNA) is a class of single-stranded small (19-24nt) regulatory RNA that silences gene expression post-transcriptionally. miRNAs regulate a wide range of biological processes through the recognition of complementary sequences between miRNAs and their target genes. Profiling studies in livestock have revealed that many miRNAs are species- and tissue-specific, indicating that miRNAs play important roles in essential biological processes in livestock, such as muscle and organ development, the immune response and metabolism. The allelic variation of miRNA target sites and possibly in miRNAs themselves are also likely to be contributing factors to many phenotypic differences in livestock. In this review, we summarize the current miRNA studies undertaken in livestock.

Proteomic Dissection of Viral Pathogenesis

Many factors play into the complexity of viral pathogenesis. Understanding viral pathogenesis is key to developing vaccines and treatments for viral diseases. One emerging area of research is proteomics, which is the study of the protein complement and functions of the genome. Many different proteomic approaches have been utilized by researchers worldwide to further elucidate viral pathogenesis. For example, a high throughput MALDI-MS approach was recently employed to study the antigenicity of the influenza virus. Another study utilized MALDI-TOF MS and liquid chromatography MS/MS of proteins present in lipid droplets of hepatoma cell lines to identify proteins involved in the pathogenesis of hepatitis C virus that contribute to its carcinogenic properties. In conjugation with MS, yeast two-hybrid systems have also been shown to be useful in identifying potential host receptors of various viruses as well as revealing the interaction of viral proteins with other host proteins and viral proteins. In this review, the focus is on various proteomic approaches to dissecting the mechanisms of viral pathogenesis.

Involvement of Eukaryotic Small RNA Pathways in Host Defense and Viral Pathogenesis

Post-transcriptional gene regulation by small RNAs is now established as an important branch of the gene regulatory system. Many different classes of small RNAs have been discovered; among these are short interfering RNAs (siRNAs) and microRNA (miRNAs). Though differences in the processing and function of small RNAs exist between plants and animals, both groups utilize small RNA-mediated gene regulation in response to pathogens. Host encoded miRNAs and siRNAs are generated from viral RNA function in host defense and pathogenic resistance in plants. In animals, miRNAs are key regulators in both immune system development and in immune function. Pathogens, in particular viruses, have evolved mechanisms to usurp the host’s small RNA-mediated regulatory system. Overall, small RNAs are a major component of host defense and immunity in eukaryotes. The goal of this review is to summarize our current knowledge of the involvement of eukaryotic small RNA pathways in host defense and viral pathogenesis.

Characterization of miR-10a mediated gene regulation in avian splenocytes.

It is well established that microRNAs (miRNAs) are an important class of post-transcriptional regulators of gene expression. Although numerous miRNA expression profiles have been generated for many eukaryotic organisms, little is known about the specific functions of individual miRNAs in regulating gene expression. We previously reported that the miRNA, miR-10a, is highly expressed during spleen development in embryonic chicks. In this current study we have identified genes and potential pathways that are both directly and indirectly influenced by miR-10a expression. To achieve this goal, miRNA Real-Time (RT) PCR analysis was first utilized to examine miR-10a expression across tissues during both embryonic and post-hatch chick development. Next, microarray analysis was employed to determine alterations in global gene expression associated with miR-10a in embryonic chick splenocytes subjected to an in vitro miR-10a inhibitor treatment. Finally the miRNA target prediction algorithm miRanda was used to predict potential chicken genes directly targeted by miR-10a. A select group of potential miR-10a target genes was validated using an RCAS-miRNA expression based luciferase assay. Our results indicate that miR-10a is highly expressed in the avian spleen, lung, kidneys, and fat tissues. Functional analysis suggests that miR-10a is involved in regulating gene expression in pathways associated with Ras signaling, intracellular trafficking, and development of immune functions. Additionally, we confirmed that chicken HOXA1 is a miR-10a target gene, suggesting a conserved role for miR-10a in the regulation of hematopoiesis across vertebrates.

