Project description:The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of FOXO4 and its DNA binding element (DBE).
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:Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is a widely used approach to study DNA methylation genome-wide. Here, we present a novel MeDIP-Seq protocol compatible with the Ion Torrent semiconductor-based sequencing platform that is scalable and accurately identifies sites of differential DNA methylation. Additionally, we demonstrate that the high-throughput data derived from MeDIP-Seq on the Ion Torrent platform provides adequate coverage of CpG cytosines, the methylation states of which we validated at single-base resolution on the Infinium HumanMethylation450K Beadchip array. We applied this integrative approach to further investigate the role of DNA methylation in alternative splicing and to profile 5-mC and 5-hmC variants of DNA methylation in normal human brain tissue that we observed localize over distinct genomic regions. These applications of MeDIP-Seq on the Ion Torrent platform have broad utility and add to the current methodologies for profiling genome-wide DNA methylation states in normal and disease conditions. MeDIP-Seq on Ion Torrent Platform in HCT116 and Human Brain
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:Recurrent non-medullary thyroid carcinoma (NMTC) is a rare disease. We initially characterized 27 recurrent NMTC: 13 papillary thyroid cancers (PTC), 10 oncocytic follicular carcinomas (FTC-OV), and 4 non-oncocytic follicular carcinomas (FTC). A validation cohort composed of benign and malignant (both recurrent and non-recurrent) thyroid tumours was subsequently analysed (n = 20). Methods Data from genome-wide SNP arrays and flow cytometry were combined to determine the chromosomal dosage (allelic state) in these tumours, including mutation analysis of components of PIK3CA/AKT and MAPK pathways. Results All FTC-OVs showed a very distinct pattern of genomic alterations. Ten out of 10 FTC-OV cases showed near-haploidisation with or without subsequent genome endoreduplication. Near-haploidisation was seen in 5/10 as extensive chromosome-wide monosomy (allelic state [A]) with near-haploid DNA indices and retention of especially chromosome 7 (seen as a heterozygous allelic state [AB]). In the remaining 5/10 chromosomal allelic states AA with near diploid DNA indices were seen with allelic state AABB of chromosome 7, suggesting endoreduplication after preceding haploidisation. The latter was supported by the presence of both near-haploid and endoreduplicated tumour fractions in some of the cases. Results were confirmed using FISH analysis. Relatively to FTC-OV limited numbers of genomic alterations were identified in other types of recurrent NMTC studied, except for chromosome 22q which showed alterations in 6 of 13 PTCs. Only two HRAS, but no mutations of EGFR or BRAF were found in FTC-OV. The validation cohort showed two additional tumours with the distinct pattern of genomic alterations (both with oncocytic features and recurrent). Conclusions We demonstrate that recurrent FTC-OV is frequently characterised by genome-wide DNA haploidisation, heterozygous retention of chromosome 7, and endoreduplication of a near-haploid genome. Whether normal gene dosage on especially chromosome 7 (containing EGFR, BRAF, cMET) is crucial for FTC-OV tumour survival is an important topic for future research. 28 thyroid tumors from 27 patients were profiled by SNP array. Comparisons between different types were made.
Project description:The aim of this study was to investigate the presence of intact O- and N-glycopeptides in profiles of normal human urine analyzed by capillary electrophoresis coupled to mass spectrometry (CE-MS).CE and liquid chromatography coupled to tandem mass spectrometry (CE- and LC-MS/MS) were both used to identify glycopeptide sequences. Furthermore, we estimated the abundance levels of these glycopeptides in urine from five different cancer types i.e. bladder cancer (BCa), prostate cancer (PCa), pancreatic cancer, cholangiocarcinoma (CC) and renal cell carcinoma (RCC).
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:Detergents and salts are widely used in lysis buffer to enhance protein extraction from biological samples, facilitating in-depth proteome analysis, however, to efficiently remove these detergent and salt additives from the digested peptides is essential for generating high quality mass spectra in LC-MS/MS analysis. The sample preparation methods, such as Filter-Aided Sample Preparation (FASP), acetone precipitation followed by in-solution digestion (AP), strong cation exchange-based centrifugal proteomic reactor (CPR), are commonly used for proteomic sample process, but the additives removal efficiency and the application scope of these methods need to be further investigated. In this study, we demonstrated an integrative work flow for systematical small molecular additives quantification as well as peptide/protein identification to provide a comprehensive evaluation for additive cleanup effect of proteomic sample preparation pipelines. The multiple reaction monitoring (MRM)-based LC-MS approach was developed for six additives (i.e., Tris, Urea, CHAPS, SDS, SDC and Triton X-100) quantification. For the peptide and protein identification, although FASP outperformed the other two methods, these three methods were complementary to each other, in terms of peptide/ protein identification, as well as GRAVY index and amino acids distributions. By integrating analysis of small molecular quantification and protein identification datasets, we first bridged the quantitative influence of additive concentration to the peptide analysis performance. Our results provide a valuable dataset for appropriate sample preparation method selection and a guideline for evaluation of small molecular additives cleanup efficiency in the sample preparation procedures.
Project description:In this study the gene expression differences between two titanium surfaces produced at Maastricht University were investigated. These two surfaces were: flat titanium-coated polystyrene and a titanium-coated polystyrene surface imprinted with a pattern selected from an earlier screening study (Ti1018). This pattern was selected based on the osteoinductive properties observed. As a positive control cells on the flat surface were treated with dexamethasone.