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Find video protocols related to scientific articles indexed in Pubmed.
An engineered affibody molecule with pH-dependent binding to FcRn mediates extended circulatory half-life of a fusion protein.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-20-2014
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Proteins endocytosed from serum are degraded in the lysosomes. However, serum albumin (SA) and IgG, through its Fc part, bind to the neonatal Fc receptor (FcRn) at low pH in the endosome after endocytosis, and are transported back to the cellular surface, where they are released into the bloodstream, resulting in an extended serum circulation time. Association with Fc or SA has been used to prolong the in vivo half-life of biopharmaceuticals, using the interaction with FcRn to improve treatment regimens. This has been achieved either directly, by fusion or conjugation to Fc or SA, or indirectly, using SA-binding proteins. The present work takes this principle one step further, presenting small affinity proteins that bind directly to FcRn, mediating extension of the serum half-life of fused biomolecules. Phage display technology was used to select affibody molecules that can bind to FcRn in the pH-dependent manner required for rescue by FcRn. The biophysical and binding properties were characterized in vitro, and the affibody molecules were found to bind to FcRn more strongly at low pH than at neutral pH. Attachment of the affibody molecules to a recombinant protein, already engineered for increased half-life, resulted in a nearly threefold longer half-life in mice. These tags should have general use as fusion partners to biopharmaceuticals to extend their half-lives in vivo.
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Use of ENCODE Resources to Characterize Novel Proteoforms and Missing Proteins in the Human Proteome.
J. Proteome Res.
PUBLISHED: 11-05-2014
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We describe integrated strategies that employ both translation of ENCODE data and major proteomic technology pillars to improve the identification of the missing proteins, protein isoforms, and PTMs. The results from proteoENCODEdb searches with experimental mass spectral data indicate that some novel splice forms detected at the transcript level are in fact translated to proteins. Our results provide a step toward the directives of the C-HPP initiative and related biomedical research.
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Defining the human gallbladder proteome by transcriptomics and affinity proteomics.
Proteomics
PUBLISHED: 10-09-2014
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Global protein analysis of human gallbladder tissue is vital for identification of molecular regulators and effectors of its physiological activity. Here, we employed a genome-wide deep RNA sequencing analysis in 28 human tissues to identify the genes overrepresented in the gallbladder and complemented it with antibody-based immunohistochemistry in 48 human tissues. We characterized human gallbladder proteins and identified 140 gallbladder-specific proteins with an elevated expression in the gallbladder as compared to the other analyzed tissues. Five genes were categorized as enriched, with at least fivefold higher levels in gallbladder, 60 genes were categorized as group enriched with elevated transcript levels in gallbladder shared with at least one other tissue and 75 genes were categorized as enhanced with higher expression than the average expression in other tissues. We explored the localization of the genes within the gallbladder through cell-type specific antibody-based protein profiling and the subcellular localization of the genes through immunofluorescent-based profiling. Finally, we revealed the biological processes and metabolic functions carried out by these genes through the use of GO, KEGG Pathway, and HMR2.0 that is compilation of the human metabolic reactions. We demonstrated the results of the combined analysis of the transcriptomics and affinity proteomics.
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Affinity Proteomic Profiling of Plasma, Cerebrospinal Fluid, and Brain Tissue within Multiple Sclerosis.
J. Proteome Res.
PUBLISHED: 10-01-2014
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The brain is a vital organ and because it is well shielded from the outside environment, possibilities for noninvasive analysis are often limited. Instead, fluids taken from the spinal cord or circulatory system are preferred sources for the discovery of candidate markers within neurological diseases. In the context of multiple sclerosis (MS), we applied an affinity proteomic strategy and screened 22 plasma samples with 4595 antibodies (3450 genes) on bead arrays, then defined 375 antibodies (334 genes) for targeted analysis in a set of 172 samples and finally used 101 antibodies (43 genes) on 443 plasma as well as 573 cerebrospinal spinal fluid (CSF) samples. This revealed alteration of protein profiles in relation to MS subtypes for IRF8, IL7, METTL14, SLC30A7, and GAP43. Respective antibodies were subsequently used for immunofluorescence on human post-mortem brain tissue with MS pathology for expression and association analysis. There, antibodies for IRF8, IL7, and METTL14 stained neurons in proximity of lesions, which highlighted these candidate protein targets for further studies within MS and brain tissue. The affinity proteomic translation of profiles discovered by profiling human body fluids and tissue provides a powerful strategy to suggest additional candidates to studies of neurological disorders.
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Investigating the applicability of antibodies generated within the human protein atlas as capture agents in immunoenrichment coupled to mass spectrometry.
J. Proteome Res.
PUBLISHED: 09-25-2014
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For identification and characterization of proteins in complex samples, immunoenrichment coupled to mass spectrometry is a good alternative due to the sensitivity of the affinity enrichment and the specificity of mass spectrometry analysis. Antibodies are commonly used affinity agents; however, for high-throughput analysis, antibody availability is usually a bottleneck. Here we present a protocol for immunoenrichment coupled to mass spectrometry in a high-throughput setup, where all steps from bead coupling to mass spectrometry sample preparation are performed in parallel in a 96-well format. Antibodies generated within the Human Protein Atlas project were tested for applicability as capture agents. The antibodies were covalently attached to protein A beads, making it possible to reuse the coupled beads at least three times without destroying the antibody binding efficiency. Target proteins were captured from a U251 MG cell lysate, eluted, digested, and analyzed using mass spectrometry. Of 30 investigated antibodies, around 50% could successfully capture the corresponding native target protein, making the available library of more than 21?000 antibodies a valuable resource for immunoenrichment assays. Due to the diversity of different antibodies regarding affinity and specificity, analyzing antibodies in a high-throughput format is challenging. Even though protocol optimization for individual antibodies can be advantageous for future studies, our method enables a fast screening strategy to determine the usefulness of antibodies in immunoenrichment setups. In addition, we show that the specificity of the antibodies can be investigated by using label-free quantification.
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Defining the human adipose tissue proteome to reveal metabolic alterations in obesity.
J. Proteome Res.
PUBLISHED: 09-25-2014
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White adipose tissue (WAT) has a major role in the progression of obesity. Here, we combined data from RNA-Seq and antibody-based immunohistochemistry to describe the normal physiology of human WAT obtained from three female subjects and explored WAT-specific genes by comparing WAT to 26 other major human tissues. Using the protein evidence in WAT, we validated the content of a genome-scale metabolic model for adipocytes. We employed this high-quality model for the analysis of subcutaneous adipose tissue (SAT) gene expression data obtained from subjects included in the Swedish Obese Subjects Sib Pair study to reveal molecular differences between lean and obese individuals. We integrated SAT gene expression and plasma metabolomics data, investigated the contribution of the metabolic differences in the mitochondria of SAT to the occurrence of obesity, and eventually identified cytosolic branched-chain amino acid (BCAA) transaminase 1 as a potential target that can be used for drug development. We observed decreased glutaminolysis and alterations in the BCAAs metabolism in SAT of obese subjects compared to lean subjects. We also provided mechanistic explanations for the changes in the plasma level of BCAAs, glutamate, pyruvate, and ?-ketoglutarate in obese subjects. Finally, we validated a subset of our model-based predictions in 20 SAT samples obtained from 10 lean and 10 obese male and female subjects.
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The lung-specific proteome defined by integration of transcriptomics and antibody-based profiling.
FASEB J.
PUBLISHED: 08-28-2014
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The combined action of multiple cell types is essential for the physiological function of the lung, and increased awareness of the molecular constituents characterizing each cell type is likely to advance the understanding of lung biology and disease. In the current study, we used genome-wide RNA sequencing of normal lung parenchyma and 26 additional tissue types, combined with antibody-based protein profiling, to localize the expression to specific cell types. Altogether, 221 genes were found to be elevated in the lung compared with their expression in other analyzed tissues. Among the gene products were several well-known markers, but also several proteins previously not described in the context of the lung. To link the lung-specific molecular repertoire to human disease, survival associations of pneumocyte-specific genes were assessed by using transcriptomics data from 7 non-small-cell lung cancer (NSCLC) cohorts. Transcript levels of 10 genes (SFTPB, SFTPC, SFTPD, SLC34A2, LAMP3, CACNA2D2, AGER, EMP2, NKX2-1, and NAPSA) were significantly associated with survival in the adenocarcinoma subgroup, thus qualifying as promising biomarker candidates. In summary, based on an integrated omics approach, we identified genes with elevated expression in lung and localized corresponding protein expression to different cell types. As biomarker candidates, these proteins may represent intriguing starting points for further exploration in health and disease.-Lindskog, C., Fagerberg, L., Hallström, B., Edlund, K., Hellwig, B., Rahnenführer, J., Kampf, C., Uhlén, M., Pontén, F., Micke, P. The lung-specific proteome defined by integration of transcriptomics and antibody-based profiling.
