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LNCaP GSEA analyses RNAseq data.

Purpose: Loss of cell-cycle control is a hallmark of cancer, which can be targeted with agents, including cyclin-dependent kinase-4/6 (CDK4/6) kinase inhibitors that impinge upon the G1–S cell-cycle checkpoint via maintaining activity of the retinoblastoma tumor suppressor (RB). This class of drugs is under clinical investigation for various solid tumor types and has recently been FDA-approved for treatment of breast cancer. However, development of therapeutic resistance is not uncommon.Experimental Design: In this study, palbociclib (a CDK4/6 inhibitor) resistance was established in models of early stage, RB-positive cancer.Results: This study demonstrates that acquired palbociclib resistance renders cancer cells broadly resistant to CDK4/6 inhibitors. Acquired resistance was associated with aggressive in vitro and in vivo phenotypes, including proliferation, migration, and invasion. Integration of RNA sequencing analysis and phosphoproteomics profiling revealed rewiring of the kinome, with a strong enrichment for enhanced MAPK signaling across all resistance models, which resulted in aggressive in vitro and in vivo phenotypes and prometastatic signaling. However, CDK4/6 inhibitor–resistant models were sensitized to MEK inhibitors, revealing reliance on active MAPK signaling to promote tumor cell growth and invasion.Conclusions: In sum, these studies identify MAPK reliance in acquired CDK4/6 inhibitor resistance that promotes aggressive disease, while nominating MEK inhibition as putative novel therapeutic strategy to treat or prevent CDK4/6 inhibitor resistance in cancer. Clin Cancer Res; 24(17); 4201–14. ©2018 AACR.

LAPC4 phosphoproteomic data

Cbioportal analyses

LAPC4 aggressive phenotypes

LAPC4 GSEA analyses RNAseq data

LAPC4 CDK4/6 inhibitor resistance

MAPK reliance in LAPC4

Background Aflatoxin B1 (AFB1) a mycotoxin produced by Aspergillus flavus and A. parasiticus is a potent carcinogen and causative agent of hepatocellular carcinoma (HCC). It is a food contaminant which presents a major risk to human health. AFB1 contamination poses a significant economic burden, as 25% of the world's food crops need to be destroyed annually. The mechanism of action (MOA) of aflatoxins remains to be fully elucidated. Recent findings suggest that AFB1 mediated endocrine disruption may occur in the population of regions with high contamination, even without evidence of direct dietary intake. Objective An integrative systems biology approach was undertaken to decipher the estrogenic component of the mechanism of action (MOA) of AFB1. Methods Molecular Docking and Molecular dynamics simulations were performed to examine the binding affinity of AFB1 and its metabolite aflatoxin Q1 (AFQ1) with the Estrogen Receptors (ERs). Differential gene expression (DGE), gene ontology (GO) and pathway analyses were carried out on hepatic transcriptomic data generated from in vivo AFB1 exposures. In parallel exposures to the synthetic estrogen ethinylestradiol (EE2) were examined for overlapping effects. Finally, protein–protein interaction (PPI) network analysis assessed the involvement of estrogen responsive targets (ERTs) associated with aflatoxin exposure. Results The free energies of binding affinity and estimated equilibrium dissociation constants (KD) demonstrated that AFB1 and AFQ1 can interact with the ERα and ERβ. DGE and GO analyses highlighted overlap in the responses between AFB1 and EE2 treatments with the activation of key processes involved in estrogenic signaling. PPI network analyses after AFBI exposure revealed a dynamic response to AFB1 treatments with the solid involvement of ERTs in regulatory networks. Conclusions This study revealed molecular interactions between aflatoxins (AFB1, AFQ1) and ERs in addition to overlap in differentially expressed genes and biological processes following AFB1 and EE2 exposures. The estrogenic components at the core of the PPI networks suggest that ER-mediated signaling pathways are a major component in the MOA of aflatoxins.

Background Autosomal dominant polycystic kidney disease is caused by genetic mutations in PKD1 or PKD2. Macrophages and their associated inflammatory cytokines promote cyst progression; however, transcription factors within macrophages that control cytokine production and cystic disease are unknown. Methods In these studies, we used conditional Pkd1 mice to test the hypothesis that macrophage-localized interferon regulatory factor-5 (IRF5), a transcription factor associated with production of cyst-promoting cytokines (TNFα, IL-6), is required for accelerated cyst progression in a unilateral nephrectomy (1K) model. Analyses of quantitative real-time PCR (qRT-PCR) and flow-cytometry data 3 weeks post nephrectomy, a time point before the onset of severe cystogenesis, indicate an accumulation of inflammatory infiltrating and resident macrophages in 1K Pkd1 mice compared with controls. qRT-PCR data from FACS cells at this time demonstrate that macrophages from 1K Pkd1 mice have increased expression of Irf5 compared with controls. To determine the importance of macrophage-localized Irf5 in cyst progression, we injected scrambled or IRF5 antisense oligonucleotide (ASO) in 1K Pkd1 mice and analyzed the effect on macrophage numbers, cytokine production, and renal cystogenesis 6 weeks post nephrectomy. Results Analyses of qRT-PCR and IRF5 ASO treatmentsignificantly reduced macrophage numbers, Irf5 expression in resident—but not infiltrating—macrophages, and the severity of cystic disease. In addition, IRF5 ASO treatment in 1K Pkd1 mice reduced Il6 expression in resident macrophages, which was correlated with reduced STAT3 phosphorylation and downstream p-STAT3 target gene expression. Conclusions These data suggest that Irf5 promotes inflammatory cytokine production in resident macrophages resulting in accelerated cystogenesis.