Interdisciplinary Parkinson’s Disease Deep Brain Stimulation Screening and the Relationship to Unintended Hospitalizations and Quality of Life

Objective To investigate the impact of pre-operative deep brain stimulation (DBS) interdisciplinary assessments on post-operative hospitalizations and quality of life (QoL). Background DBS has been utilized successfully in Parkinson’s disease (PD) for the treatment of tremor, rigidity, bradykinesia, off time, and motor fluctuations. Although DBS is becoming a more common management approach there are no standardized criteria for selection of DBS candidates, and sparse data exist to guide the use of interdisciplinary evaluations for DBS screening. We reviewed the outcomes of the use of an interdisciplinary model which utilized seven specialties to pre-operatively evaluate potential DBS candidates. Methods The University of Florida (UF) INFORM database was queried for PD patients who had DBS implantations performed at UF between January 2011 and February 2013. Records were reviewed to identify unintended hospitalizations, falls, and infections. Minor and major concerns or reservations from each specialty were previously documented and quantified. Clinical outcomes were assessed through the use of the Parkinson disease quality of life questionnaire (PDQ-39), and the Unified Parkinson’s Disease Rating Score (UPDRS) Part III. Results A total of 164 cases were evaluated for possible DBS candidacy. There were 133 subjects who were approved for DBS surgery (81%) following interdisciplinary screening. There were 28 cases (21%) who experienced an unintended hospitalization within the first 12 months following the DBS operation. The patients identified during interdisciplinary evaluation with major or minor concerns from any specialty service had more unintended hospitalizations (93%) when compared to those without concerns (7%). When the preoperative “concern” shifted from “major” to “minor” to “no concerns,” the rate of hospitalization decreased from 89% to 33% to 3%. A strong relationship was uncovered between worsened PDQ-39 at 12 months and increased hospitalization. Conclusions Unintended hospitalizations and worsened QOL scores correlated with the number and severity of concerns raised by interdisciplinary DBS evaluations. The data suggest that detailed screenings by interdisciplinary teams may be useful for more than just patient selection. These evaluations may help to stratify risk for post-operative hospitalization and QoL outcomes.

Interaction of porcine reproductive and respiratory syndrome virus major envelope proteins GP5 and M with the cellular protein Snapin

BACKGROUND Porcine reproductive and respiratory syndrome (PRRS) is characterized by abortions in pregnant sows and respiratory disease, particularly in young pigs. The causative agent is porcine reproductive and respiratory syndrome virus (PRRSV), a member of the arterivirus family. GP5 and M are the major envelope proteins encoded by PRRSV. To further characterize these two viral proteins, a yeast two-hybrid approach was utilized to identify interacting partners of PRRSV GP5 and M proteins. METHODS Interacting partners of PRRSV GP5 and M were identified using a porcine macrophage cDNA library yeast two-hybrid screen. Subsequently, the interactions between PRRSV GP5/M and the cellular protein Snapin were mapped using truncated versions of the GP5 and M proteins in a yeast two-hybrid assay to localize the interactions. The Snapin gene from the African green monkey kidney cell line MARC-145, which is permissive to PRRSV, was cloned and sequenced, and compared to porcine Snapin. Cellular Snapin expression was reduced in PRRSV-infected cells via Snapin-specific siRNA targeting. RESULTS Here we show that the cellular Snap-Associated Protein (Snapin), an accessory protein of the SNARE membrane fusion network and also a member of the BLOC-1 complex, specifically interacts with GP5 and M. Inhibition of Snapin expression via siRNA targeting of Snapin results in the reduction of PRRSV replication. CONCLUSIONS The PRRSV GP5 and M proteins are known to form a heterodimeric complex which is important for viral structure and infectivity, and both PRRSV proteins can interact with cellular Snapin. Snapin knock-down suggests these interactions could be important in the PRRSV lifecycle. GP5 and M proteins may interact with Snapin to exploit its roles in intracellular transport and membrane fusion.

Alterations in cellular and viral microRNA and cellular gene expression in Marek's disease virus-transformed T-cell lines treated with sodium butyrate

ABSTRACT A shared feature of herpesviruses is their ability to enter a latent state following an initially lytic infection. Mareks disease virus serotype 1 (MDV‐1) is an oncogenic avian herpesvirus. Small RNA profiling studies have suggested that microRNAs (miRNAs) are involved in viral latency. Sodium butyrate treatment is known to induce herpesvirus reactivation. The present study was undertaken to determine transcriptome and miRNome changes induced by sodium butyrate in 2 MDV‐transformed cell lines, RP2 and CU115. In the first 24 h post‐treatment, microarray analysis of transcriptional changes in cell lines RP2 and CU115 identified 137 and 114 differentially expressed genes, respectively. Small RNA deep‐sequencing analysis identified 17 cellular miRNAs that were differentially expressed. The expression of MDV‐encoded miRNAs was also altered upon treatment. Many of the genes and miRNAs that are differentially expressed are involved in regulation of the cell cycle, mitosis, DNA metabolism, and lymphocyte differentiation.