We generated a blood-derived transcriptional signature that discriminates patients with lung cancer from non-affected smokers. When applied to blood samples from one of the largest prospective population-based cancer studies (the European Prospective Investigation into Cancer and Nutrition), this signature accurately predicted the occurrence of lung cancer in smokers within two years before the onset of clinical symptoms. Such a blood test could be used as a screening tool to enable early diagnosis of lung cancer at a curable stage.
Blood-based gene expression signatures in non-small cell lung cancer.
Specimen part
View SamplesARC (NSC 188491, SMA-491), 4-amino-6-hydrazino-7-beta-d-ribofuranosyl-7H-pyrrolo-(2,3-d)-pyrimidine-5-carboxamide, is a nucleoside analog with profound in vitro anti-cancer activity. First identified in a high-throughput screen for inhibitors of p21 mRNA expression, subsequent experiments showed that ARC also repressed expression of hdm2 and survivin, leading to its classification as a global inhibitor of transcription 1. The following Hu U133 plus 2.0 arrays represent single time point (24 hour) gene expression analysis of transcripts altered by ARC treatment. Arrays for the other compounds (sangivamycin and doxorubicin) are included as comparators.
ARC (NSC 188491) has identical activity to Sangivamycin (NSC 65346) including inhibition of both P-TEFb and PKC.
No sample metadata fields
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Translational regulation of specific mRNAs controls feedback inhibition and survival during macrophage activation.
Specimen part, Time
View SamplesWhen macrophages encounter pathogens, they transiently induce an orchestrated cascade of pro- and anti-inflammatory genes. We systematically analyzed the contribution of translational regulation to the early phase of macrophage activation. While the expression of most cytokines is regulated by changes in mRNA levels, de-repression of translation was found to permit expression of many feedback inhibitors of the inflammatory response. This includes NF-kB inhibitors (IkBd, IkBz, Nr4a1, Ier3), a p38 MAPK antagonist (Dusp1) and post-transcriptional suppressors of cytokine expression (TTP and Zc3h12a). Ier3 is tightly co-regulated with TNF at the level of mRNA abundance and translation. Macrophages lacking Ier3 show reduced survival upon activation, indicating that induction of Ier3 is required to protect macrophages from lipopolysaccharide-induced cell death. Our analysis reveals an important role of translational regulation in the resolution of inflammation and macrophage survival.
Translational regulation of specific mRNAs controls feedback inhibition and survival during macrophage activation.
Specimen part
View SamplesThe expression of adipogenic genes is decreased in obesity and diabetes mellitus
The expression of adipogenic genes is decreased in obesity and diabetes mellitus.
No sample metadata fields
View SamplesWhen macrophages encounter pathogens, they transiently induce an orchestrated cascade of pro- and anti-inflammatory genes. To obtain a precise picture of transcriptome-wide mRNA expression patterns, we performed RNA-Seq of total RNA at a high temporal resolution during the first two hours of macrophage activation. We systematically analyzed the contribution of translational regulation to the early phase of macrophage activation. While the expression of most cytokines is pre-dominanatly regulated by changes in mRNA levels, de-repression of translation was found to permit expression of many feedback inhibitors of the inflammatory response. Overall design: Expression profiles of LPS-treated Raw264.7 cells (0, 15, 30, 45, 60, 75, 90 and 120 min after stimulation) were generated by deep sequencing using Illumina HiSeq 2000.
Translational regulation of specific mRNAs controls feedback inhibition and survival during macrophage activation.
No sample metadata fields
View SamplesChronic non-healing venous leg ulcers (VLUs) are a widespread debilitating disease with high morbidity and associated costs, as approximately $15 billion annually are spent on the care of VLUs. Despite their socioeconomic burden, there is a paucity of novel treatments targeted towards healing VLUs, which can be attributed to both lack of pathophysiologic insight into VLU development as well as lack of knowledge regarding biologic actions of VLU-targeted therapies. Currently, the bioengineered bilayered living cellular construct (BLCC) skin substitute is the only FDA-approved biologic treatment for healing VLUs. To elucidate the mechanisms through which the BLCC promotes healing of chronic VLUs, we conducted a clinical trial (NCT01327937) in which patients with non-healing VLUs were treated with either standard care (compression therapy) or with BLCC together with standard care. Tissue was collected from the VLU edge before and 1 week after treatment, and samples underwent comprehensive microarray, mRNA and protein analyses. Ulcers treated with BLCC skin substitute displayed three distinct patterns suggesting the mechanisms by which BLCC shifted a non-healing into a healing tissue response: it modulated inflammatory and growth factor signaling; it activated keratinocytes; and it attenuated Wnt/-catenin signaling. In these ways, BLCC application orchestrated a shift of the chronic non-healing ulcer microenvironment into a distinctive healing milieu resembling that of an acute, healing wound. Our findings also provide first patient-derived in vivo evidence of specific biologic processes that can be targeted in the design of therapies to promote healing of chronic VLUs.