An emerging theory about racial differences in cancer risk and outcomes is that psychological and social stressors influence cellular stress responses, however, limited empirical data are available on racial differences in cellular stress responses among men who are at risk for adverse prostate cancer outcomes. In this study, we undertook a systems approach to examine molecular profiles and cellular stress responses in an important segment of African American (AA) and European American (EA) men: men undergoing prostate biopsy. We assessed the prostate transcriptome with a single biopsy core via high throughput RNA sequencing (RNA-Seq). Transcriptomic analyses uncovered impacted biological pathways including PI3K-Akt signaling pathway, Neuroactive ligand-receptor interaction pathway, and ECM-receptor interaction. Additionally, 187 genes mapping to the Gene Ontology (GO) terms RNA binding, structural constituent of ribosome, SRP-dependent co-translational protein targeting to membrane and the biological pathways, translation, L13a-mediated translational silencing of Ceruloplasmin expression were differentially expressed (DE) between EA and AA. This signature allowed separation of AA and EA patients, and AA patients with the most severe clinical characteristics. AA patients with elevated expression levels of this genomic signature presented with higher Gleason scores, a greater number of positive core biopsies, elevated dehydroepiandrosterone sulfate levels and serum vitamin D deficiency. Protein-protein interaction (PPI) network analysis revealed a high degree of connectivity between these 187 proteins.

The sensory system of animals detects a massive and unknown array of chemical cues that evoke a diversity of physiological and behavioural responses. One group of nitrogen-containing carbon ring chemicals—nucleobases—are thought to be involved in numerous behaviours yet have received little attention. We took a top-down approach to examine responses evoked by nucleobases at behavioural, tissue, and gene expression levels. Fish generally avoided nucleobases, and this behaviour, when observed, was driven by purines but not pyrimidines. At the tissue level, olfactory neuron generator potential responses tended to be concentration specific and robust at concentrations lower than amino acid detection ranges. In terms of gene expression, more than 2000 genes were significantly upregulated following nucleobase exposure, some of which were expected (e.g., genes involved in purine binding) and some of which were not (e.g., tubulin-related genes). Humanized RNA pathway analysis showed that we had exposed the animal to a nucleobase. Our data indicate that responses to nucleobase-containing compounds may be highly structure based and are evident from changes in behaviour to mRNA expression. Many of these responses were surprising, and all provide numerous routes for further research endeavour.

Purpose: Loss of cell-cycle control is a hallmark of cancer, which can be targeted with agents, including cyclin-dependent kinase-4/6 (CDK4/6) kinase inhibitors that impinge upon the G1–S cell-cycle checkpoint via maintaining activity of the retinoblastoma tumor suppressor (RB). This class of drugs is under clinical investigation for various solid tumor types and has recently been FDA-approved for treatment of breast cancer. However, development of therapeutic resistance is not uncommon. Experimental Design: In this study, palbociclib (a CDK4/6 inhibitor) resistance was established in models of early stage, RB-positive cancer. Results: This study demonstrates that acquired palbociclib resistance renders cancer cells broadly resistant to CDK4/6 inhibitors. Acquired resistance was associated with aggressive in vitro and in vivo phenotypes, including proliferation, migration, and invasion. Integration of RNA sequencing analysis and phosphoproteomics profiling revealed rewiring of the kinome, with a strong enrichment for enhanced MAPK signaling across all resistance models, which resulted in aggressive in vitro and in vivo phenotypes and prometastatic signaling. However, CDK4/6 inhibitor–resistant models were sensitized to MEK inhibitors, revealing reliance on active MAPK signaling to promote tumor cell growth and invasion. Conclusions: In sum, these studies identify MAPK reliance in acquired CDK4/6 inhibitor resistance that promotes aggressive disease, while nominating MEK inhibition as putative novel therapeutic strategy to treat or prevent CDK4/6 inhibitor resistance in cancer. Clin Cancer Res; 24(17); 4201–14. ©2018 AACR.

Adult fish produce new cells throughout their central nervous system during the course of their lives and maintain a tremendous capacity to repair damaged neural tissue. Much of the focus on understanding brain repair and regeneration in adult fish has been directed at regions of the brainstem and forebrain; however, the mesencephalon (midbrain) and diencephalon have received little attention. We sought to examine differential gene expression in the midbrain/diencephalon in response to injury in the adult fish using RNA-seq. Using the mummichog (Fundulus heteroclitus), we administered a mechanical lesion to the midbrain/diencephalon and examined differentially expressed genes (DEGs) at an acute recovery time of 1 h post-injury. Comparisons of whole transcriptomes derived from isolated RNA of intact and injured midbrain/diencephalic tissue identified 404 DEGs with the vast majority being upregulated. Using qPCR, we validated the upregulation of DEGs pim-2-like, syndecan-4-like, and cd83. Based on genes both familiar and novel regarding the adult brain response to injury, these data provide an extensive molecular profile giving insight into a range of cellular processes involved in the injury response of a brain regenerative-capable vertebrate.