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Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice.
Nat. Med.
PUBLISHED: 08-10-2014
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Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject-derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5' exons of DMD.
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Prognostic and treatment predictive significance of SATB1 and SATB2 expression in pancreatic and periampullary adenocarcinoma.
J Transl Med
PUBLISHED: 08-04-2014
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BackgroundPancreatic cancer and other pancreaticobiliary type periampullary adenocarcinomas have a dismal prognosis even after resection and neoadjuvant chemotherapy. Intestinal type periampullary adenocarcinomas generally have a better prognosis, but little is known on optimal neoadjuvant and adjuvant treatment. New prognostic and treatment predictive biomarkers are needed for improved treatment stratification of patients with both types of periampullary adenocarcinoma. Expression of the Special AT-rich sequence-binding protein 1 (SATB1) has been demonstrated to confer a worse prognosis in several tumour types, whereas its close homologue SATB2 is a proposed diagnostic and favourable prognostic marker for colorectal cancer. The prognostic value of SATB1 and SATB2 expression in periampullary adenocarcinoma has not yet been described.MethodsImmunohistochemical expression of SATB1 and SATB2 was analysed in tissue microarrays with primary tumours and a subset of paired lymph node metastases from 175 patients operated with pancreaticoduodenectomy for periampullary adenocarcinoma. Kaplan-Meier and Cox regression analysis were applied to explore the impact of SATB1 and SATB2 expression on recurrence free survival (RFS) and overall survival (OS).ResultsPositive expression of SATB1 was denoted in 16/106 primary pancreatobiliary type tumours and 11/65 metastases, and in 15/63 primary intestinal type tumours and 4/26 metastases, respectively. Expression of SATB1 was an independent predictor of a significantly shorter RFS and OS in pancreatobiliary type, but not in intestinal type adenocarcinomas. Moreover, SATB1 expression predicted an improved response to adjuvant chemotherapy in both tumour types. SATB2-expression was seen in 3/107 pancreatobiliary type primary tumours, and in 8/61 intestinal type primary tumours. The small number of cases with positive SATB2 expression did not allow for any firm conclusions on its prognostic value.ConclusionsThese findings demonstrate the potential utility of SATB1 as a prognostic and predictive biomarker for chemotherapy response in both intestinal type and pancreatobiliary type periampullary adenocarcinomas, including pancreatic cancer.
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Analysis of candidate genes for lineage-specific expression changes in humans and primates.
J. Proteome Res.
PUBLISHED: 07-15-2014
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RUNX2, a gene involved in skeletal development, has previously been shown to be potentially affected by positive selection during recent human evolution. Here we have used antibody-based proteomics to characterize potential differences in expression patterns of RUNX2 interacting partners during primate evolution. Tissue microarrays consisting of a large set of normal tissues from human and macaque were used for protein profiling of 50 RUNX2 partners with immunohistochemistry. Eleven proteins (AR, CREBBP, EP300, FGF2, HDAC3, JUN, PRKD3, RUNX1, SATB2, TCF3, and YAP1) showed differences in expression between humans and macaques. These proteins were further profiled in tissues from chimpanzee, gorilla, and orangutan, and the corresponding genes were analyzed with regard to genomic features. Moreover, protein expression data were compared with previously obtained RNA sequencing data from six different organs. One gene (TCF3) showed significant expression differences between human and macaque at both the protein and RNA level, with higher expression in a subset of germ cells in human testis compared with macaque. In conclusion, normal tissues from macaque and human showed differences in expression of some RUNX2 partners that could be mapped to various defined cell types. The applied strategy appears advantageous to characterize the consequences of altered genes selected during evolution.
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Peroxiredoxin-1 protects estrogen receptor ? from oxidative stress-induced suppression and is a protein biomarker of favorable prognosis in breast cancer.
Breast Cancer Res.
PUBLISHED: 07-01-2014
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Peroxiredoxin-1 (PRDX1) is a multifunctional protein, acting as a hydrogen peroxide (H2O2) scavenger, molecular chaperone and immune modulator. Although differential PRDX1 expression has been described in many tumors, the potential role of PRDX1 in breast cancer remains highly ambiguous. Using a comprehensive antibody-based proteomics approach, we interrogated PRDX1 protein as a putative biomarker in estrogen receptor (ER)-positive breast cancer.
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Improved production of a heterologous amylase in Saccharomyces cerevisiae by inverse metabolic engineering.
Appl. Environ. Microbiol.
PUBLISHED: 06-27-2014
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The increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeast Saccharomyces cerevisiae is widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. In this study, we applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in the VTA1 gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis.
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Combination of novel HER2-targeting antibody 1E11 with trastuzumab shows synergistic antitumor activity in HER2-positive gastric cancer.
Mol Oncol
PUBLISHED: 06-24-2014
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The synergistic interaction of two antibodies targeting the same protein could be developed as an effective anti-cancer therapy. Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20-25% of breast and gastric cancer patients, and HER2-targeted antibody therapy using trastuzumab is effective in many of these patients. Nonetheless, improving therapeutic efficacy and patient survival is important, particularly in patients with HER2-positive gastric cancer. Here, we describe the development of 1E11, a HER2-targeted humanized monoclonal antibody showing increased efficacy in a highly synergistic manner in combination with trastuzumab in the HER2-overexpressing gastric cancer cell lines NCI-N87 and OE-19. The two antibodies bind to sub-domain IV of the receptor, but have non-overlapping epitopes, allowing them to simultaneously bind HER2. Treatment with 1E11 alone induced apoptosis in HER2-positive cancer cells, and this effect was enhanced by combination treatment with trastuzumab. Combination treatment with 1E11 and trastuzumab reduced the levels of total HER2 protein and those of aberrant HER2 signaling molecules including phosphorylated HER3 and EGFR. The synergistic antitumor activity of 1E11 in combination with trastuzumab indicates that it could be a novel potent therapeutic antibody for the treatment of HER2-overexpressing gastric cancers.
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Affinity proteomics within rare diseases: a BIO-NMD study for blood biomarkers of muscular dystrophies.
EMBO Mol Med
PUBLISHED: 06-13-2014
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Despite the recent progress in the broad-scaled analysis of proteins in body fluids, there is still a lack in protein profiling approaches for biomarkers of rare diseases. Scarcity of samples is the main obstacle hindering attempts to apply discovery driven protein profiling in rare diseases. We addressed this challenge by combining samples collected within the BIO-NMD consortium from four geographically dispersed clinical sites to identify protein markers associated with muscular dystrophy using an antibody bead array platform with 384 antibodies. Based on concordance in statistical significance and confirmatory results obtained from analysis of both serum and plasma, we identified eleven proteins associated with muscular dystrophy, among which four proteins were elevated in blood from muscular dystrophy patients: carbonic anhydrase III (CA3) and myosin light chain 3 (MYL3), both specifically expressed in slow-twitch muscle fibers and mitochondrial malate dehydrogenase 2 (MDH2) and electron transfer flavoprotein A (ETFA). Using age-matched sub-cohorts, 9 protein profiles correlating with disease progression and severity were identified, which hold promise for the development of new clinical tools for management of dystrophinopathies.
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Plasma profiling reveals three proteins associated to amyotrophic lateral sclerosis.
Ann Clin Transl Neurol
PUBLISHED: 05-30-2014
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Amyotrophic lateral sclerosis (ALS) is the most common adult motor neuron disease leading to muscular paralysis and death within 3-5 years from onset. Currently, there are no reliable and sensitive markers able to substantially shorten the diagnosis delay. The objective of the study was to analyze a large number of proteins in plasma from patients with various clinical phenotypes of ALS in search for novel proteins or protein profiles that could serve as potential indicators of disease.
