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Deep plasma proteome profiling by enrichment of extracellular vesicles

ABSTRACT: This work show that enrichment of extracellular vesicles by ultracentrifugation increases the plasma proteome depth by an order of magnitude. Following the enrichment protocol, more than two thousand proteins can routinely be quantified reproducibly by label-free quantification and data independent acquisition (DIA) in single-shot LC-MSMS runs of less than one hour. The project also present an optimized workflow for plasma proteomics that enables high-throughput with very short chromatographic gradients analyzing hundred samples per day with deep proteome coverage, especially when including study-specific spectral libraries generated by repeated injection and gas-phase fractionation of pooled samples

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

DISEASE(S): Disease Free

SUBMITTER: Ole Østergaard

LAB HEAD: Jesper Velgaard Olsen

PROVIDER: PXD037340 | Pride | 2022-12-04

REPOSITORIES: Pride

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Ontogeny, distribution and potential roles of hydroxymethylcytosine in human liver function

Project description:Background: Interindividual differences in liver functions such as protein synthesis, lipid and carbohydrate metabolism and drug metabolism are influenced by epigenetic factors. The role of the epigenetic machinery in such processes has, however, been barely investigated. 5-hydroxymethylcytosine (5hmC) is a recently re-discovered epigenetic DNA modification that plays an important role in the control of gene expression. Results: In this study, we investigate 5hmC occurrence and genomic distribution in 8 fetal and 7 adult human liver samples in relation to ontogeny and function. LC-MS analysis shows that in the adult liver samples 5hmc comprises up to 1% of the total cytosine content, whereas in all fetal livers it is below 0.125%. Immunohistostaining of liver sections with a polyclonal anti-5hmC antibody shows that 5hmC is detected in most of the hepatocytes. Genome-wide mapping of the distribution of 5hmC in human liver samples by next-generation sequencing shows significant differences between fetal and adult livers. In adult livers, 5hmC occupancy is overrepresented in genes involved in active catabolic and metabolic processes, whereas 5hmC elements which are found in genes exclusively in fetal livers and disappear in the adult state, are more specific to pathways for differentiation and development. Conclusions: Our findings suggest that 5-hydroxymethylcytosine plays an important role in the development and function of the human liver and might be an important determinant for development of liver diseases as well as of the interindividual differences in drug metabolism and toxicity. 8 fetal and 7 adult human liver samples

2013-07-29 | E-GEOD-49291 | biostudies-arrayexpress

Deep proteome profiling of human cerebrospinal fluid from patients diagnosed with Multiple System Atrophy, Parkinson’s Disease, Dementia with Lewy Bodies, Progressive Supranuclear Palsy, and from Controls

Project description:Human cerebrospinal fluid was collected from patients diagnosed with neurodegenerative diseases including multiple system atrophy (n=28), Parkinson’s disease (n=40), dementia with Lewy bodies (n=20), progressive supranuclear palsy (n=39) and from controls (n=17) in order to perform a comparative quantitative proteome profiling of cerebrospinal fluids from the five groups.

2022-08-12 | PXD026802 | Pride

Deubiquitinating enzymes and the proteasome regulate unique sets of ubiquitin substrates.

Project description:The ubiquitin-proteasome axis has been extensively explored at a system-wide level, but the impact of deubiquitinating enzymes (DUBs) on the ubiquitinome remains largely unknown. Using UbiSite technology and inhibitors, we have compared the contributions of the proteasome and DUBs on the ubiquitinome. We uncovered large differential dynamic Ub signalling networks with substrates and sites uniquely regulated by DUBs or by the proteasome, highlighting the role of DUBs in degradation-independent ubiquitin signalling. DUBs regulate substrates via nearly 40,000 unique sites. Moreover, we found that ubiquitin conjugated to SUMO2/3 forms a unidirectional proteasomal degradation signal with strikingly rapid kinetics compared to ubiquitin polymers only. We found that PARP1, is hyper ubiquitinated in response to DUB inhibition, increasing its enzymatic activity. Our findings highlight the key regulatory roles of DUBs on ubiquitin dynamics.