The Deepwater Horizon (DWH) incident resulted in extensive oiling of the pelagic zone and shoreline habitats of many commercially important fish species. Exposure to the water-accommodated fraction (WAF) of oil from the spill causes developmental toxicity through cardiac defects in pelagic fish species. However, few studies have evaluated the effects of the oil on near-shore estuarine fish species such as red drum (Sciaenops ocellatus). Following exposure to a certified weathered slick oil (4.74 μg/L ∑PAH50) from the DWH event, significant sublethal impacts were observed ranging from impaired nervous system development [average 17 and 22% reductions in brain and eye area at 48 h postfertilization (hpf), respectively] to abnormal cardiac morphology (100% incidence at 24, 48, and 72 hpf) in red drum larvae. Consistent with the phenotypic responses, significantly differentially expressed transcripts, enriched gene ontology, and altered functions and canonical pathways predicted adverse outcomes in nervous an...

We identified unique molecular mechanisms of resistance to CDK4/6 inhibitors, an area of intense biomedical investigation., The cyclin-dependent kinase 4/6 (CDK4/6) kinase is dysregulated in melanoma, highlighting it as a potential therapeutic target. CDK4/6 inhibitors are being evaluated in trials for melanoma and additional cancers. While beneficial, resistance to therapy is a concern, and the molecular mechanisms of such resistance remain undefined. We demonstrate that reactivation of mammalian target of rapamycin 1 (mTORC1) signaling through increased expression of the amino acid transporter, solute carrier family 36 member 1 (SLC36A1), drives resistance to CDK4/6 inhibitors. Increased expression of SLC36A1 reflects two distinct mechanisms: (i) Rb loss, which drives SLC36A1 via reduced suppression of E2f; (ii) fragile X mental retardation syndrome–associated protein 1 overexpression, which promotes SLC36A1 translation and subsequently mTORC1. Last, we demonstrate that a combination of a CDK4/6 inhibitor with an mTORC1 inhibitor has increased therapeutic efficacy in vivo, providing an important avenue for improved therapeutic intervention in aggressive melanoma.

Mitral valve prolapse (MVP) affects 1 in 40 people and is the most common indication for mitral valve surgery. MVP can cause arrhythmias, heart failure, and sudden cardiac death, and to date, the causes of this disease are poorly understood. We now demonstrate that defects in primary cilia genes and their regulated pathways can cause MVP in familial and sporadic nonsyndromic MVP cases. Our expression studies and genetic ablation experiments confirmed a role for primary cilia in regulating ECM deposition during cardiac development. Loss of primary cilia during development resulted in progressive myxomatous degeneration and profound mitral valve pathology in the adult setting. Analysis of a large family with inherited, autosomal dominant nonsyndromic MVP identified a deleterious missense mutation in a cilia gene, DZIP1 A mouse model harboring this variant confirmed the pathogenicity of this mutation and revealed impaired ciliogenesis during development, which progressed to adult myxomatous valve disease and functional MVP. Relevance of primary cilia in common forms of MVP was tested using pathway enrichment in a large population of patients with MVP and controls from previously generated genome-wide association studies (GWAS), which confirmed the involvement of primary cilia genes in MVP. Together, our studies establish a developmental basis for MVP through altered cilia-dependent regulation of ECM and suggest that defects in primary cilia genes can be causative to disease phenotype in some patients with MVP.

RNAseq is a powerful technique enabling global profiles of transcriptomes in healthy and diseased states. In this chapter we review pipelines to analyze the data generated by sequencing RNA, from raw data to a system level analysis. We first give an overview of workflow to generate mapped reads from FASTQ files, including quality control of FASTQ, filtering and trimming of reads, and alignment of reads to a genome. Then, we compare and contrast three popular options to determine differentially expressed (DE) transcripts (The Tuxedo Pipeline, DESeq2, and Limma/voom). Finally, we examine four tool sets to extrapolate biological meaning from the list of DE genes (Genecards, The Human Protein Atlas, GSEA, and ToppGene). We emphasize the need to ask a concise scientific question and to clearly under stand the strengths and limitations of the methods.

RNA sequencing (RNA-seq) is revolutionizing the study of cancer by providing a highly sensitive and robust tool to interrogate the transcriptome. It leverages the power of deep sequencing technology and provides global and multidimensional views of transcriptional landscapes in healthy and tumor tissues. Such information is contributing innovative insights to our understanding of the genetic basis of cancer and the progression of the disease. RNA-seq is a superior technology to DNA microarrays in that it provides digital rather than analog information on transcripts and their isoforms. The front end (sequencing library preparation and validation) is technically complex and time intensive. The primary objective in preparing a sequencing library is to eliminate or minimize bias, so that the library is reflective of the input RNA sample in terms of both sequence content and transcript abundance. This chapter describes the RNA-seq approach, and reviews methods and good practices for library preparation and sequencing.

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 17β-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).

RNA sequencing (RNA-seq) is revolutionizing the study of cancer by providing a highly sensitive and robust tool to interrogate the transcriptome. It leverages the power of deep sequencing technology and provides global and multidimensional views of transcriptional landscapes in healthy and tumor tissues. Such information is contributing innovative insights to our understanding of the genetic basis of cancer and the progression of the disease. RNA-seq is a superior technology to DNA microarrays in that it provides digital rather than analog information on transcripts and their isoforms. The front end (sequencing library preparation and validation) is technically complex and time intensive. The primary objective in preparing a sequencing library is to eliminate or minimize bias, so that the library is reflective of the input RNA sample in terms of both sequence content and transcript abundance. This chapter describes the RNA-seq approach, and reviews methods and good practices for library preparation and sequencing.

