Investigating the effects of chronic low-dose radiation exposure in the liver of a hypothermic zebrafish model.

Mankind's quest for a manned mission to Mars is placing increased emphasis on the development of innovative radio-protective countermeasures for long-term space travel. Hibernation confers radio-protective effects in hibernating animals, and this has led to the investigation of synthetic torpor to mitigate the deleterious effects of chronic low-dose-rate radiation exposure. Here we describe an induced torpor model we developed using the zebrafish. We explored the effects of radiation exposure on this model with a focus on the liver. Transcriptomic and behavioural analyses were performed. Radiation exposure resulted in transcriptomic perturbations in lipid metabolism and absorption, wound healing, immune response, and fibrogenic pathways. Induced torpor reduced metabolism and increased pro-survival, anti-apoptotic, and DNA repair pathways. Coupled with radiation exposure, induced torpor led to a stress response but also revealed maintenance of DNA repair mechanisms, pro-survival and anti-apoptotic signals. To further characterise our model of induced torpor, the zebrafish model was compared with hepatic transcriptomic data from hibernating grizzly bears (Ursus arctos horribilis) and active controls revealing conserved responses in gene expression associated with anti-apoptotic processes, DNA damage repair, cell survival, proliferation, and antioxidant response. Similarly, the radiation group was compared with space-flown mice revealing shared changes in lipid metabolism.

miRNA and lncRNA Expression Networks Modulate Cell Cycle and DNA Repair Inhibition in Senescent Prostate Cells.

Cellular senescence is a state of permanent growth arrest that arises once cells reach the limit of their proliferative capacity. It creates an inflammatory microenvironment favouring the initiation and progression of various age-related diseases, including prostate cancer. Non-coding RNAs (ncRNAs) have emerged as important regulators of cellular gene expression. Nonetheless, very little is known about the interplay of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) and how deregulation of ncRNA networks promotes cellular senescence. To investigate this, human prostate epithelial cells were cultured through different passages until senescent, and their RNA was extracted and sequenced using RNA sequencing (RNAseq) and microRNA sequencing (miRNA-seq) miRNAseq. Differential expression (DE) gene analysis was performed to compare senescent and proliferating cells with Limma, miRNA-target interactions with multiMiR, lncRNA-target interactions using TCGA data and network evaluation with miRmapper. We found that miR-335-3p, miR-543 and the lncRNAs H19 and SMIM10L2A all play central roles in the regulation of cell cycle and DNA repair processes. Expression of most genes belonging to these pathways were down-regulated by senescence. Using the concept of network centrality, we determined the top 10 miRNAs and lncRNAs, with miR-335-3p and H19 identified as the biggest hubs for miRNAs and lncRNA respectively. These ncRNAs regulate key genes belonging to pathways involved in cell senescence and prostate cancer demonstrating their central role in these processes and opening the possibility for their use as biomarkers or therapeutic targets to mitigate against prostate ageing and carcinogenesis.

PDGF Promotes Dermal Fibroblast Activation via a Novel Mechanism Mediated by Signaling Through MCHR1.

Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy and excessive fibrosis of the skin and internal organs. To this day, no effective treatments to prevent the progression of fibrosis exist, and SSc patients have disabilities and reduced life expectancy. The need to better understand pathways that drive SSc and to find therapeutic targets is urgent. RNA sequencing data from SSc dermal fibroblasts suggested that melanin-concentrating hormone receptor 1 (MCHR1), one of the G protein-coupled receptors regulating emotion and energy metabolism, is abnormally deregulated in SSc. Platelet-derived growth factor (PDGF)-BB stimulation upregulated MCHR1 mRNA and protein levels in normal human dermal fibroblasts (NHDF), and MCHR1 silencing prevented the PDGF-BB-induced expression of the profibrotic factors transforming growth factor beta 1 (TGFß1) and connective tissue growth factor (CTGF). PDGF-BB bound MCHR1 in membrane fractions of NHDF, and the binding was confirmed using surface plasmon resonance (SPR). MCHR1 inhibition blocked PDGF-BB modulation of intracellular cyclic adenosine monophosphate (cAMP). MCHR1 silencing in NHDF reduced PDGF-BB signaling. In summary, MCHR1 promoted the fibrotic response in NHDF through modulation of TGFß1 and CTGF production, intracellular cAMP levels, and PDGF-BB-induced signaling pathways, suggesting that MCHR1 plays an important role in mediating the response to PDGF-BB and in the pathogenesis of SSc. Inhibition of MCHR1 should be considered as a novel therapeutic strategy in SSc-associated fibrosis.