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Reduced expression of ezrin in urothelial bladder cancer signifies more advanced tumours and an impaired survival: validatory study of two independent patient cohorts.
BMC Urol
PUBLISHED: 05-09-2014
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Reduced membranous expression of the cytoskeleton-associated protein ezrin has previously been demonstrated to correlate with tumour progression and poor prognosis in patients with T1G3 urothelial cell carcinoma of the bladder treated with non-maintenance Bacillus Calmette-Guérin (n = 92), and the associations with adverse clinicopathological factors have been validated in another, unselected, cohort (n = 104). In the present study, we examined the prognostic significance of ezrin expression in urothelial bladder cancer in a total number of 442 tumours from two independent patient cohorts.
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SATB1 is an independent prognostic factor in radically resected upper gastrointestinal tract adenocarcinoma.
Virchows Arch.
PUBLISHED: 04-17-2014
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Gastric cancer is the second most common cause of cancer-related death worldwide, and the incidence of esophageal adenocarcinoma is rising. While some progress has been made in treatment strategies, overall survival remains very poor for patients with adenocarcinoma in the upper gastrointestinal tract. Special AT-rich sequence binding protein 1 (SATB1) is a global genome organizer that has been demonstrated to promote aggressive tumor behavior in several different types of cancer, including gastric cancer. The prognostic value of SATB1 expression in esophageal cancer has, however, not yet been described. In this study, expression of SATB1 was examined by immunohistochemistry on tissue microarrays prepared from tissue samples from 175 patients with adenocarcinoma of the esophagus, cardia, or stomach and containing normal tissue, intestinal metaplasia, primary tumors, and metastases. A well-validated antibody was used. We found SATB1 to be an independent prognostic factor in patients with a radically resected tumor, correlating with shorter overall survival as well as with shorter recurrence-free survival. SATB1 expression was also found to be significantly lower in primary tumors associated with intestinal metaplasia than those without intestinal metaplasia. This observation is of potential biological interest as it has been proposed that intestinal metaplasia-associated tumors constitute a less aggressive phenotype.
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Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins.
Mol. Cell Proteomics
PUBLISHED: 04-10-2014
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The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.
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The role of SATB2 as a diagnostic marker for tumors of colorectal origin: Results of a pathology-based clinical prospective study.
Am. J. Clin. Pathol.
PUBLISHED: 04-10-2014
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Immunohistochemistry is an important extension to clinical information and morphology, and prevails as an invaluable tool for establishing a correct cancer diagnosis in clinical diagnostic pathology. The applicability of immunohistochemistry is limited by the availability of validated cell- and cancer-type specific antibodies, rendering an unmet need to discover, test, and validate novel markers. The SATB2 protein is selectively expressed in glandular cells from the lower gastrointestinal tract and expression is retained in a large majority of primary and metastatic colorectal cancers.
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The human gastrointestinal tract-specific transcriptome and proteome as defined by RNA sequencing and antibody-based profiling.
J. Gastroenterol.
PUBLISHED: 04-07-2014
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The gastrointestinal tract (GIT) is subdivided into different anatomical organs with many shared functions and characteristics, but also distinct differences. We have combined a genome-wide transcriptomics analysis with immunohistochemistry-based protein profiling to describe the gene and protein expression patterns that define the human GIT.
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Proteome-wide epitope mapping of antibodies using ultra-dense peptide arrays.
Mol. Cell Proteomics
PUBLISHED: 04-04-2014
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Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody's linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on- and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.
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Characterization of Leukocyte Formin FMNL1 Expression in Human Tissues.
J. Histochem. Cytochem.
PUBLISHED: 04-03-2014
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Formins are cytoskeleton regulating proteins characterized by a common FH2 structural domain. As key players in the assembly of actin filaments, formins direct dynamic cytoskeletal processes that influence cell shape, movement and adhesion. The large number of formin genes, fifteen in the human, suggests distinct tasks and expression patterns for individual family members, in addition to overlapping functions. Several formins have been associated with invasive cell properties in experimental models, linking them to cancer biology. One example is FMNL1, which is considered to be a leukocyte formin and is known to be overexpressed in lymphomas. Studies on FMNL1 and many other formins have been hampered by a lack of research tools, especially antibodies suitable for staining paraffin-embedded formalin-fixed tissues. Here we characterize, using bioinformatics tools and a validated antibody, the expression pattern of FMNL1 in human tissues and study its subcellular distribution. Our results indicate that FMNL1 expression is not restricted to hematopoietic tissues and that neoexpression of FMNL1 can be seen in epithelial cancer.
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Affinity proteomics reveals elevated muscle proteins in plasma of children with cerebral malaria.
PLoS Pathog.
PUBLISHED: 04-01-2014
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Systemic inflammation and sequestration of parasitized erythrocytes are central processes in the pathophysiology of severe Plasmodium falciparum childhood malaria. However, it is still not understood why some children are more at risks to develop malaria complications than others. To identify human proteins in plasma related to childhood malaria syndromes, multiplex antibody suspension bead arrays were employed. Out of the 1,015 proteins analyzed in plasma from more than 700 children, 41 differed between malaria infected children and community controls, whereas 13 discriminated uncomplicated malaria from severe malaria syndromes. Markers of oxidative stress were found related to severe malaria anemia while markers of endothelial activation, platelet adhesion and muscular damage were identified in relation to children with cerebral malaria. These findings suggest the presence of generalized vascular inflammation, vascular wall modulations, activation of endothelium and unbalanced glucose metabolism in severe malaria. The increased levels of specific muscle proteins in plasma implicate potential muscle damage and microvasculature lesions during the course of cerebral malaria.
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HMG-CoA reductase expression in primary colorectal cancer correlates with favourable clinicopathological characteristics and an improved clinical outcome.
Diagn Pathol
PUBLISHED: 03-29-2014
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An association between tumor-specific HMG-CoA reductase (HMGCR) expression and good prognosis has previously been demonstrated in breast and ovarian cancer. In this study, the expression, clinicopathological correlates and prognostic value of HMGCR expression in colorectal cancer was examined.
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Identification of anticancer drugs for hepatocellular carcinoma through personalized genome-scale metabolic modeling.
Mol. Syst. Biol.
PUBLISHED: 03-21-2014
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Genome-scale metabolic models (GEMs) have proven useful as scaffolds for the integration of omics data for understanding the genotype-phenotype relationship in a mechanistic manner. Here, we evaluated the presence/absence of proteins encoded by 15,841 genes in 27 hepatocellular carcinoma (HCC) patients using immunohistochemistry. We used this information to reconstruct personalized GEMs for six HCC patients based on the proteomics data, HMR 2.0, and a task-driven model reconstruction algorithm (tINIT). The personalized GEMs were employed to identify anticancer drugs using the concept of antimetabolites; i.e., drugs that are structural analogs to metabolites. The toxicity of each antimetabolite was predicted by assessing the in silico functionality of 83 healthy cell type-specific GEMs, which were also reconstructed with the tINIT algorithm. We predicted 101 antimetabolites that could be effective in preventing tumor growth in all HCC patients, and 46 antimetabolites which were specific to individual patients. Twenty-two of the 101 predicted antimetabolites have already been used in different cancer treatment strategies, while the remaining antimetabolites represent new potential drugs. Finally, one of the identified targets was validated experimentally, and it was confirmed to attenuate growth of the HepG2 cell line.
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The human liver-specific proteome defined by transcriptomics and antibody-based profiling.
FASEB J.
PUBLISHED: 03-19-2014
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Human liver physiology and the genetic etiology of the liver diseases can potentially be elucidated through the identification of proteins with enriched expression in the liver. Here, we combined data from RNA sequencing (RNA-Seq) and antibody-based immunohistochemistry across all major human tissues to explore the human liver proteome with enriched expression, as well as the cell type-enriched expression in hepatocyte and bile duct cells. We identified in total 477 protein-coding genes with elevated expression in the liver: 179 genes have higher expression as compared to all the other analyzed tissues; 164 genes have elevated transcript levels in the liver shared with at least one other tissue type; and an additional 134 genes have a mild level of increased expression in the liver. We identified the precise localization of these proteins through antibody-based protein profiling and the subcellular localization of these proteins through immunofluorescent-based profiling. We also identified the biological processes and metabolic functions associated with these proteins, investigated their contribution in the occurrence of liver diseases, and identified potential targets for their treatment. Our study demonstrates the use of RNA-Seq and antibody-based immunohistochemistry for characterizing the human liver proteome, as well as the use of tissue-specific proteins in identification of novel drug targets and discovery of biomarkers.-Kampf, C., Mardinoglu, A., Fagerberg, L., Hallström, B. M., Edlund, K., Lundberg, E., Pontén, F., Nielsen, J., Uhlen, M. The human liver-specific proteome defined by transcriptomics and antibody-based profiling.
