ABSTRACT: We previously reported the isolation and in vitro propagation of highly tumorigenic mammospheres (MCFS) isolated from MCF7 breast cancer cell line. In this study we obtained gene expression profiles of MCFS and parental MCF7 cell lines using SOLiD RNA-sequencing after linear isothermal DNA amplification.
Project description:Pulmonary Arterial Hypertension (PAH) is characterized by progressive increase in pulmonary vascular resistance, right ventricular failure and premature death. Owing to severe complications, lung biopsies cannot be envisioned to characterize the disease. Based on the prominent role of inflammation in PAH development, we used Peripheral Blood Mononuclear Cells (PBMCs) as cell source, and relied on the SOLiD platform of Serial Analysis of Gene Expression (SAGE) for transcriptional profiling
Project description:A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT) which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified, but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments relying on isobaric TMT reporter ion quantification.
Project description:Background: Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer but there are no comprehensive proteomic studies on this entity. Materials and Methods: We employed liquid chromatography coupled with tandem mass spectrometry (MS/MS) using formalin-fixed paraffin-embedded (FFPE) cSCC material to study the tumor and normal skin tissue proteomes. Ingenuity Pathway Analysis (IPA) was used to interpret the role of altered proteins in cSCC pathophysiology. Results were validated using the Human Protein Atlas and Oncomine database in silico. Results: Of 1,310 unique proteins identified, expression of an average of 144 and 88 proteins were significantly (p<0.05) increased and decreased, respectively, in the tumor samples compared to their normal counterparts. IPA analysis revealed disruptions in proteins associated with cell proliferation, apoptosis, and migration. In silico analysis confirmed that proteins corresponding to 12 antibodies, and genes corresponding to 18 proteins were differentially expressed between the two categories, validating our proteomic measurements. Conclusion: Label-free MS-based proteomics is useful for analyzing FFPE cSCC tissues.
Project description:Anaplasma phagocytophilum infects a wide variety of host species and causes the diseases granulocytic anaplasmosis in humans, horses and dogs and tick-borne fever in ruminants. The objective of this research was to characterize differential gene expression in wild boar naturally infected with A. phagocytophilum by microarray hybridization using the GeneChip® Porcine Genome Array Differential gene expression in wild boar naturally infected with A. phagocytophilum was chacarterized by microarray hybridization using the GeneChip® Porcine Genome Array and real-time RT-PCR.
Project description:MicroRNAs regulate several aspects of tumorigenesis and cancer progression. Most cancer tissues are archived formalin-fixed and paraffin-embedded (FFPE). While microRNAs are a more stable form of RNA thought to withstand FFPE-processing and degradation there is only limited evidence for the latter assumption. We examined whether microRNA profiling can be successfully conducted on FFPE cancer tissues using SOLiD ligation based sequencing. Tissue storage times (3-9 years) appeared to not affect the number of detected microRNAs in FFPE samples compared to matched frozen samples (paired t-test p>0.7). Correlations of microRNA expression values were very high across microRNAs in a given sample (PearsonM-bM-^@M-^Ys r=0.71-0.95). Higher variance of expression values among samples was associated with higher correlation coefficients between FFPE and frozen tissues. One of the FFPE samples in this study was degraded for unknown reasons with a peak read length of 17 nucleotides compared to 21 in all other samples. The number of detected microRNAs in this sample was within the range of microRNAs detected in all other samples. Ligation-based microRNA deep sequencing on FFPE cancer tissues is feasible and RNA degradation to the degree observed in our study appears to not affect the number of microRNAs that can be quantified. we analyzed eight matched frozen and FFPE tissues of different histologies for which detailed processing and storage information.
Project description:Men and women with type 2 diabetes, all born in Europe, were recruited to this phase II clinical trial to examine the possible effects of tadalafil on insulin resistance. The study had a cross-over design and participants were randomised to either start with tadalafil or placebo for 6 weeks. At the end of the tretment period participants were extensively examined, including a hyperinsulinemic clamp. Initial treatment period was followed by 8 weeks of wash-out, before participants started with the other treatment for 6 weeks, and a new examination. Tadalafil showed no effect on the primary endpoint (clamp), but did reduce HbA1c, a secondary endpoint.
Project description:Thyroid carcinoma (TC) is generally associated with good prognosis, nevertheless no effective treatments are available for aggressive forms not cured by current therapies. We previously identified the coatomer protein complex zeta 1 (COPZ1), as a new putative therapeutic target for TC, since its depletion impairs the viability of tumor cells, leads to abortive autophagy, ER stress, unfolded protein response and apoptosis, and reduces the tumor growth of TC xenograft models. In this study, by combining genomic, proteomic and functional approaches, we provided evidence that COPZ1 silencing stimulates a type I IFN-mediated viral mimicry response, boosts the production of several inflammatory molecules and finally induces immunogenic cell death, which, in turn, promotes dendritic cell maturation and subsequent activation of T cells. Collectively, our findings support the notion that COPZ1 targeting can be exploited as a new strategy to kill cancer cells with the subsequent involvement of an anti-tumor immune response.