RNAseq is a powerful technique enabling global profiles of transcriptomes in healthy and diseased states. In this chapter we review pipelines to analyze the data generated by sequencing RNA, from raw data to a system level analysis. We first give an overview of workflow to generate mapped reads from FASTQ files, including quality control of FASTQ, filtering and trimming of reads, and alignment of reads to a genome. Then, we compare and contrast three popular options to determine differentially expressed (DE) transcripts (The Tuxedo Pipeline, DESeq2, and Limma/voom). Finally, we examine four tool sets to extrapolate biological meaning from the list of DE genes (Genecards, The Human Protein Atlas, GSEA, and ToppGene). We emphasize the need to ask a concise scientific question and to clearly under stand the strengths and limitations of the methods.

The etiology and reasons underlying the ethnic disparities in systemic sclerosis (SSc) remain unknown. African Americans are disproportionally affected by SSc and yet are underrepresented in research. The aim of this study was to comprehensively investigate the association of DNA methylation levels with SSc in dermal fibroblasts from patients of African ancestry. Reduced representation bisulfite sequencing (RRBS) was performed on primary dermal fibroblasts from 15 SSc patients and 15 controls of African ancestry, and over 3.8 million CpG sites were tested for differential methylation patterns between cases and controls. The dermal fibroblasts from African American patients exhibited widespread reduced DNA methylation. Differentially methylated CpG sites were most enriched in introns and intergenic regions while depleted in 5&prime, UTR, promoters, and CpG islands. Seventeen genes and eleven promoters showed significant differential methylation, mostly in non-coding RNA genes and pseudogenes. Gene set enrichment analysis (GSEA) and gene ontology (GO) analyses revealed an enrichment of pathways related to interferon signaling and mesenchymal differentiation. The hypomethylation of DLX5 and TMEM140 was accompanied by these genes&rsquo, overexpression in patients but underexpression for lncRNA MGC12916. These data show that differential methylation occurs in dermal fibroblasts from African American patients with SSc and identifies novel coding and non-coding genes.

The Deepwater Horizon (DWH) oil spill contaminated the spawning habitats for numerous commercially and ecologically important fishes. Exposure to the water accommodated fraction (WAF) of oil from the spill has been shown to cause cardiac toxicity during early developmental stages across fishes. To better understand the molecular events and explore new pathways responsible for toxicity, RNA sequencing was performed in conjunction with physiological and morphological assessments to analyze the time-course (24, 48, and 96 h post fertilization (hpf)) of transcriptional and developmental responses in embryos/larvae of mahi-mahi exposed to WAF of weathered (slick) and source DWH oils. Slick oil exposure induced more pronounced changes in gene expression over time than source oil exposure. Predominant transcriptomic responses included alteration of EIF2 signaling, steroid biosynthesis, ribosome biogenesis and activation of the cytochrome P450 pathway. At 96 hpf, slick oil exposure resulted in significant perturbations in eye development and peripheral nervous system, suggesting novel targets in addition to the heart may be involved in the developmental toxicity of DHW oil. Comparisons of changes of cardiac genes with phenotypic responses were consistent with reduced heart rate and increased pericardial edema in larvae exposed to slick oil but not source oil.

Aim: African–Americans (AA) have increased prostate cancer risk and a greater mortality rate than European–Americans (EA). AA exhibit a high prevalence of vitamin D deficiency. We examined the global prostate transcriptome in AA and EA, and the effect of vitamin D3 supplementation. Patients & methods: Twenty-seven male subjects (ten AA and 17 EA), slated to undergo prostatectomy were enrolled in the study. Fourteen subjects received vitamin D3 (4000 IU daily) and 13 subjects received placebo for 2 months prior to surgery. Results: AA show higher expression of genes associated with immune response and inflammation. Conclusion: Systems level analyses support the concept that Inflammatory processes may contribute to disease progression in AA. These transcripts can be modulated by a short course of vitamin D3 supplementation.

The monitoring of marine species as sentinels for ecosystem health has long been a valuable tool worldwide, providing insight into how both anthropogenic pollution and naturally occurring phenomena (i.e., harmful algal blooms) may lead to human and animal dietary concerns. The marine environments contain many contaminants of anthropogenic origin that have sufficient similarities to steroid and thyroid hormones, to potentially disrupt normal endocrine physiology in humans, fish, and other animals. An appropriate understanding of the effects of these endocrine disrupting chemicals (EDCs) on forage fish (e.g., sardine, anchovy, mackerel) can lead to significant insight into how these contaminants may affect local ecosystems in addition to their potential impacts on human health. With advancements in molecular tools (e.g., high-throughput sequencing, HTS), a genomics approach offers a robust toolkit to discover putative genetic biomarkers in fish exposed to these chemicals. However, the lack of available sequence information for non-model species has limited the development of these genomic toolkits. Using HTS and de novo assembly technology, the present study aimed to establish, for the first time for Sardinops sagax (Pacific sardine), Scomber japonicas (Pacific chub mackerel) and Pleuronichthys verticalis (hornyhead turbot), a de novo global transcriptome database of the liver, the primary organ involved in detoxification. The assembled transcriptomes provide a foundation for further downstream validation, comparative genomic analysis and biomarker development for future applications in ecotoxicogenomic studies, as well as environmental evaluation (e.g., climate change) and public health safety (e.g., dietary screening).