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RNA- and antibody-based profiling of the human proteome with focus on chromosome 19.
J. Proteome Res.
PUBLISHED: 03-12-2014
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An important part of the Human Proteome Project is to characterize the protein complement of the genome with antibody-based profiling. Within the framework of this effort, a new version 12 of the Human Protein Atlas ( www.proteinatlas.org ) has been launched, including transcriptomics data for 27 tissues and 44 cell lines to complement the protein expression data from antibody-based profiling. Besides the extensive addition of transcriptomics data, the Human Protein Atlas now contains antibody-based protein profiles for 82% of the 20?329 putative protein-coding genes. The comprehensive data resulting from RNA-seq analysis and antibody-based profiling performed within the Human Protein Atlas as well as information from UniProt were used to generate evidence summary scores for each of the 20?329 genes, of which 94% now have experimental evidence at least at transcript level. The evidence scores for all individual genes are displayed with regards to both RNA- and antibody-based protein profiles, including chromosome-centric visualizations. An analysis of the human chromosome 19 shows that ?43% of the genes are expressed at the transcript level in all 27 tissues analyzed, suggesting a "house-keeping" function, while 12% of the genes show a more tissue-specific pattern with enriched expression in one of the analyzed tissues only.
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Neuronal calcium-binding proteins 1/2 localize to dorsal root ganglia and excitatory spinal neurons and are regulated by nerve injury.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-10-2014
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Neuronal calcium (Ca(2+))-binding proteins 1 and 2 (NECAB1/2) are members of the phylogenetically conserved EF-hand Ca(2+)-binding protein superfamily. To date, NECABs have been explored only to a limited extent and, so far, not at all at the spinal level. Here, we describe the distribution, phenotype, and nerve injury-induced regulation of NECAB1/NECAB2 in mouse dorsal root ganglia (DRGs) and spinal cord. In DRGs, NECAB1/2 are expressed in around 70% of mainly small- and medium-sized neurons. Many colocalize with calcitonin gene-related peptide and isolectin B4, and thus represent nociceptors. NECAB1/2 neurons are much more abundant in DRGs than the Ca(2+)-binding proteins (parvalbumin, calbindin, calretinin, and secretagogin) studied to date. In the spinal cord, the NECAB1/2 distribution is mainly complementary. NECAB1 labels interneurons and a plexus of processes in superficial layers of the dorsal horn, commissural neurons in the intermediate area, and motor neurons in the ventral horn. Using CLARITY, a novel, bilaterally connected neuronal system with dendrites that embrace the dorsal columns like palisades is observed. NECAB2 is present in cell bodies and presynaptic boutons across the spinal cord. In the dorsal horn, most NECAB1/2 neurons are glutamatergic. Both NECAB1/2 are transported into dorsal roots and peripheral nerves. Peripheral nerve injury reduces NECAB2, but not NECAB1, expression in DRG neurons. Our study identifies NECAB1/2 as abundant Ca(2+)-binding proteins in pain-related DRG neurons and a variety of spinal systems, providing molecular markers for known and unknown neuron populations of mechanosensory and pain circuits in the spinal cord.
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A chromosome-centric analysis of antibodies directed toward the human proteome using Antibodypedia.
J. Proteome Res.
PUBLISHED: 02-17-2014
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Antibodies are crucial for the study of human proteins and have been defined as one of the three pillars in the human chromosome-centric Human Proteome Project (C-HPP). In this article the chromosome-centric structure has been used to analyze the availability of antibodies as judged by the presence within the portal Antibodypedia, a database designed to allow comparisons and scoring of publicly available antibodies toward human protein targets. This public database displays antibody data from more than one million antibodies toward human protein targets. A summary of the content in this knowledge resource reveals that there exist more than 10 antibodies to over 70% of all the putative human genes, evenly distributed over the 24 human chromosomes. The analysis also shows that at present, less than 10% of the putative human protein-coding genes (n = 1882) predicted from the genome sequence lack antibodies, suggesting that focused efforts from the antibody-based and mass spectrometry-based proteomic communities should be encouraged to pursue the analysis of these missing proteins. We show that Antibodypedia may be used to track the development of available and validated antibodies to the individual chromosomes, and thus the database is an attractive tool to identify proteins with no or few antibodies yet generated.
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Expression and prognostic significance of the polymeric immunoglobulin receptor in esophageal and gastric adenocarcinoma.
J Transl Med
PUBLISHED: 01-25-2014
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The polymeric immunoglobulin receptor (PIGR) has been proposed to be a candidate prognostic biomarker in a few cancer forms, and one previous study reported that reduced PIGR expression signifies more aggressive tumours of the distal esophagus and gastroesophageal junction (GEJ). In the present study, we examined the expression, clinicopathological correlates and prognostic significance of PIGR expression in an extended cohort of adenocarcinoma of the upper gastrointestinal tract.
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Expression and prognostic significance of the polymeric immunoglobulin receptor in epithelial ovarian cancer.
J Ovarian Res
PUBLISHED: 01-20-2014
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High expression of the polymeric immunoglobulin receptor (PIGR) has previously been associated with a favourable prognosis in a few cancer forms, but its expression and relationship with clinical outcome in epithelial ovarian cancer (EOC) has not yet been reported. The aim of this study was therefore to examine the clinicopathological correlates and prognostic significance of PIGR expression in EOC.
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Genome-scale metabolic modelling of hepatocytes reveals serine deficiency in patients with non-alcoholic fatty liver disease.
Nat Commun
PUBLISHED: 01-15-2014
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Several liver disorders result from perturbations in the metabolism of hepatocytes, and their underlying mechanisms can be outlined through the use of genome-scale metabolic models (GEMs). Here we reconstruct a consensus GEM for hepatocytes, which we call iHepatocytes2322, that extends previous models by including an extensive description of lipid metabolism. We build iHepatocytes2322 using Human Metabolic Reaction 2.0 database and proteomics data in Human Protein Atlas, which experimentally validates the incorporated reactions. The reconstruction process enables improved annotation of the proteomics data using the network centric view of iHepatocytes2322. We then use iHepatocytes2322 to analyse transcriptomics data obtained from patients with non-alcoholic fatty liver disease. We show that blood concentrations of chondroitin and heparan sulphates are suitable for diagnosing non-alcoholic steatohepatitis and for the staging of non-alcoholic fatty liver disease. Furthermore, we observe serine deficiency in patients with NASH and identify PSPH, SHMT1 and BCAT1 as potential therapeutic targets for the treatment of non-alcoholic steatohepatitis.
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Antibody performance in western blot applications is context-dependent.
Biotechnol J
PUBLISHED: 01-03-2014
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An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well-designed conditions. Here, we have analyzed 13?000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application-specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full-length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full-length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context- and application-dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross-reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data.
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Reduced expression of the polymeric immunoglobulin receptor in pancreatic and periampullary adenocarcinoma signifies tumour progression and poor prognosis.