A bioengineered living cell construct activates an acute wound healing response in venous leg ulcers.
Specimen part, Disease stage, Time
View SamplesAcetyltransferases and histone deacetylases regulate gene expression at the level of chromatin, mainly by affecting transcription. In this study, we report that hyperacetylation induced by inhibition of histone deacetylases (HDACs) causes massive degradation of mRNA. The effect is promoter-independent and affects poly-A mRNA globally. HDAC inhibition leads to the removal of poly-A tails from mRNAs through activation of the deadenylase CAF1a, which we find to be acetylated together with its activator BTG2 by the histone acetyl transferases (HATs) p300 and CBP. By mutation of critical lysine residues, we provide evidence that acetylation of CAF1a and BTG2 induces enhanced poly-A mRNA degradation. Our study reveals a fundamental mechanism by which cells coordinate epigenetic and transcriptional control of gene expression with posttranscriptional control of poly-A mRNA stability. In this experiment, HeLa cells were exposed to the HDAC inhibitor trichostatin A (TSA) for 16 hours, followed by treatment with actinomycin D. Total RNA was isolated after 0, 2, 4 and 6 hours, and analysed by RNA sequencing. The half-lives of 7431 RNAs were calculated after normalization to rRNA (18S + 28S) levels. The experiment shows that TSA treatment causes a general reduction of poly-A RNA stability, while replication-dependent histone mRNA stability is not affected. Overall design: RNA half-lives were measured in TSA-treated or untreated HeLa cells by RNA-Seq using Illumina HiSeq 2000.
Acetylation-Dependent Control of Global Poly(A) RNA Degradation by CBP/p300 and HDAC1/2.
No sample metadata fields
View SamplesAnimal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide target sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible targets and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knock-down, protein but not mRNA expression of the 439 targets was specifically and highly significantly upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites nearby the start codon of target genes. We propose that GLD-1 interacts with the translation machinery nearby the start codon, a so far unknown mode of gene regulation in eukaryotes. Overall design: Arrested L1 worms were grown in liquid culture supplemented with 2mM 4SU or 6SG. 250,000 worms were sufficient for one iPAR-CLIP experiment. Living adult worms were transferred to NGM plates and crosslinked on ice using a Stratalinker (Stratagene) with customized 365nm UV-lamps (energy setting: 2J/cm2). Worms were lysed on ice by douncing in NP40 lysis buffer (50 mM HEPES-K pH 7.5, 150 mM KCl, 2 mM EDTA, 0.5% (v/v) NP-40, 0.5 mM DTT, protease inhibitor cocktail (Roche)). Cleared lysates were treated with RNase T1 (Fermentas) (final concentration 1U/?l) for 15 min at 22ºC. GLD-1::GFP::FLAG fusion proteins were immunoprecipitated for 1h at 4ºC using anti-FLAG antibody (Sigma, F3165) coupled to Protein G magnetic beads (Invitrogen). For one iPAR-CLIP experiment (1ml cleared lysate obtained from 250,000 worms), 300µl beads and 150µg antibody were used. Immunoprecipitates were treated with RNase T1 (100U/?l) for exactly 12 min at 22 ºC. Subsequently, PAR-CLIP was carried out as described previously (Hafner et al, 2010). cDNA libraries were sequenced on a Genome Analyzer II (Illumina).
In vivo and transcriptome-wide identification of RNA binding protein target sites.
Cell line, Subject
View SamplesAnimal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide target sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible targets and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knock-down, protein but not mRNA expression of the 439 targets was specifically and highly significantly upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites nearby the start codon of target genes. We propose that GLD-1 interacts with the translation machinery nearby the start codon, a so far unknown mode of gene regulation in eukaryotes. Overall design: PolyA mRNA was extracted from young adult wildtype (N2) worms and young adult germline less worms (glp-4(bn2) TS) to identify and quantify genes expressed in the young adult germline by sequencing. 2x100 paired end sequencing was performed according to the protocol on the Illumina HiSeq 2000.
In vivo and transcriptome-wide identification of RNA binding protein target sites.
Cell line, Subject
View Samples