Environmental factors are the largest contributors to cardiovascular disease. Here we show that cardiac organoids that incorporate an oxygen-diffusion gradient and that are stimulated with the neurotransmitter noradrenaline model the structure of the human heart after myocardial infarction (by mimicking the infarcted, border and remote zones), and recapitulate hallmarks of myocardial infarction (in particular, pathological metabolic shifts, fibrosis and calcium handling) at the transcriptomic, structural and functional levels. We also show that the organoids can model hypoxia-enhanced doxorubicin cardiotoxicity. Human organoids that model diseases with non-genetic pathological factors could help with drug screening and development.

Endocrine disrupting chemicals (EDCs) are substances which disrupt normal functioning of the endocrine system by interfering with hormone regulated physiological pathways. Aquatic environments provide the ultimate reservoir for many EDCs as they enter rivers and the ocean via effluent discharges and accumulate in sediments. One EDC widely dispersed in municipal wastewater effluent discharges is 17α-ethynylestradiol (EE2), which is one of the most widely prescribed medicines. EE2 is a bio-active estrogen employed in the majority of oral contraceptive pill formulations. As evidence of the health risks posed by EDCs mount, there is an urgent need to improve diagnostic tools for monitoring the effects of pollutants. As the cost of high throughput sequencing (HTS) diminishes, transcriptional profiling of an organism in response to EDC perturbation presents a cost-effective way of screening a wide range of endocrine responses. Coastal pelagic filter feeding fish species analyzed using HTS provide an excellent tool for EDC risk assessment in the marine environment. Unfortunately, there are limited genome sequence data and annotation for many of these species including Pacific sardine (Sardinops sagax) and chub mackerel (Scomber japonicus), which limits the utility of molecular tools such as HTS to interrogate the effects of endocrine disruption. In this study, we carried out RNA sequencing (RNAseq) of liver RNA harvested from wild sardine and mackerel exposed for 5 h under laboratory conditions to a concentration of 12.5 pM EE2 in the tank water. We developed an analytical framework for transcriptomic analyses of species with limited genomic information. EE2 exposure altered expression patterns of key genes involved in important metabolic and physiological processes. The systems approach presented here provides a powerful tool for obtaining a comprehensive picture of endocrine disruption in aquatic organisms. The development of a framework for RNAseq analysis in sentinel species that lack genomic sequence allowed us to determine that environmental levels of EE2 disrupted basic biological processes and pathways in both male sardine and mackerel, leading to molecular signatures of metabolic, hormonal and immune dysfunction, as well as carcinogenesis in exposed fish and provided novel biomarkers for diagnostic use in monitoring ocean environmental health.

The RNA binding protein CELF1 (also known as CUGBP1) is emerging as a critical regulator of cancer cell proliferation and apoptosis. Here, to provide a global prospective of CELF1 regulation of oral squamous cell carcinoma, we performed RNA-sequencing in oral cancer cells and CELF1 overexpression analysis in non-malignant human oral keratinocytes. Our approaches identified 1283 mRNAs differentially regulated as a function of CELF1 expression and more importantly CELF1 promoted alternative splicing of several target pre-mRNAs, which are known to be involved in various cancer biological processes. Overexpression of CELF1 in non-malignant human oral keratinocytes protected cells against oxidative damage and altered gene expression patterns. Finally, we provide evidence that reduction of CELF1 protein using a xenograft tumorigenesis mouse model decreased tumor growth. Altogether, these data provided a comprehensive view of the CELF1 mRNA regulatory network in oral cancer and suggests that CELF1 and/or its target mRNAs are viable candidates for therapeutic intervention.

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.

The cancer-associated RNA-binding protein La is posttranslationally modified by phosphorylation and sumoylation. Sumoylation of La regulates not only the trafficking of La in neuronal axons but also its association with specific mRNAs. Depletion of La in various types of cancer cell lines impairs cell proliferation; however, the molecular mechanism whereby La supports cell proliferation is not clearly understood. In this study, we address the question of whether sumoylation of La contributes to cell proliferation of HEK293 cells. We show that HEK293 cells stably expressing green fluorescent protein (GFP)-tagged wild-type La (GFP-LaWT) grow faster than cells expressing a sumoylation-deficient mutant La (GFP-LaSD), suggesting a proproliferative function of La in HEK293 cells. Further, we found that STAT3 protein levels were reduced in GFP-LaSD cells due to an increase in STAT3 ubiquitination and that overexpression of STAT3 partially restored cell proliferation. Finally, we present RNA sequencing data from RNA immunoprecipitations (RIPs) and report that mRNAs associated with the cell cycle and ubiquitination are preferentially bound by GFP-LaWT and are less enriched in GFP-LaSD RIPs. Taken together, results of our study support a novel mechanism whereby sumoylation of La promotes cell proliferation by averting ubiquitination-mediated degradation of the STAT3 protein.