PLoS ONE
PUBLISHED: 01-01-2014
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The polymeric immunoglobulin receptor (pIgR) is a key component of the mucosal immune system that mediates epithelial transcytosis of immunoglobulins. High pIgR expression has been reported to correlate with a less aggressive tumour phenotype and an improved prognosis in several human cancer types. Here, we examined the expression and prognostic significance of pIgR in pancreatic and periampullary adenocarcinoma. The study cohort encompasses a consecutive series of 175 patients surgically treated with pancreaticoduodenectomy for pancreatic and periampullary adenocarcinoma in Malmö and Lund University Hospitals, Sweden, between 2001-2011. Tissue microarrays were constructed from primary tumours (n?=?175) and paired lymph node metastases (n?=?105). A multiplied score was calculated from the fraction and intensity of pIgR staining. Classification and regression tree analysis was used to select the prognostic cut-off. Unadjusted and adjusted hazard ratios (HR) for death and recurrence within 5 years were calculated. pIgR expression could be evaluated in 172/175 (98.3%) primary tumours and in 96/105 (91.4%) lymph node metastases. pIgR expression was significantly down-regulated in lymph node metastases as compared with primary tumours (p?=?0.018). Low pIgR expression was significantly associated with poor differentiation grade (p<0.001), perineural growth (p?=?0.027), lymphatic invasion (p?=?0.016), vascular invasion (p?=?0.033) and infiltration of the peripancreatic fat (p?=?0.039). In the entire cohort, low pIgR expression was significantly associated with an impaired 5-year survival (HR?=?2.99, 95% confidence interval (CI) 1.71-5.25) and early recurrence (HR?=?2.89, 95% CI 1.67-4.98). This association remained significant for survival after adjustment for conventional clinicopathological factors, tumour origin and adjuvant treatment (HR?=?1.98, 95% CI 1.10-3.57). These results demonstrate, for the first time, that high tumour-specific pIgR expression signifies a more favourable tumour phenotype and that low expression independently predicts a shorter survival in patients with pancreatic and periampullary cancer. The mechanistic basis for the putative tumour suppressing properties of pIgR in these cancers merits further study.
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High expression of RNA-binding motif protein 3 in esophageal and gastric adenocarcinoma correlates with intestinal metaplasia-associated tumours and independently predicts a reduced risk of recurrence and death.
Biomark Res
PUBLISHED: 01-01-2014
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High nuclear expression of the RNA-binding motif protein 3 (RBM3) has previously been found to correlate with favourable clinicopathological characteristics and a prolonged survival in several cancer forms. Here, we examined the clinicopathological correlates and prognostic significance of RBM3 expression in tumours from a consecutive cohort of upper gastrointestinal adenocarcinoma.
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Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics.
Mol. Cell Proteomics
PUBLISHED: 12-05-2013
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Global classification of the human proteins with regards to spatial expression patterns across organs and tissues is important for studies of human biology and disease. Here, we used a quantitative transcriptomics analysis (RNA-Seq) to classify the tissue-specific expression of genes across a representative set of all major human organs and tissues and combined this analysis with antibody-based profiling of the same tissues. To present the data, we launch a new version of the Human Protein Atlas that integrates RNA and protein expression data corresponding to approximately 80% of the human protein-coding genes with access to the primary data for both the RNA and the protein analysis on an individual gene level. We present a classification of all human protein-coding genes with regards to tissue-specificity and spatial expression pattern. The integrative human expression map can be used as a starting point to explore the molecular constituents of the human body.
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Using transcriptomics to improve butanol tolerance of Synechocystis sp. strain PCC 6803.
Appl. Environ. Microbiol.
PUBLISHED: 09-20-2013
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Cyanobacteria are emerging as promising hosts for production of advanced biofuels such as n-butanol and alkanes. However, cyanobacteria suffer from the same product inhibition problems as those that plague other microbial biofuel hosts. High concentrations of butanol severely reduce growth, and even small amounts can negatively affect metabolic processes. An understanding of how cyanobacteria are affected by their biofuel product can enable identification of engineering strategies for improving their tolerance. Here we used transcriptome sequencing (RNA-Seq) to assess the transcriptome response of Synechocystis sp. strain PCC 6803 to two concentrations of exogenous n-butanol. Approximately 80 transcripts were differentially expressed at 40 mg/liter butanol, and 280 transcripts were different at 1 g/liter butanol. Our results suggest a compromised cell membrane, impaired photosynthetic electron transport, and reduced biosynthesis. Accumulation of intracellular reactive oxygen species (ROS) scaled with butanol concentration. Using the physiology and transcriptomics data, we selected several genes for overexpression in an attempt to improve butanol tolerance. We found that overexpression of several proteins, notably, the small heat shock protein HspA, improved tolerance to butanol. Transcriptomics-guided engineering created more solvent-tolerant cyanobacteria strains that could be the foundation for a more productive biofuel host.
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Integration of cardiac proteome biology and medicine by a specialized knowledgebase.
Circ. Res.
PUBLISHED: 08-21-2013
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Omics sciences enable a systems-level perspective in characterizing cardiovascular biology. Integration of diverse proteomics data via a computational strategy will catalyze the assembly of contextualized knowledge, foster discoveries through multidisciplinary investigations, and minimize unnecessary redundancy in research efforts.
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Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody.
N Biotechnol
PUBLISHED: 07-11-2013
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One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues.
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Autoantibody profiling in multiple sclerosis using arrays of human protein fragments.
Mol. Cell Proteomics
PUBLISHED: 06-03-2013
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Profiling the autoantibody repertoire with large antigen collections is emerging as a powerful tool for the identification of biomarkers for autoimmune diseases. Here, a systematic and undirected approach was taken to screen for profiles of IgG in human plasma from 90 individuals with multiple sclerosis related diagnoses. Reactivity pattern of 11,520 protein fragments (representing ?38% of all human protein encoding genes) were generated on planar protein microarrays built within the Human Protein Atlas. For more than 2,000 antigens IgG reactivity was observed, among which 64% were found only in single individuals. We used reactivity distributions among multiple sclerosis subgroups to select 384 antigens, which were then re-evaluated on planar microarrays, corroborated with suspension bead arrays in a larger cohort (n = 376) and confirmed for specificity in inhibition assays. Among the heterogeneous pattern within and across multiple sclerosis subtypes, differences in recognition frequencies were found for 51 antigens, which were enriched for proteins of transcriptional regulation. In conclusion, using protein fragments and complementary high-throughput protein array platforms facilitated an alternative route to discovery and verification of potentially disease-associated autoimmunity signatures, that are now proposed as additional antigens for large-scale validation studies across multiple sclerosis biobanks.
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A systematic analysis of commonly used antibodies in cancer diagnostics.
Histopathology
PUBLISHED: 05-14-2013
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Immunohistochemistry plays a pivotal role in cancer differential diagnostics. To identify the primary tumour from a metastasis specimen remains a significant challenge, despite the availability of an increasing number of antibodies. The aim of the present study was to provide evidence-based data on the diagnostic power of antibodies used frequently for clinical differential diagnostics.
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Psychiatric patient stratification using biosignatures based on cerebrospinal fluid protein expression clusters.
J Psychiatr Res
PUBLISHED: 05-13-2013
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Psychiatric disorders are caused by perturbed molecular pathways that affect brain circuitries. The identification of specific biosignatures that are the result of altered pathway activities in major depression, bipolar disorder and schizophrenia can contribute to a better understanding of disease etiology and aid in the implementation of diagnostic assays. In the present study we identified disease-specific protein biosignatures in cerebrospinal fluid of depressed (n: 36), bipolar (n: 27) and schizophrenic (n: 35) patients using the Reverse Phase Protein Microarray technology. These biosignatures were able to stratify patient groups in an objective manner according to cerebrospinal fluid protein expression patterns. Correct classification rates were over 90%. At the same time several protein sets that play a role in neuronal growth, proliferation and differentiation (NEGR1, NPDC1), neurotransmission (SEZ6) and protection from oxidative damage (GPX3) were able to distinguish diseased from healthy individuals (n: 35) indicating a molecular signature overlap for the different psychiatric phenotypes. Our study is a first step toward implementing a psychiatric patient stratification system based on molecular biosignatures. Protein signatures may eventually be of use as specific and sensitive biomarkers in clinical trials not only for patient diagnostic and subgroup stratification but also to follow treatment response.
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CDK-mediated activation of the SCF(FBXO) (28) ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer.
EMBO Mol Med
PUBLISHED: 05-09-2013
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SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCF(FBXO28) activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCF(FBXO28) plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer.
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Podocalyxin-like protein expression in primary colorectal cancer and synchronous lymph node metastases.
Diagn Pathol
PUBLISHED: 04-09-2013
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Previous studies have shown that membranous expression of podocalyxin-like protein (PODXL) is associated with poor prognosis in colorectal cancer (CRC). In this study, we compared PODXL expression in primary CRC and synchronous lymph node metastases. We further analyzed whether its expression changed in rectal tumours after neoadjuvant radiation therapy.