Project description:Background Worldwide, gastric cancer is the fourth most common malignancy and the most common cancer in East Asia. Development of targeted therapies for this disease has focused on a few known oncogenes but has had limited effects. Objective To determine oncogenic mechanisms and novel therapeutic targets specific for gastric cancer by identifying commonly dys-regulated genes from the tumors of both Asian-Pacific and Caucasian patients. Design We generated transcriptomic profiles of 22 Caucasian gastric cancer tumors and their matched non-cancerous samples, and performed an integrative analysis across different gastric cancer gene expression datasets. We examined the inhibition of commonly overexpressed oncogenes and their constituent signaling pathways by RNAi and/or pharmacologic inhibition. Results We found that HNF4α upregulation was a key signaling event in gastric tumors from both Caucasian and Asian patients, and HNF4α antagonism was antineoplastic. Perturbation experiments in GC tumor cell lines and xenograft models further demonstrated that HNF4α is downregulated by AMPKα signaling and the AMPK agonist metformin; blockade of HNF4α activity resulted in cyclin downregulation, cell cycle arrest, and tumor growth inhibition. HNF4α also regulated WNT signaling through its target gene WNT5A, a potential prognostic marker of diffuse type gastric tumors. Conclusions Our results indicate that HNF4α is a targetable oncoprotein in gastric cancer, is regulated by AMPK signaling through AMPKα, and resides upstream of WNT signaling. HNF4α may regulate “metabolic switch” characteristic of a general malignant phenotype and its target WNT5A has potential prognostic values. The AMPKα-HNF4α-WNT5A signaling cascade represents a potentially targetable pathway for drug development. Integrative analysis of Caucasian and Asian-Pacific gastric tumor expression datasets (including newly generated transcriptomic profiling of 22 tumors in this study) revealed a relatively small common sets of highly overexpressed genes.
Project description:Rifampin causes drug interactions by altering hepatic drug metabolism. Because microRNAs (miRNAs) have been shown to regulate genes involved in drug metabolism, we determined the effect of rifampin on the expression of hepatic miRNAs. Primary human hepatocytes from seven subjects were treated with rifampin, and the expression of miRNA and cytochrome P450 (P450) mRNAs was measured by TaqMan assays and RNA-seq, respectively. Rifampin induced the expression of 10 clinically important and 13 additional P450 genes and repressed the expression of 9 other P450 genes (P < 0.05). Rifampin induced the expression of 33 miRNAs and repressed the expression of 35 miRNAs (P < 0.05). Several of these changes were highly negatively correlated with the rifampin-induced changes in the expression of their predicted target P450 mRNAs, supporting the possibility of miRNA-induced regulation of P450 mRNA expression. In addition, several other miRNA changes were positively correlated with the changes in P450 mRNA expression, suggesting similar regulatory mechanisms. Despite the interindividual variability in the rifampin effects on miRNA expression, principal components analysis clearly separated the rifampin-treated samples from the controls. In conclusion, rifampin treatment alters miRNA expression patterns in human hepatocytes, and some of the changes were correlated with the rifampin-induced changes in expression of the P450 mRNAs they are predicted to target. Primary human hepatocytes were treated rifampin or vehicle for 24 hours. Rifampin-treated samples are not available for the 30Jul10 patient or the Hep2 patient due to raw data file corruption.
Project description:Given the possible critical importance of placental gene imprinting and random monoallelic expression on fetal and infant health, most of those genes must be identified, in order to understand the risks that the baby might meet during pregnancy and after birth. Therefore, the aim of the current study was to introduce a workflow and tools for analyzing imprinted and random monoallelic gene expression in human placenta, by applying whole-transcriptome (WT) RNA sequencing of placental tissue and genotyping of coding DNA variants in family trios. Ten family trios, each with a healthy spontaneous single-term pregnancy, were recruited. Total RNA was extracted for WT analysis, providing the full sequence information for the placental transcriptome. Parental and child blood DNA genotypes were analyzed by exome SNP genotyping microarrays, mapping the inheritance and estimating the abundance of parental expressed alleles. Imprinted genes showed consistent expression from either parental allele, as demonstrated by the SNP content of sequenced transcripts, while monoallelically expressed genes had random activity of parental alleles. We revealed 4 novel possible imprinted genes (LGALS8, LGALS14, PAPPA2 and SPTLC3) and confirmed the imprinting of 4 genes (AIM1, PEG10, RHOBTB3 and ZFAT-AS1) in human placenta. The major finding was the identification of 4 genes (ABP1, BCLAF1, IFI30 and ZFAT) with random allelic bias, expressing one of the parental alleles preferentially. The main functions of the imprinted and monoallelically expressed genes included: i) mediating cellular apoptosis and tissue development; ii) regulating inflammation and immune system; iii) facilitating metabolic processes; and iv) regulating cell cycle. Placentas from ten family trios were analysed using RNA-Seq.