The impacts of Deepwater Horizon (DWH) oil on morphology and function during embryonic development have been documented for a number of fish species, including the economically and ecologically important pelagic species, mahi-mahi (Coryphaena hippurus). However, further investigations on molecular events and pathways responsible for developmental toxicity have been largely restricted due to the limited molecular data available for this species. We sought to establish the de novo transcriptomic database from the embryos and larvae of mahi-mahi exposed to water accommodated fractions (HEWAFs) of two DWH oil types (weathered and source oil), in an effort to advance our understanding of the molecular aspects involved during specific toxicity responses. By high throughput sequencing (HTS), we obtained the first de novo transcriptome of mahi-mahi, with 60,842 assembled transcripts and 30,518 BLAST hits. Among them, 2,345 genes were significantly regulated in 96hpf larvae after exposure to weathered oil. With comparative analysis to a reference-transcriptome-guided approach on gene ontology and tox-pathways, we confirmed the novel approach effective for exploring tox-pathways in non-model species, and also identified a list of co-expressed genes as potential biomarkers which will provide information for the construction of an Adverse Outcome Pathway which could be useful in Ecological Risk Assessments.

Mahi-mahi (Coryphaena hippurus) is a commercially and ecologically important species of fish occurring in tropical and temperate waters worldwide. Understanding early life events is crucial for predicting effects of environmental stress, which is largely restricted by a lack of genetic resources regarding expression of early developmental genes and regulation of pathways. The need for anchoring developmental stages to transcriptional activities is highlighted by increasing evidence on the impacts of recurrent worldwide oil spills in this sensitive species during early development. By means of high throughput sequencing, we characterized the developmental transcriptome of mahi-mahi at three critical developmental stages, from pharyngula embryonic stage (24 hpf) to 48 hpf yolk-sac larva (transition 1), and to 96 hpf free-swimming larva (transition 2). With comparative analysis by multiple bioinfor-matic tools, a larger number of significantly altered genes and more diverse gene ontology terms were observed during transition 2 than transition 1. Cellular and tissue development terms were more significantly enriched in transition 1, while metabolism related terms were more enriched in transition 2, indicating a switch progressing from general embryonic development to metabolism during the two transitions. Special focus was given on the most significant common canonical pathways (e.g. calcium signaling, glutamate receptor signaling, cAMP response element-binding protein signaling, cardiac β-adrenergic signaling, etc.) and expression of developmental genes (e.g. collagens, myosin, notch, glutamate metabotropic receptor etc.), which were associated with morphological changes of nervous, muscular, and cardiovascular system. These data will provide an important basis for understanding embryonic development and identifying molecular mechanisms of abnormal development in fish species.

The emergence and mass utilization of high-throughput (HT) technologies, including sequencing technologies (genomics) and mass spectrometry (proteomics, metabolomics, lipids), has allowed geneticists, biologists, and biostatisticians to bridge the gap between genotype and phenotype on a massive scale. These new technologies have brought rapid advances in our understanding of cell biology, evolutionary history, microbial environments, and are increasingly providing new insights and applications towards clinical care and personalized medicine. Areas covered: The very success of this industry also translates into daunting big data challenges for researchers and institutions that extend beyond the traditional academic focus of algorithms and tools. The main obstacles revolve around analysis provenance, data management of massive datasets, ease of use of software, interpretability and reproducibility of results. Expert commentary: The authors review the challenges associated with implementing bioinformatics best practices in a large-scale setting, and highlight the opportunity for establishing bioinformatics pipelines that incorporate data tracking and auditing, enabling greater consistency and reproducibility for basic research, translational or clinical settings.

Cyclin dependent kinase-4/6 (CDK4/6) kinase inhibitors have shown clinical benefit in treatment of solid tumor types, including breast cancer. However, resistance is common, and the underpinning mechanisms of action are not well understood. Given the dependence of CDK4/6 inhibitors on retinoblastoma tumor suppressor (RB) function for activity, this class of agents may be particularly effective in tumor types for which RB loss is infrequent or occurs late in tumor progression. Here, models of acquired palbociclib resistance were generated in early-stage, RB-positive cancers, wherein it was shown that acquired palbociclib resistance resulted in cross-resistance to other CDK4/6 inhibitors under clinical testing. Furthermore, cells showing acquired resistance exhibited aggressive in vitro and in vivo phenotypes without genetic loss of RB or RB pathway members, including enhanced proliferative capacity, migratory potential, and characteristics of epithelial-to-mesenchymal transition. Further analyses through integration of RNA sequencing and phospho-proteomics identified activation of the MAPK signaling pathway as a mediator of CDK4/6 inhibitor resistance, capable of bypassing CDK4/6 activity. However, this altered kinase dependence resulted in sensitization to MEK inhibitors, suggestive of new clinical opportunities in CDK4/6-resistant tumors. In sum, the studies herein not only identify activation of the MAPK pathway as capable of bypassing the CDK4/6 requirement and promoting aggressive tumor characteristics, but also nominate MEK inhibitors as potential mechanisms to treat or prevent CDK4/6 inhibitor resistance. Citation Format: Renee de Leeuw, Christopher M. McNair, Matthew J. Schiewer, Neermala P. Neupane, Michael A. Augello, Zhen Li, Larry Cheng, Akihiro Yoshida, J. Alan Diehl, E Starr Hazard, Sean M. McCourtney, Gary Hardiman, Maha H. Hussain, Justin M. Drake, Wm. Kevin Kelly, Karen E. Knudsen. Bypass kinase pathways lead to acquired CDK4/6 inhibitor resistance in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A043.

