Project description:Cancer-associated fibroblasts (CAFs) have been recognized as important contributors to cancer development and progression. However, opposing evidence has been published whether CAFs, in addition to epigenetic, also undergo somatic genetic alterations and whether these changes contribute to carcinogenesis and tumour progression. We combined multiparameter DNA flow cytometry, flow-sorting and 6K SNP-arrays to study DNA aneuploidy, % S-phase, loss of heterozygosity (LOH) and copy number alterations (CNAs) to study somatic genetic alterations in cervical cancer-associated stromal cell fractions (n = 58) from formalin-fixed, paraffin-embedded (FFPE) samples. Tissue sections were examined for the presence of CAFs. Microsatellite analysis was used to study LOH. By flow cytometry we found no proof for DNA aneuploidy in the vimentin-positive stromal cell fractions of any samples (CV G0G1 population 3.7% +/- 1.2; S-phase 1.4% +/- 1.8). The genotype concordance between the stromal cells and the paired normal endometrium samples was > 99.9%. No evidence for CNAs or LOH was found in the stromal cell fractions. In contrast, high frequencies of DNA content abnormalities (43/57), a significant higher S-phase (14.6% +/- 8.5)(p = 0.0001) and substantial numbers of CNAs and LOH were identified in the keratin-positive epithelial cell fractions (CV G0G1 population 4.1% +/- 1.0). Smooth muscle actin and vimentin immunohistochemistry verified the presence of CAFs in all cases tested. LOH hot-spots on chromosomes 3p, 4p and 6p were confirmed by microsatellite analysis but the stromal cell fractions showed retention of heterozygosity only. From our study we conclude that stromal cell fractions from cervical carcinomas are DNA diploid, have a genotype undistinguishable from patient-matched normal tissue and are genetically stable. Stromal genetic changes do not seem to play a role during cervical carcinogenesis and progression. In addition, the stromal cell fraction of cervical carcinomas can be used as reference allowing large retrospective studies of archival FFPE tissues for which no normal reference tissue is available. Paired experiment, Endometrial (non-tumor) cells vs stromal cells from cervical tumors. Biological replicates: 58 patients. From 5 tumors also the tumor fraction was profiled.
Project description:We report for the first time movement of Correia Repeat Enclosed Elements, through inversion of the element at its chromosomal location. Analysis of Ion Torrent generated genome sequence data from Neisseria gonorrhoeae strain NCCP11945 passaged for 8 weeks in the laboratory under standard conditions and stress conditions revealed a total of 37 inversions: 24 were exclusively seen in the stressed sample; 7 in the control sample; and the remaining 3 were seen in both samples. These inversions have the capability to alter gene expression in N. gonorrhoeae through the previously determined activities of the sequence features of these elements. In addition, the locations of predicted non-coding RNAs were investigated to identify potential associations with CREE. Associations varied between strains, as did the number of each element identified. The analysis indicates a role for CREE in disrupting ancestral regulatory networks, including non-coding RNAs. RNA-Seq was used to examine expression changes related to Correia repeats in the strain
Project description:In order to determine whether dis-regulation of a genetic pathway could explain the increased apoptosis of parp-2-/- double positive thymocytes, the gene expression profiles in double positive thymocytes derived from wild-type and parp-2-/- mice were analysed using Affymetrix oligonucleotide chips (mouse genome 430 2.0).
Project description:Ossification of the posterior longitudinal ligament (OPLL) is formed by heterogeneous ossification of posterior longitudinal ligament. The patho-mechanism of OPLL is still largely unknown. MicroRNAs are small nucleatides that function as regulators of gene expression in almost any biological process. However, few microRNAs are reported to have a role in the pathological process of OPLL. Therefore, we performed high-throughput microRNA sequencing and transcriptome sequencing of primary OPLL and PLL cells in order to decipher the interacting network of microRNAs in OPLL. MRNA and microRNA profiles were done using primary culture cells of human ossification of the posterior longitudinal ligament (OPLL) tissue and normal posterior longitudinal ligament (PLL) tissue.
Project description:Ossification of the posterior longitudinal ligament (OPLL) is formed by heterogeneous ossification of posterior longitudinal ligament. The patho-mechanism of OPLL is still largely unknown. Recently, disorders of metabolism are thought to be the center of many diseases such as OPLL. Advanced glycation end product (AGE) are accumulated in many extracellular matrixes such as ligament fibers, and it can functions as cellular signal through its receptor (RAGE), contributing to various events such as atherosclerosis or oxidative stress. However, its role in OPLL formation is not yet known. Therefore, we performed high-through-put RNA sequencing on primary posterior longitudinal ligament cells treated with different doses of AGEs (1µM, 5µM and negative control), with or without BMP2 (1µM). mRNA profiles of Primary human posterior longitudinal ligament cells stimulated with various stimuli (Control, 1µM AGE-BSA, 5µM AGE-BSA, 1µM AGE-BSA with BMP2, 5µM AGE-BSA with BMP2) were generated by deep sequencing on Ion Proton
Project description:MicroRNAs are important negative regulators of protein coding gene expression, and have been studied intensively over the last few years. To this purpose, different measurement platforms to determine their RNA abundance levels in biological samples have been developed. In this study, we have systematically compared 12 commercially available microRNA expression platforms by measuring an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples, and synthetic spikes from homologous microRNA family members. We developed novel quality metrics in order to objectively assess platform performance of very different technologies such as small RNA sequencing, RT-qPCR and (microarray) hybridization. We assessed reproducibility, sensitivity, quantitative performance, and specificity. The results indicate that each method has its strengths and weaknesses, which helps guiding informed selection of a quantitative microRNA gene expression platform in function of particular study goals.