Induced Torpor as a Countermeasure for Low Dose Radiation Exposure in a Zebrafish Model.

The development of the Artemis programme with the goal of returning to the moon is spurring technology advances that will eventually take humans to Mars and herald a new era of interplanetary space travel. However, long-term space travel poses unique challenges including exposure to ionising radiation from galactic cosmic rays and potential solar particle events, exposure to microgravity and specific nutritional challenges arising from earth independent exploration. Ionising radiation is one of the major obstacles facing future space travel as it can generate oxidative stress and directly damage cellular structures such as DNA, in turn causing genomic instability, telomere shortening, extracellular-matrix remodelling and persistent inflammation. In the gastrointestinal tract (GIT) this can lead to leaky gut syndrome, perforations and motility issues, which impact GIT functionality and affect nutritional status. While current countermeasures such as shielding from the spacecraft can attenuate harmful biological effects, they produce harmful secondary particles that contribute to radiation exposure. We hypothesised that induction of a torpor-like state would confer a radioprotective effect given the evidence that hibernation extends survival times in irradiated squirrels compared to active controls. To test this hypothesis, a torpor-like state was induced in zebrafish using melatonin treatment and reduced temperature, and radiation exposure was administered twice over the course of 10 days. The protective effects of induced-torpor were assessed via RNA sequencing and qPCR of mRNA extracted from the GIT. Pathway and network analysis were performed on the transcriptomic data to characterise the genomic signatures in radiation, torpor and torpor + radiation groups. Phenotypic analyses revealed that melatonin and reduced temperature successfully induced a torpor-like state in zebrafish as shown by decreased metabolism and activity levels. Genomic analyses indicated that low dose radiation caused DNA damage and oxidative stress triggering a stress response, including steroidal signalling and changes to metabolism, and cell cycle arrest. Torpor attenuated the stress response through an increase in pro-survival signals, reduced oxidative stress via the oxygen effect and detection and removal of misfolded proteins. This proof-of-concept model provides compelling initial evidence for utilizing an induced torpor-like state as a potential countermeasure for radiation exposure.

Prominence of IL6, IGF, TLR, and Bioenergetics Pathway Perturbation in Lung Tissues of Scleroderma Patients With Pulmonary Fibrosis.

Scleroderma-associated pulmonary fibrosis (SSc-PF) and idiopathic pulmonary fibrosis (IPF) are two of many chronic fibroproliferative diseases that are responsible for nearly 45% of all deaths in developed countries. While sharing several pathobiological characteristics, they also have very distinct features. Currently no effective anti-fibrotic treatments exist that can halt the progression of PF or reverse it. Our goal is to uncover potential gene targets for the development of anti-fibrotic therapies efficacious in both diseases, and those specific to SSc-PF, by identifying universal pathways and molecules driving fibrosis in SSc-PF and IPF tissues as well as those unique to SSc-PF. Using DNA microarray data, a meta-analysis of the differentially expressed (DE) genes in SSc-PF and IPF lung tissues (diseased vs. normal) was performed followed by a full systems level analysis of the common and unique transcriptomic signatures obtained. Protein-protein interaction networks were generated to identify hub proteins and explore the data using the centrality principle. Our results suggest that therapeutic strategies targeting IL6 trans-signaling, IGFBP2, IGFL2, and the coagulation cascade may be efficacious in both SSc-PF and IPF. Further, our data suggest that the expression of matrikine-producing collagens is also perturbed in PF. Lastly, an overall perturbation of bioenergetics, specifically between glycolysis and fatty acid metabolism, was uncovered in SSc-PF. Our findings provide insights into potential targets for the development of anti-fibrotic therapies that could be effective in both IPF and SSc-PF.

Genome-Wide Analysis of Low Dose Bisphenol-A (BPA) Exposure in Human Prostate Cells.