2022-05-23 | PXD030644 | Pride

Molecular Characterization of Three Gametoclones of Citrus clementina

Project description:Gene deletion analysis of three independent gametoclones of Citrus clementina by comparative genomic hybridization

2013-11-12 | E-MEXP-3926 | biostudies-arrayexpress

The HDAC inhibitor SAHA regulates CBX2 stability via a SUMO-triggered ubiquitin-mediated pathway in leukemia

Project description:Leukemias are characterized by bone marrow failure due to oncogenic mutations of hematopoietic stem cells (HSC) or blood precursor cells. HSC differentiation and self-renewal properties are tightly regulated by Polycomb group (PcG) proteins, a well-characterized family of transcriptional epigenetic regulators. PcG proteins form two canonical complexes: Polycomb repressive complex 1 (PRC1), and Polycomb repressive complex 2 (PRC2).CBX proteins link the activity of PRC1 with PRC2, serving as critical regulators of PcG-mediating activity. While the functional role of some CBX proteins in cancer has been largely explored, recent reports support the specific role of CBX2 in human tumors, thus it represent a promising new target for anti-cancer strategies. To date, chromodomain inhibitors have been identified for CBX7 , but no molecules inhibiting CBX2 have been described. Nevertheless, different chromatin-modulating drugs such as histone deacetylase inhibitors (HDACi) are reported to regulate CBX2 targets on chromatin, suggesting that HDACi might be used to indirectly modulate aberrant effects of CBX2 in cancer. We describe a novel SAHA-mediated mechanism of CBX2 post-translational regulation. We found that CBX2 undergoes SAHA-induced SUMO2/3 modification and that CBX2 SUMOylation promotes its ubiquitination and proteasome-dependent degradation. We also identified the specific molecular pathway and key players regulating CBX2 stability, demonstrating that CBX4 and RNF4 act as the E3 SUMO and E3 ubiquitin ligase, respectively. Additionally, CBX2-depleted leukemic cells display impaired proliferation, showing that CBX2 is required for leukemia cell clonogenicity. Our study provides the first evidence of a non-canonical SAHA-mediated anti-tumorigenic activity via CBX2 SUMOylation and degradation

2018-02-27 | PXD008287 | Pride

BRD9 regulates leukemic cell survival via STAT5 pathway inhibition

Project description:Leukemia is characterized by genetic and epigenetic mutations resulting in selection of cancer stem cells, which are unable to differentiate. While genetic alterations are difficult to target, the epigenome is intrinsically dynamic and readily offers new therapeutic strategies. Thus, identifying cancer-specific context-dependent targets and unraveling their biological function may open up new therapeutic perspectives. Here, we identify bromodomain-containing protein 9 (BRD9) as a critical target required in acute myeloid leukemia (AML). We show that BRD9 is overexpressed in AML cells including ex vivo primary blasts compared to CD34+. By targeting BRD9 expression in AML, we observed an alteration in proliferation and survival, ultimately resulting in the induction of apoptosis. Intriguingly, genome-wide profiling revealed that BRD9 binds enhancer regions in a cell type-specific manner, regulating cell type-related processes. We unveil a novel BRD9-sustained STAT5 pathway activation via regulation of SOC3 expression levels. Our findings identify a previously undescribed BRD9-STAT5 axis as critical for leukemia maintenance, suggesting BRD9 as a potential therapeutic target.