Project description:Metabolite or substrate channeling is a direct transfer of metabolites from one enzyme to the next enzyme in a cascade. Among many potential advantages of substrate channeling, acceleration of the total reaction rate is considered as one of the most important and self-evident. However, using a simple model, supported by stochastic simulations, we show that it is not always the case; particularly at long times (i.e. in steady state) and high substrate concentrations, a channeled reaction cannot be faster, and can even be slower, than the original non-channeled cascade reaction. In addition we show that increasing the degree of channeling may lead to an increase of the metabolite pool size. We substantiate that the main advantage of channeling likely lies in protecting metabolites from degradation or competing side reactions.
Project description:MotivationMany enzymes are not absolutely specific, or even promiscuous: they can catalyze transformations of more compounds than the traditional ones as listed in, e.g. KEGG. This information is currently only available in databases, such as the BRENDA enzyme activity database. In this article, we propose to model enzyme aspecificity by predicting whether an input compound is likely to be transformed by a certain enzyme. Such a predictor has many applications, for example, to complete reconstructed metabolic networks, to aid in metabolic engineering or to help identify unknown peaks in mass spectra.ResultsWe have developed a system for metabolite and reaction inference based on enzyme specificities (MaRIboES). It employs structural and stereochemistry similarity measures and molecular fingerprints to generalize enzymatic reactions based on data available in BRENDA. Leave-one-out cross-validation shows that 80% of known reactions are predicted well. Application to the yeast glycolytic and pentose phosphate pathways predicts a large number of known and new reactions, often leading to the formation of novel compounds, as well as a number of interesting bypasses and cross-links.AvailabilityMatlab and C++ code is freely available at https://gforge.nbic.nl/projects/mariboes/
Project description:In this study, RNA-Seq was used to reveal the differences of molecular pathways in hepatopancreas of O. niloticus adapated to water with salinity of 8 or 16 practical salinity (psu), respectively, with fish at freshwater as the control,. Significantly changed pathways were mainly related to lipid metabolism, glucose utilization, protein consumption, osmotic regulation, signal transduction and immunology. Based on the tendencies from freshwater to 8 or 16 psu, the differentially expressed gene unions were categorized into eight unique models, which were further classified into three categories which were constant-change (either keep increasing or decreasing), change-then-stable and stable-then-change. In constant-change category, steroid biosynthesis, steroid hormone biosynthesis, fat digestion and absorption, complement and coagulation cascades were extremely significantly affected by ambient salinity (P < 0.01), indicating that these pathways play pivotal roles in molecular response to salinity acclimation from freshwater to saline water in O. niloticus, and should be the main research focus in the future. In change-then-stable category, ribosome, oxidative phosphorylation, peroxisome proliferator-activated receptors (PPAR) signaling pathway, fat digestion and absorption changed significantly with ambient increasing salinity (P < 0.01), showing these pathways were sensitive to environmental salinity variation, but had a response threshold, and would stop changing once salinity exceeds the threshold. In stable-then-change category, protein export, protein processing in endoplasmic reticulum, tight junction, thyroid hormone synthesis, antigen processing and presentation, glycolysis/gluconeogenesis and glycosaminoglycan biosynthesis - keratan sulfate were the top changed pathways (P < 0.01), suggesting that these pathways were not sensitive to salinity variation, but these pathways will respond significantly under salinity exceeding a certain level. The pathways and genes reported in this study laid on a solid foundation for future studies in understanding the underlying mechanism for salinity adaptation of freshwater fish. Examination of 3 different salinities treated hepatopancreas in Nile tilapia
Project description:Long non-coding RNAs (lncRNAs) are recently characterized players that are involved in the regulatory circuitry of self-renewal in human embryonic stem cells (hESCs). However, the specific roles of lncRNAs in this circuitry are poorly understood. Here, we determined that growth-arrest-specific transcript 5 (GAS5), which is a known tumor suppressor and growth arrest gene, is abundantly expressed in the cytoplasm of hESCs and essential for hESC self-renewal. GAS5 depletion in hESCs significantly impaired their pluripotency and self-renewal ability, whereas GAS5 overexpression in hESCs accelerated the cell cycle, enhanced their colony formation ability and increased pluripotency marker expression. By RNA sequencing and bioinformatics analysis, we determined that GAS5 activates NODAL-SMAD2/3 signaling by sustaining the expression of NODAL, which plays a key role in hESC self-renewal but not in somatic cell growth. Further studies indicated that GAS5 functions as a competing endogenous RNA (ceRNA) to protect NODAL mRNA against degradation and that GAS5 transcription is directly controlled by the core pluripotency transcriptional factors (TFs). Taken together, we suggest that the core TFs, GAS5 and NODAL-SMAD2/3 form a feed-forward loop to maintain the hESC self-renewal process. These findings are specific to ESCs and did not occur in the somatic cell lines we tested; therefore, our findings also provide evidence that the functions of lncRNAs vary in different biological contexts. We analyzed long non-coding RNAs in two hESC cell lines (X-01 and H1), and found GAS5 is highly expressed and functional in maintaining hESC self-renewal. We generate stable overexpressed or knockdown hESC cell lines using lentiviral approach. We transfected cells initialy after passage, and lentiviruses are added with daily medium change for three days (at a final concentration of 10^5 IU/ml). Puromycin is added for selection and supplied with daily medium change. Stable cell lines are established after two passages and verified under fluorescence scope. Total RNAs and miRNAs are extracted separately of all three cell lines (LV-NC, LV-GAS5 and LV-shGAS5) and put to sequencing.