Endocrine disrupting compounds (EDCs) have the potential to cause adverse effects on wild-life and human health. Two important EDCs are the synthetic estrogen 17α-ethynylestradiol (EE2) and bisphenol-A (BPA) both of which are xenoestrogens (XEs) as they bind the estrogen receptor and dis-rupt estrogen physiology in mammals and other vertebrates. In the recent years the influence of XEs on oncogenes, specifically in relation to breast and prostate cancer has been the subject of considerable study. METHODOLOGY: In this study, healthy primary human prostate epithelial cells (PrECs) were exposed to environmentally relevant concentrations of BPA (5nM and 25nM BPA) and interrogated using a whole genome microarray. RESULTS: Exposure to 5 and 25nM BPA resulted in 7,182 and 7,650 differentially expressed (DE) genes, respectively in treated PrECs. Exposure to EE2 had the greatest effect on the PrEC transcriptome (8,891 DE genes). CONCLUSION: We dissected and investigated the nature of the non-estrogenic gene signature associated with BPA with a focus on transcripts relevant to epigenetic modifications. The expression of transcripts encoding nuclear hormone receptors as well as histone and DNA methylation, modifying enzymes were significantly perturbed by exposure to BPA.

Systems analysis of the liver transcriptome in adult male zebrafish exposed to the non-ionic surfactant nonylphenol.

Nonylphenol (NP) arises from the environmental degradation of nonylphenol ethoxylates. It is a ubiquitous environmental contaminant and has been detected at levels up to 167 nM in rivers in the United States. NP is an endocrine disruptor (ED) that can act as an agonist for estrogen receptors. The Adverse Outcome Pathway (AOP) framework defines an adverse outcome as the causal result of a series of molecular initiating events (MIEs) and key events (KEs) that lead to altered phenotypes. This study examined the liver transcriptome after a 21 day exposure to NP and 17ß-estradiol (E2) by exploiting the zebrafish (Danio rerio) as a systems toxicology model. The goal of this study was to tease out non-estrogenic genomic signatures associated with NP exposure using DNA microarray and RNA sequencing. Our experimental design included E2 as a positive and potent estrogenic control in order to effectively compare and contrast the 2 compounds. This approach allowed us to identify hepatic transcriptomic perturbations that could serve as MIEs for adverse health outcomes in response to NP. Our results revealed that exposure to NP was associated with differential expression (DE) of genes associated with the development of steatosis, disruption of metabolism, altered immune response, and metabolism of reactive oxygen species, further highlighting NP as a chemical of emerging concern (CEC).

miRmapper: A Tool for Interpretation of miRNA⁻mRNA Interaction Networks.

It is estimated that 30% of all genes in the mammalian cells are regulated by microRNA (miRNAs). The most relevant miRNAs in a cellular context are not necessarily those with the greatest change in expression levels between healthy and diseased tissue. Differentially expressed (DE) miRNAs that modulate a large number of messenger RNA (mRNA) transcripts ultimately have a greater influence in determining phenotypic outcomes and are more important in a global biological context than miRNAs that modulate just a few mRNA transcripts. Here, we describe the development of a tool, "miRmapper", which identifies the most dominant miRNAs in a miRNA⁻mRNA network and recognizes similarities between miRNAs based on commonly regulated mRNAs. Using a list of miRNA⁻target gene interactions and a list of DE transcripts, miRmapper provides several outputs: (1) an adjacency matrix that is used to calculate miRNA similarity utilizing the Jaccard distance; (2) a dendrogram and (3) an identity heatmap displaying miRNA clusters based on their effect on mRNA expression; (4) a miRNA impact table and (5) a barplot that provides a visual illustration of this impact. We tested this tool using nonmetastatic and metastatic bladder cancer cell lines and demonstrated that the most relevant miRNAs in a cellular context are not necessarily those with the greatest fold change. Additionally, by exploiting the Jaccard distance, we unraveled novel cooperative interactions between miRNAs from independent families in regulating common target mRNAs; i.e., five of the top 10 miRNAs act in synergy.

The Plasticizer Bisphenol A Perturbs the Hepatic Epigenome: A Systems Level Analysis of the miRNome.