Karenia brevis (C. C. Davis) G. Hansen et Moestrup is a dinoflagellate responsible for red tides in the Gulf of Mexico. The signaling pathways regulating its cell cycle are of interest because they are the key to the formation of toxic blooms that cause mass marine animal die-offs and human illness. Karenia brevis displays phased cell division, in which cells enter S phase at precise times relative to the onset of light. Here, we demonstrate that a circadian rhythm underlies this behavior and that light quality affects the rate of cell-cycle progression: in blue light, K. brevis entered the S phase early relative to its behavior in white light of similar intensity, whereas in red light, K. brevis was not affected. A data base of 25,000 K. brevis expressed sequence tags (ESTs) revealed several sequences with similarity to cryptochrome blue-light receptors, but none related to known red-light receptors. We characterized the K. brevis cryptochrome (Kb CRY) and modeled its three-dimensional protein structure. Phylogenetic analysis of the photolyase/CRY gene family showed that Kb CRY is a member of the cryptochrome DASH (CRY DASH) clade. Western blotting with an antibody designed to bind a conserved peptide within Kb CRY identified a single band at ∼55 kDa. Immunolocalization showed that Kb CRY, like CRY DASH in Arabidopsis, is localized to the chloroplast. This is the first blue-light receptor to be characterized in a dinoflagellate. As the Kb CRY appears to be the only blue-light receptor expressed, it is a likely candidate for circadian entrainment of the cell cycle.

Lung cancer is the leading cause of cancer-related deaths worldwide. Biomarker discovery has become increasingly important for the effective diagnosis, prognosis and treatment of the disease. The analysis of differential gene expression data has been the primary method for biomarker discovery. Our research demonstrates that alternative splicing events (ASE) can be another source of data for predictive model creation by identifying putative biomarkers that are complementary to those found from traditional gene expression. RNASeq data from 21 patients diagnosed with lung adenocarcinoma, a non-small cell lung carcinoma (11 of which relapsed) were analyzed. After quantifying splice variants and gene expression with a bioinformatics pipeline, we were able to create predictive models, using orthogonal projections to latent structures discriminate analysis (OPLS-DA) that recognize two clinical phenotypes (disease free and relapse); thus distinguishing between more indolent and aggressive disease. Hierarchical clustering of samples pre and post predictive model feature selection showed that clustering based on ASE was more indicative of the relapse phenotype. A novel hybrid multiple objective genetic algorithm combining alternative splicing events with gene expression was used for discriminate feature selection. A post-processing examination of the putative biomarkers found by the genetic algorithm and ranked correlation tests demonstrate that the analysis of alternative splicing events provide complementary and non-redundant predictive power by identifying biologically relevant patterns that do not result in differential gene expression.

The visual pigment from the ultraviolet (UV) cone photoreceptor of the tiger salamander has been cloned, expressed, and characterized. The cDNA contains a full-length open reading frame encoding 347 amino acids. The phylogenetic analysis indicates that the highest sequence homology is to the visual pigments in the S group. The UV opsin was tagged at the carboxy-terminus with the sequence for the 1D4 epitope. This fusion opsin was expressed in COS-1 cells, regenerated with 11-cis retinal (A1) and immuno-purified, yielding a pigment with an absorbance maximum (λmax) of 356 nm which is blue shifted from the absorption of retinal itself. The transducin activation assay demonstrated that this pigment is able to activate rod transducin in a light-dependent manner. Regeneration with 11-cis 3,4-dehydroretinal (A2) yielded a pigment with a λmax of 360 nm, only 4 nm red shifted from that of the A1 pigment, while bovine rhodopsin generated with A2 showed a 16-nm red shift from the corresponding A1 pigment. These results demonstrate that the trend for a shorter wavelength pigment to have a smaller shift of λmax between the A1 and A2 pigments also fits UV pigments. We hypothesize that the small red shift with A2 could be due to a twist in the chromophore that essentially isolates the ring double bond(s) from conjugation with the rest of the polyene chain.

The structure of bacteriorhodopsin (bR) has been probed by a large number of experimental methods. In earlier work distance constraints measured from the 1BRD Brookhaven structure (1, 2) were used to guide site-directed mutagenesis/affinity labeling experiments (3-5). In the present study we report on the use of limited molecular dynamics (MD) investigations of the same bR/affinity label system. We show here that the chiral center introduced when 4-bromo-all-trans retinal is synthesized produces variable impact on potential crosslinking. Our MD analysis suggests the following ranking of binding site mutants in order of reactivity: R118C > S118C >> S121C > R141C >> S141C >>> R121C, R138C, S138C. Chirality appears to have limited effect for the M118C mutants but shows more dramatic impact for the T121C and S141C mutants. These results are in excellent agreement with the experimental observations and offer encouragement that MD can be a useful component of experimental design with considerable predictive power.

— Halobacterium halobium, grown in a defined medium where tyrosine had been largely replaced with m-fluorotyrosine, biosynthetically produced purple membrane. Analysis of this membrane by high pressure liquid chromatography of phenylthiocarbamyl derivatized amino acids of membrane acid hydrolysates revealed that up to 50% of the tyrosine was present as the m-fluorotyrosine form. Yields of the purple membrane decreased as the level of incorporation increased. The experimental purple membrane showed a single 19F NMR resonance at -61.983 ppm (relative to trifluoroacetic acid). The bacteriorhodopsin (bR) in the purple membrane was normal as assayed by gel electrophor-esis, isoelectric focusing, circular dichroic spectra, and UV-visible spectra. However, the fluorinated tyrosine bacteriorhodopsins at near neutral pH exhibited slightly slower rates of proton uptake and a slower M-state decay with biphasic kinetics reminiscent of alkaline solutions of bR (pH > 9). These results imply that the tyrosines in bacteriorhodopsin may play a role in the photoactivated proton translocation process of this pigment.