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Majority of differentially expressed genes are down-regulated during malignant transformation in a four-stage model.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-08-2013
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The transformation of normal cells to malignant, metastatic tumor cells is a multistep process caused by the sequential acquirement of genetic changes. To identify these changes, we compared the transcriptomes and levels and distribution of proteins in a four-stage cell model of isogenically matched normal, immortalized, transformed, and metastatic human cells, using deep transcriptome sequencing and immunofluorescence microscopy. The data show that ?6% (n = 1,357) of the human protein-coding genes are differentially expressed across the stages in the model. Interestingly, the majority of these genes are down-regulated, linking malignant transformation to dedifferentiation. The up-regulated genes are mainly components that control cellular proliferation, whereas the down-regulated genes consist of proteins exposed on or secreted from the cell surface. As many of the identified gene products control basic cellular functions that are defective in cancers, the data provide candidates for follow-up studies to investigate their functional roles in tumor formation. When we further compared the expression levels of four of the identified proteins in clinical cancer cohorts, similar differences were observed between benign and cancer cells, as in the cell model. This shows that this comprehensive demonstration of the molecular changes underlying malignant transformation is a relevant model to study the process of tumor formation.
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Profiling post-centrifugation delay of serum and plasma with antibody bead arrays.
J Proteomics
PUBLISHED: 03-22-2013
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Several biobanking initiatives have emerged to create extensive collections of specimen for biomedical studies and various analytical platforms. An affinity proteomic analysis with antibody suspension bead arrays was conducted to investigate the influence of the pre-analytical time and temperature conditions on blood derived samples. Serum and EDTA plasma prepared from 16 individuals was centrifuged and aliquots were kept either at 4°C or in ambient temperature for 1h and up to 36h prior to first storage. Multiplexed protein profiles of post-centrifugation delay were generated in 384 biotinylated samples using 373 antibodies that targeted 343 unique proteins. Very few profiles were observed as significantly altered by the studied temperature and time intervals. Single binder and sandwich assays revealed decreasing levels of caldesmon 1 (CALD1) related to EDTA standard tubes and prolonged post-centrifugation delay of 36h. Indications from changes in CALD1 levels require further confirmation in independent material, but the current data suggests that samples should preferentially be frozen during the day of collection when to be profiled with antibody arrays selected for this study.
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Antibody-based profiling of cerebrospinal fluid within multiple sclerosis.
Proteomics
PUBLISHED: 03-19-2013
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Antibody suspension bead arrays have proven to enable multiplexed and high-throughput protein profiling in unfractionated plasma and serum samples through a direct labeling approach. We here describe the development and application of an assay for protein profiling of cerebrospinal fluid (CSF). While setting up the assay, systematic intensity differences between sample groups were observed that reflected inherent sample specific total protein amounts. Supplementing the labeling reaction with BSA and IgG diminished these differences without impairing the apparent sensitivity of the assay. We also assessed the effects of heat treatment on the analysis of CSF proteins and applied the assay to profile 43 selected proteins by 101 antibodies in 339 CSF samples from a multiple sclerosis (MS) cohort. Two proteins, GAP43 and SERPINA3 were found to have a discriminating potential with altered intensity levels between sample groups. GAP43 was detected at significantly lower levels in secondary progressive MS compared to early stages of MS and the control group of other neurological diseases. SERPINA3 instead was detected at higher levels in all MS patients compared to controls. The developed assay procedure now offers new possibilities for broad-scale protein profiling of CSF within neurological disorders.
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Systematic antibody generation and validation via tissue microarray technology leading to identification of a novel protein prognostic panel in breast cancer.
BMC Cancer
PUBLISHED: 03-19-2013
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Although omic-based discovery approaches can provide powerful tools for biomarker identification, several reservations have been raised regarding the clinical applicability of gene expression studies, such as their prohibitive cost. However, the limited availability of antibodies is a key barrier to the development of a lower cost alternative, namely a discrete collection of immunohistochemistry (IHC)-based biomarkers. The aim of this study was to use a systematic approach to generate and screen affinity-purified, mono-specific antibodies targeting progression-related biomarkers, with a view towards developing a clinically applicable IHC-based prognostic biomarker panel for breast cancer.
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Targeting HMG-CoA reductase with statins in a window-of-opportunity breast cancer trial.
Breast Cancer Res. Treat.
PUBLISHED: 02-28-2013
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Lipophilic statins purportedly exert anti-tumoral effects on breast cancer by decreasing proliferation and increasing apoptosis. HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway, is the target of statins. However, data on statin-induced effects on HMGCR activity in cancer are limited. Thus, this pre-operative study investigated statin-induced effects on tumor proliferation and HMGCR expression while analyzing HMGCR as a predictive marker for statin response in breast cancer treatment. The study was designed as a window-of-opportunity trial and included 50 patients with primary invasive breast cancer. High-dose atorvastatin (i.e., 80 mg/day) was prescribed to patients for 2 weeks before surgery. Pre- and post-statin paired tumor samples were analyzed for Ki67 and HMGCR immunohistochemical expression. Changes in the Ki67 expression and HMGCR activity following statin treatment were the primary and secondary endpoints, respectively. Up-regulation of HMGCR following atorvastatin treatment was observed in 68 % of the paired samples with evaluable HMGCR expression (P = 0.0005). The average relative decrease in Ki67 expression following atorvastatin treatment was 7.6 % (P = 0.39) in all paired samples, whereas the corresponding decrease in Ki67 expression in tumors expressing HMGCR in the pre-treatment sample was 24 % (P = 0.02). Furthermore, post-treatment Ki67 expression was inversely correlated to post-treatment HMGCR expression (rs = -0.42; P = 0.03). Findings from this study suggest that HMGCR is targeted by statins in breast cancer cells in vivo, and that statins may have an anti-proliferative effect in HMGCR-positive tumors. Future studies are needed to evaluate HMGCR as a predictive marker for the selection of breast cancer patients who may benefit from statin treatment.
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Initial quantitative proteomic map of 28 mouse tissues using the SILAC mouse.
Mol. Cell Proteomics
PUBLISHED: 02-22-2013
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Identifying the building blocks of mammalian tissues is a precondition for understanding their function. In particular, global and quantitative analysis of the proteome of mammalian tissues would point to tissue-specific mechanisms and place the function of each protein in a whole-organism perspective. We performed proteomic analyses of 28 mouse tissues using high-resolution mass spectrometry and used a mix of mouse tissues labeled via stable isotope labeling with amino acids in cell culture as a "spike-in" internal standard for accurate protein quantification across these tissues. We identified a total of 7,349 proteins and quantified 6,974 of them. Bioinformatic data analysis showed that physiologically related tissues clustered together and that highly expressed proteins represented the characteristic tissue functions. Tissue specialization was reflected prominently in the proteomic profiles and is apparent already in their hundred most abundant proteins. The proportion of strictly tissue-specific proteins appeared to be small. However, even proteins with household functions, such as those in ribosomes and spliceosomes, can have dramatic expression differences among tissues. We describe a computational framework with which to correlate proteome profiles with physiological functions of the tissue. Our data will be useful to the broad scientific community as an initial atlas of protein expression of a mammalian species.
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Integration of clinical data with a genome-scale metabolic model of the human adipocyte.
Mol. Syst. Biol.
PUBLISHED: 02-11-2013
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We evaluated the presence/absence of proteins encoded by 14?077 genes in adipocytes obtained from different tissue samples using immunohistochemistry. By combining this with previously published adipocyte-specific proteome data, we identified proteins associated with 7340 genes in human adipocytes. This information was used to reconstruct a comprehensive and functional genome-scale metabolic model of adipocyte metabolism. The resulting metabolic model, iAdipocytes1809, enables mechanistic insights into adipocyte metabolism on a genome-wide level, and can serve as a scaffold for integration of omics data to understand the genotype-phenotype relationship in obese subjects. By integrating human transcriptome and fluxome data, we found an increase in the metabolic activity around androsterone, ganglioside GM2 and degradation products of heparan sulfate and keratan sulfate, and a decrease in mitochondrial metabolic activities in obese subjects compared with lean subjects. Our study hereby shows a path to identify new therapeutic targets for treating obesity through combination of high throughput patient data and metabolic modeling.