2019-11-11 | PXD013245 | Pride

Transcription profiling of ovarium cancer samples from patients with tumors of different stages and sensitivity to platinum chemotherapy treatment

Project description:We investigated whether prognostic information is reflected in the expression patterns of ovarian carcinoma samples. RNA obtained from seven FIGO stage I without recurrence, seven platin-sensitive advanced-stage (III or IV), and six platin-resistant advanced-stage ovarian tumors was hybridized on a complementary DNA microarray with 21,372 spotted clones. The results revealed that a considerable number of genes exhibit nonaccidental differential expression between the different tumor classes. Principal component analysis reflected the differences between the three tumor classes and their order of transition. Using a leave-one-out approach together with least squares support vector machines, we obtained an estimated classification test accuracy of 100% for the distinction between stage I and advanced-stage disease and 76.92% for the distinction between platin-resistant versus platin-sensitive disease in FIGO stage III/IV. These results indicate that gene expression patterns could be useful in clinical management of ovarian cancer.<br> KEYWORDS: clinical, FIGO stage, microarrays, ovarian cancer, platin resistance.

2007-02-10 | E-MEXP-979 | biostudies-arrayexpress

Transcription profiling of ovarium cancer samples from patients with tumors of different stages, sensitivity to platinum chemotherapy and with no disease recurrence, recurrence or progression to evaluate models of clinical behaviour

Project description:Ovarian cancer is the leading cause of death in gynaecological malignancies in women. However, currently there are no clinical or pathologic parameters available that can reliably predict clinical outcome. In a previously published pilot study we explored the performance of microarrays in predicting clinical behaviour of ovarian tumours. For this purpose we performed microarray analysis on 20 patients and estimated that we could predict disease stage with 100% accuracy and the response to platin-based chemotherapy with 76.92% accuracy using leave-one-out cross validation techniques in combination with Least Squares Support Vector Machines (LS-SVMs). In the current study we prospectively evaluate models, built on the pilot data set, on a set of 49 new patients. Principal component analysis showed that the gene expression data from stage I, platin-sensitive advanced stage and platin-resistant advanced stage tumours in the prospective study did not correspond to their respective classes in the pilot study. Additionally, LS-SVM models built using the data from the pilot study, only misclassified one of four stage I tumours and correctly classified all 45 advanced stage tumours but this model was not able to predict resistance to platin-based chemotherapy. We discuss possible reasons for prospective failure of these models and conclude that existing results based on gene expression patterns of ovarian tumours need to be thoroughly scrutinized before this technology could be considered ready for clinical use.

2007-12-31 | E-MEXP-995 | biostudies-arrayexpress

Transcription profiling of rice to identify of downstream genes regulated by RFL

Project description:To gain mechanistic insights on RFL functions in rice architecture, we have employed global expression profilling to identify the genes regulated by RFL. Transgenic rice panicle RNAs down-regulated for RFL were compared with the wild type panicle RNA of similar developmental stage. Genes downregulated in transgenic lines will be those which expression requires functional RFL whereas upregulated genes will need RFL for their repression. Experiment Overall Design: Two independent biological RNA pools of dsRNAiRFL and RFL antisense transgenic panicles were compared with two independent wild type RNA pool in two different hybridizations. Two hybridizations were performed with reciprocally dye-labeled wild type RNA and respective mutant RNA.

2008-06-11 | E-GEOD-10098 | biostudies-arrayexpress

Transcription profiling of rice panicle RNAs were compared with the wild type panicle RNA of similar developmental stage to identify of downstream genes regulated by OsMADS2

Project description:To gain mechanistic insights on OsMADS2 functions in rice spikelet development, we have employed global expression profilling to identify the genes regulated by OsMADS2. Transgenic rice panicle RNAs were compared with the wild type panicle RNA of similar developmental stage. Genes downregulated in transgenic lines will be those which expression requires functional OsMADS2 whereas upregulated genes will need OsMADS2 for their repression. Experiment Overall Design: Two independent biological RNA pools of dsRNAiOsMADS2 transgenic panicles were compared with a single wild type RNA pool in two different hybridizations. A third hybridization was performed with reciprocally dye-lebeled wild type RNA and one of the mutant RNA.

2008-06-15 | E-GEOD-7192 | biostudies-arrayexpress

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