Retinol decomposes rapidly into a number of products, including its aldehyde form, retinal, when introduced into buffer in phospholipid vesicles or ethanol. Interphotoreceptor retinoid-binding protein at low concentrations is found to protect retinol from isomerization and oxidation. The addition of alpha-tocopherol to either liposomes or an ethanolic-buffer solution also prevents decomposition. Neither of these agents interferes with the successful regeneration of pigment with 9-cis retinal in rod outer segment preparations or the restoration of sensitivity by retinoids in isolated rod photoreceptors.

Retinas can be protected from subsequent severe ischemic injury by ischemic preconditioning. Ischemic preconditioning is dependent on gene expression and protein synthesis; however, it is not clear which genes are important in this process. In this study, we have identified and characterized the rat homolog of yeast Apg3p/Aut1p, an important autophagy protein encoded by the autophagy 3-like (APG3L) gene. We have also further characterized the homologous human APG3L gene.A fragment of the rat Apg3 cDNA was identified by mRNA differential display from hypoxia-treated E1A-NR3, an immortalized cell line derived from rat retinal cells that manifests phenotypes of retinal neurons. The full length of rat Apg3 (rApg3) cDNA sequence (about 1.4 kb) encoding 341 amino acids was cloned from a rat retinal cDNA library and characterized using Southern and northern blot analysis, and a global GenBank search. Protein expression was determined by western blotting, and immunohistochemistry. Ischemic preconditioning was achieved by ligation of the retinal arteries of the right eye for 5 min followed by 5 h reperfusion. The prolonged retinal ischemia was induced by ligation of the retinal arteries for 45 min followed by 5 h reperfusion. The full-length homologous human APG3L gene was cloned and sequenced from a human genomic DNA library.The combination of genomic Southern blot analysis and a global GenBank search indicated that rat APG3L is a single copy gene. Rat Apg3 mRNA is expressed in the retina at a high level but is also detected in other tissues. In the process of comparing the rat and human APG3L genes we showed that the organization of the human APG3L gene includes a unique transcriptional start site, a coding region with 12 translated exons and 11 introns and is located on human chromosome 3q13.1. Subcellular localization studies showed that recombinant rat autophagocytosis protein (Apg3p) is a cytosolic protein. Rat Apg3 mRNA level was upregulated by ischemic preconditioning but downregulated by prolonged ischemia.Our results suggest that the upregulation of rApg3 is a specific response to ischemic preconditioning rather than to retina ischemia, and autophagy may contribute to the neuroprotective effect of ischemic preconditioning in the retina.

The purpose of this study was to clone and characterize the green rod pigment in Xenopus laevis.The cDNA for the Xenopus "green rod" pigment was cloned and sequenced from Xenopus retina mRNA by reverse transcription polymerase chain reaction and the 5' end cloned by rapid amplification of the cDNA ends. The cellular localization of the Xenopus opsin was determined by immunolabeling of flat-mounted retinas using a specific antibody against this opsin. Spectral properties of the expressed protein were determined by absorption spectroscopy using recombinant pigment.A novel Xenopus opsin cDNA containing a full-length coding region has been cloned and sequenced. The deduced amino acid sequence predicts a protein of 362 amino acids, forming 7 hydrophobic helices. Sequence analysis indicates that it belongs firmly to the SWS2 class of visual pigments and has 89%, 80%, and 75% amino acid sequence identity with bullfrog, tiger salamander, and newt SWS2 pigments, respectively. Staining of Xenopus retina with a Xenopus SWS2 opsin-specific polyclonal antibody demonstrated that the SWS2 pigment is expressed in green rods. After expression in COS cells, reconstitution with 11-cis retinal, and purification, the SWS2 pigment exhibits an absolute absorption maximum of 434 nm Thus, the name "SWS2, P434" was assigned for this opsin. The pigment decays rapidly in hydroxylamine in the dark, unlike the red rod pigment, rhodopsin.A novel green rod opsin cDNA has been cloned and sequenced from the retina of adult Xenopus laevis, which encodes a protein belonging to the SWS2 group of opsins. The expressed opsin possesses cone-opsin-like properties although it was identified only in the Xenopus green rod cells.

Flash photolysis kinetics of carbon monoxide hemoglobin show a decrease in the fraction of ligand recombination occurring as geminate when the hemoglobin has fewer ligands bound. Fully saturated samples, normally referred to as R state, show approximately 50% geminate phase, while samples at low saturation (T state) show less than 3%. The latter result was obtained by photolysis of samples with a short delay after stopped flow of solutions of deoxy hemoglobin (Hb) and ligand. The decrease in the fraction of geminate phase was also observed using a double flash technique. The transient mixture of R and T states generated by flash photolysis of Hb-CO was probed with a weaker time-delayed photolysis pulse. The kinetics of both the geminate and bimolecular phases following the second pulse were measured. The fraction geminate signal was least at delays where the maximum proportion of liganded T state tetramer is expected. The biphasic bimolecular process is also an indicator of the allosteric state of Hb. The populations of R and T may be determined from the overall ligand recombination kinetics; however, the analysis is model-dependent. The fraction geminate reaction may provide a rapid measure of the amount of liganded hemes in the R and T states.