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Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells.
Nat. Methods
PUBLISHED: 01-25-2013
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Imaging techniques such as immunofluorescence (IF) and the expression of fluorescent protein (FP) fusions are widely used to investigate the subcellular distribution of proteins. Here we report a systematic analysis of >500 human proteins comparing the localizations obtained in live versus fixed cells using FPs and IF, respectively. We identify systematic discrepancies between IF and FPs as well as between FP tagging at the N and C termini. The analysis shows that for 80% of the proteins, IF and FPs yield the same subcellular distribution, and the locations of 250 previously unlocalized proteins were determined by the overlap between the two methods. Approximately 60% of proteins localize to multiple organelles for both methods, indicating a complex subcellular protein organization. These results show that both IF and FP tagging are reliable techniques and demonstrate the usefulness of an integrative approach for a complete investigation of the subcellular human proteome.
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Decreased expression of RNA-binding motif protein 3 correlates with tumour progression and poor prognosis in urothelial bladder cancer.
BMC Urol
PUBLISHED: 01-20-2013
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Low nuclear expression of the RNA-binding motif protein 3 (RBM3) has previously been found to be associated with poor prognosis in several cancer forms e.g. breast, ovarian, colorectal, prostate cancer and malignant melanoma. The aim of this study was to examine the prognostic impact of RBM3 expression in urinary bladder cancer.
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Selectivity analysis of single binder assays used in plasma protein profiling.
Proteomics
PUBLISHED: 01-17-2013
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The increasing availability of antibodies toward human proteins enables broad explorations of the proteomic landscape in cells, tissues, and body fluids. This includes assays with antibody suspension bead arrays that generate protein profiles of plasma samples by flow cytometer analysis. However, antibody selectivity is context dependent so it is necessary to corroborate on-target detection over off-target binding. To address this, we describe a concept to directly verify interactions from antibody-coupled beads by analysis of their eluates by Western blots and MS. We demonstrate selective antibody binding in complex samples with antibodies toward a set of chosen proteins with different abundance in plasma and serum, and illustrate the need to adjust sample and bead concentrations accordingly. The presented approach will serve as an important tool for resolving differential protein profiles from antibody arrays within plasma biomarker discoveries.
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Contribution of antibody-based protein profiling to the human Chromosome-centric Proteome Project (C-HPP).
J. Proteome Res.
PUBLISHED: 01-02-2013
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A gene-centric Human Proteome Project has been proposed to characterize the human protein-coding genes in a chromosome-centered manner to understand human biology and disease. Here, we report on the protein evidence for all genes predicted from the genome sequence based on manual annotation from literature (UniProt), antibody-based profiling in cells, tissues and organs and analysis of the transcript profiles using next generation sequencing in human cell lines of different origins. We estimate that there is good evidence for protein existence for 69% (n = 13985) of the human protein-coding genes, while 23% have only evidence on the RNA level and 7% still lack experimental evidence. Analysis of the expression patterns shows few tissue-specific proteins and approximately half of the genes expressed in all the analyzed cells. The status for each gene with regards to protein evidence is visualized in a chromosome-centric manner as part of a new version of the Human Protein Atlas ( www.proteinatlas.org ).
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Robust Expression of the Human Neonatal Fc Receptor in a Truncated Soluble Form and as a Full-Length Membrane-Bound Protein in Fusion with eGFP.
PLoS ONE
PUBLISHED: 01-01-2013
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Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.
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FHOD1, a Formin Upregulated in Epithelial-Mesenchymal Transition, Participates in Cancer Cell Migration and Invasion.
PLoS ONE
PUBLISHED: 01-01-2013
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Cancer cells can obtain their ability to invade and metastasise by undergoing epithelial-to-mesenchymal transition (EMT). Exploiting this mechanism of cellular plasticity, malignant cells can remodel their actin cytoskeleton and down-regulate proteins needed for cell-cell contacts. The mechanisms of cytoskeletal reorganisation resulting in mesenchymal morphology and increased invasive potential are poorly understood. Actin nucleating formins have been implicated as key players in EMT. Here, we analysed which formins are altered in squamous cell carcinoma related EMT. FHOD1, a poorly studied formin, appeared to be markedly upregulated upon EMT. In human tissues FHOD1 was primarily expressed in mesenchymal cells, with little expression in epithelia. However, specimens from oral squamous cell cancers demonstrated consistent FHOD1 upregulation in mesenchymally transformed cells at the invasive edge. This upregulation was confirmed in an oral squamous carcinoma model, where FHOD1 expression was markedly increased upon EMT in a PI3K signalling dependent manner. In the EMT cells FHOD1 contributed to the spindle-shaped morphology and mesenchymal F-actin organization. Furthermore, functional assays demonstrated that FHOD1 contributes to cell migration and invasion. Finally, FHOD1 depletion reduced the ability of EMT cancer cells to form invadopodia and to degrade extracellular matrix. Our results indicate that FHOD1 participates in cytoskeletal changes in EMT. In addition, we show that FHOD1 upregulation occurs during cancer cell EMT in vivo, which indicates that FHOD1 may contribute to tumour progression.
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Nuclear T-STAR protein expression correlates with HER2 status, hormone receptor negativity and prolonged recurrence free survival in primary breast cancer and decreased cancer cell growth in vitro.
PLoS ONE
PUBLISHED: 01-01-2013
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T-STAR (testis-signal transduction and activation of RNA) is an RNA binding protein, containing an SH3-binding domain and thus potentially playing a role in integration of cell signaling and RNA metabolism. The specific function of T-STAR is unknown and its implication in cancer is poorly characterized. Expression of T-STAR has been reported in human testis, muscle and brain tissues, and is associated with a growth-inhibitory role in immortalized fibroblasts. The aim of this paper was to investigate the functional role of T-STAR through (i) survival analysis of patients with primary invasive breast cancer and (ii) experimental evaluation of the effect of T-STAR on breast cancer cell growth. T-STAR protein expression was analysed by immunohistochemistry (IHC) in tissue microarrays with tumors from 289 patients with primary invasive breast cancer, and correlations to clinicopathological characteristics, recurrence-free and overall survival (RFS and OS) and established tumor markers such as HER2 and ER status were evaluated. In addition, the function of T-STAR was investigated using siRNA-mediated knock-down and overexpression of the gene in six breast cancer cell lines. Of the tumors analysed, 86% showed nuclear T-STAR expression, which was significantly associated with an improved RFS and strongly associated with positive HER2 status and negative hormone receptor status. Furthermore, experimental data showed that overexpression of T-STAR decreased cellular growth while knock-down increased it, as shown both by thymidine incorporation and metabolic activity. In summary, we demonstrate that T-STAR protein expression correlates with an improved RFS in primary breast cancer. This is supported by functional data, indicating that T-STAR regulation is of importance both for breast cancer biology and clinical outcome but future studies are needed to determine a potential role in patient stratification.
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A texture based pattern recognition approach to distinguish melanoma from non-melanoma cells in histopathological tissue microarray sections.
PLoS ONE
PUBLISHED: 01-01-2013
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Immunohistochemistry is a routine practice in clinical cancer diagnostics and also an established technology for tissue-based research regarding biomarker discovery efforts. Tedious manual assessment of immunohistochemically stained tissue needs to be fully automated to take full advantage of the potential for high throughput analyses enabled by tissue microarrays and digital pathology. Such automated tools also need to be reproducible for different experimental conditions and biomarker targets. In this study we present a novel supervised melanoma specific pattern recognition approach that is fully automated and quantitative.
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Antibody-based protein profiling of the human chromosome 21.
Mol. Cell Proteomics
PUBLISHED: 10-31-2011
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The Human Proteome Project has been proposed to create a knowledge-based resource based on a systematical mapping of all human proteins, chromosome by chromosome, in a gene-centric manner. With this background, we here describe the systematic analysis of chromosome 21 using an antibody-based approach for protein profiling using both confocal microscopy and immunohistochemistry, complemented with transcript profiling using next generation sequencing data. We also describe a new approach for protein isoform analysis using a combination of antibody-based probing and isoelectric focusing. The analysis has identified several genes on chromosome 21 with no previous evidence on the protein level, and the isoform analysis indicates that a large fraction of human proteins have multiple isoforms. A chromosome-wide matrix is presented with status for all chromosome 21 genes regarding subcellular localization, tissue distribution, and molecular characterization of the corresponding proteins. The path to generate a chromosome-specific resource, including integrated data from complementary assay platforms, such as mass spectrometry and gene tagging analysis, is discussed.