Ubiquitous exposure to bisphenol A (BPA), an endocrine disruptor (ED), has raised concerns for both human and ecosystem health. Epigenetic factors, including microRNAs (miRNAs), are key regulators of gene expression during cancer. The effect of BPA exposure on the zebrafish epigenome remains poorly characterized. Zebrafish represents an excellent model to study cancer as the organism develops a disease that resembles human cancer. Using zebrafish as a systems toxicology model, we hypothesized that chronic BPA-exposure impacts the miRNome in adult zebrafish and establishes an epigenome more susceptible to cancer development. After a 3 week exposure to 100 nM BPA, RNA from the liver was extracted to perform high throughput mRNA and miRNA sequencing. Differential expression (DE) analyses comparing BPA-exposed to control specimens were performed using established bioinformatics pipelines. In the BPA-exposed liver, 6188 mRNAs and 15 miRNAs were differently expressed (q ≤ 0.1). By analyzing human orthologs of the DE zebrafish genes, signatures associated with non-alcoholic fatty liver disease (NAFLD), oxidative phosphorylation, mitochondrial dysfunction and cell cycle were uncovered. Chronic exposure to BPA has a significant impact on the liver miRNome and transcriptome in adult zebrafish with the potential to cause adverse health outcomes including cancer.

beta-Adrenergic receptor stimulated Ncx1 upregulation is mediated via a CaMKII/AP-1 signaling pathway in adult cardiomyocytes.

The Na(+)-Ca(2+) exchanger gene (Ncx1) is upregulated in hypertrophy and is often found elevated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. beta-Adrenergic receptor (beta-AR) signaling plays an important role in the regulation of calcium homeostasis in the cardiomyocyte, but chronic activation in periods of cardiac stress contributes to heart failure by mechanisms which include Ncx1 upregulation. Here, using a Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIdelta(c)) null mouse, we demonstrate that beta-AR-stimulated Ncx1 upregulation is dependent on CaMKII. beta-AR-stimulated Ncx1 expression is mediated by activator protein 1 (AP-1) factors and is independent of cAMP-response element-binding protein (CREB) activation. The MAP kinases (ERK1/2, JNK and p38) are not required for AP-1 factor activation. Chromatin immunoprecipitation demonstrates that beta-AR stimulation activates the ordered recruitment of JunB homodimers, which then are replaced by c-Jun homodimers binding to the proximal AP-1 elements of the endogenous Ncx1 promoter. In conclusion, this work has provided insight into the intracellular signaling pathways and transcription factors regulating Ncx1 gene expression in a chronically beta-AR-stimulated heart.

Regulation of Ncx1 expression. Identification of regulatory elements mediating cardiac-specific expression and up-regulation.

The Na+-Ca2+ exchanger (NCX1) is up-regulated in hypertrophy and is often found up-regulated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. We have previously shown that the 1831-bp Ncx1 H1 (1831Ncx1) promoter directs cardiac-specific expression of the exchanger in both development and in the adult, and is sufficient for the up-regulation of Ncx1 in response to pressure overload. Here, we utilized adenoviral mediated gene transfer and transgenics to identify minimal regions and response elements that mediate Ncx1 expression in the heart. We demonstrate that the proximal 184 bp of the Ncx1 H1 (184Ncx1) promoter is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for up-regulation in response to pressure overload. Mutational analysis revealed that both the -80 CArG and the -50 GATA elements were required for expression in isolated adult cardiomyocytes. Chromatin immunoprecipitation assays in adult cardiocytes demonstrate that SRF and GATA4 are associated with the proximal region of the endogenous Ncx1 promoter. Transgenic lines were established for the 1831Ncx1 promoter-luciferase containing mutations in the -80 CArG or -50 GATA element. No luciferase activity was detected during development, in the adult, or after pressure overload in any of the -80 CArG transgenic lines. The Ncx1 -50 GATA mutant promoter was sufficient for driving the normal spatiotemporal pattern of Ncx1 expression in development and for up-regulation in response to pressure overload but importantly, expression was no longer cardiac restricted. This work is the first in vivo study that demonstrates which cis elements are important for Ncx1 regulation.