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Characterization of MRFAP1 turnover and interactions downstream of the NEDD8 pathway.
Mol. Cell Proteomics
PUBLISHED: 10-29-2011
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The NEDD8-Cullin E3 ligase pathway plays an important role in protein homeostasis, in particular the degradation of cell cycle regulators and transcriptional control networks. To characterize NEDD8-cullin target proteins, we performed a quantitative proteomic analysis of cells treated with MLN4924, a small molecule inhibitor of the NEDD8 conjugation pathway. MRFAP1 and its interaction partner, MORF4L1, were among the most up-regulated proteins after NEDD8 inhibition in multiple human cell lines. We show that MRFAP1 has a fast turnover rate in the absence of MLN4924 and is degraded via the ubiquitin-proteasome system. The increased abundance of MRFAP1 after MLN4924 treatment results from a decreased rate of degradation. Characterization of the binding partners of both MRFAP1 and MORF4L1 revealed a complex protein-protein interaction network. MRFAP1 bound to a number of E3 ubiquitin ligases, including CUL4B, but not to components of the NuA4 complex, including MRGBP, which bound to MORF4L1. These data indicate that MRFAP1 may regulate the ability of MORF4L1 to interact with chromatin-modifying enzymes by binding to MORF4L1 in a mutually exclusive manner with MRGBP. Analysis of MRFAP1 expression in human tissues by immunostaining with a MRFAP1-specific antibody revealed that it was detectable in only a small number of tissues, in particular testis and brain. Strikingly, analysis of the seminiferous tubules of the testis showed the highest nuclear staining in the spermatogonia and much weaker staining in the spermatocytes and spermatids. MRGBP was inversely correlated with MRFAP1 expression in these cell types, consistent with an exchange of MORF4L1 interaction partners as cells progress through meiosis in the testis. These data highlight an important new arm of the NEDD8-cullin pathway.
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Hypothalamic mitochondrial dysfunction associated with anorexia in the anx/anx mouse.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 10-24-2011
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The anorectic anx/anx mouse exhibits disturbed feeding behavior and aberrances, including neurodegeneration, in peptidergic neurons in the appetite regulating hypothalamic arcuate nucleus. Poor feeding in infants, as well as neurodegeneration, are common phenotypes in human disorders caused by dysfunction of the mitochondrial oxidative phosphorylation system (OXPHOS). We therefore hypothesized that the anorexia and degenerative phenotypes in the anx/anx mouse could be related to defects in the OXPHOS. In this study, we found reduced efficiency of hypothalamic OXPHOS complex I assembly and activity in the anx/anx mouse. We also recorded signs of increased oxidative stress in anx/anx hypothalamus, possibly as an effect of the decreased hypothalamic levels of fully assembled complex I, that were demonstrated by native Western blots. Furthermore, the Ndufaf1 gene, encoding a complex I assembly factor, was genetically mapped to the anx interval and found to be down-regulated in anx/anx mice. These results suggest that the anorexia and hypothalamic neurodegeneration of the anx/anx mouse are associated with dysfunction of mitochondrial complex I.
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Systematic analysis of protein pools, isoforms, and modifications affecting turnover and subcellular localization.
Mol. Cell Proteomics
PUBLISHED: 10-16-2011
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In higher eukaryotes many genes encode protein isoforms whose properties and biological roles are often poorly characterized. Here we describe systematic approaches for detection of either distinct isoforms, or separate pools of the same isoform, with differential biological properties. Using information from ion intensities we have estimated protein abundance levels and using rates of change in stable isotope labeling with amino acids in cell culture isotope ratios we measured turnover rates and subcellular distribution for the HeLa cell proteome. Protein isoforms were detected using three data analysis strategies that evaluate differences between stable isotope labeling with amino acids in cell culture isotope ratios for specific groups of peptides within the total set of peptides assigned to a protein. The candidate approach compares stable isotope labeling with amino acids in cell culture isotope ratios for predicted isoform-specific peptides, with ratio values for peptides shared by all the isoforms. The rule of thirds approach compares the mean isotope ratio values for all peptides in each of three equal segments along the linear length of the protein, assessing differences between segment values. The three in a row approach compares mean isotope ratio values for each sequential group of three adjacent peptides, assessing differences with the mean value for all peptides assigned to the protein. Protein isoforms were also detected and their properties evaluated by fractionating cell extracts on one-dimensional SDS-PAGE prior to trypsin digestion and MS analysis and independently evaluating isotope ratio values for the same peptides isolated from different gel slices. The effect of protein phosphorylation on turnover rates was analyzed by comparing mean turnover values calculated for all peptides assigned to a protein, either including, or excluding, values for cognate phosphopeptides. Collectively, these experimental and analytical approaches provide a framework for expanding the functional annotation of the genome.
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A Protein Epitope Signature Tag (PrEST) library allows SILAC-based absolute quantification and multiplexed determination of protein copy numbers in cell lines.
Mol. Cell Proteomics
PUBLISHED: 09-30-2011
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Mass spectrometry-based proteomics increasingly relies on relative or absolute quantification. In relative quantification, stable isotope based methods often allow mixing at early stages of sample preparation, whereas for absolute quantification this has generally required recombinant expression of full length, labeled protein standards. Here we make use of a very large library of Protein Epitope Signature Tags (PrESTs) that has been developed in the course of the Human Protein Atlas Project. These PrESTs are expressed recombinantly in E. coli and they consist of a short and unique region of the protein of interest as well as purification and solubility tags. We first quantify a highly purified, stable isotope labeling of amino acids in cell culture (SILAC)-labeled version of the solubility tag and use it determine the precise amount of each PrEST by its SILAC ratios. The PrESTs are then spiked into cell lysates and the SILAC ratios of PrEST peptides to peptides from endogenous target proteins yield their cellular quantities. The procedure can readily be multiplexed, as we demonstrate by simultaneously determining the copy number of 40 proteins in HeLa cells. Among the proteins analyzed, the cytoskeletal protein vimentin was found to be most abundant with 20 million copies per cell, while the transcription factor and oncogene FOS only had 6000 copies. Direct quantification of the absolute amount of single proteins is possible via a SILAC experiment in which labeled cell lysate is mixed both with the heavy labeled solubility tag and with the corresponding PrEST. The SILAC-PrEST combination allows accurate and streamlined quantification of the absolute or relative amount of proteins of interest in a wide variety of applications.
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High nuclear RBM3 expression is associated with an improved prognosis in colorectal cancer.
Proteomics Clin Appl
PUBLISHED: 09-30-2011
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In this study, we investigated the prognostic impact of human RBM3 expression in colorectal cancer using tissue microarray-based immunohistochemical analysis.
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Plasma profiling reveals human fibulin-1 as candidate marker for renal impairment.
J. Proteome Res.
PUBLISHED: 09-29-2011
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There is a need for reliable and sensitive biomarkers for renal impairments to detect early signs of kidney toxicity and to monitor progression of disease. Here, antibody suspension bead arrays were applied to profile plasma samples from patients with four types of kidney disorders: glomerulonephritis, diabetic nephropathy, obstructive uropathy, and analgesic abuse. In total, 200 clinical renal-associated cases and control plasma samples from different cohorts were profiled. Parallel plasma protein profiles were obtained using biotinylated and nonfractionated samples and a selected set of 94 proteins targeted by 129 antigen-purified polyclonal antibodies. Out of the analyzed target proteins, human fibulin-1 was detected at significantly higher levels in the glomerulonephritis patient group compared to the controls and with elevated levels in patient samples for all other renal disorders investigated. Two polyclonal antibodies and one monoclonal antibody directed toward separate, nonoverlapping epitopes showed the same trend in the discovery cohorts. A technical verification using Western blot analysis of selected patient plasma confirmed the trends toward higher abundance of the target protein in disease samples. Furthermore, a verification study was carried out in the context of glomerulonephritis using an independent case and control cohort, and this confirmed the results from the discovery cohort, suggesting that plasma levels of fibulin-1 could serve as a potential indicator to monitor kidney malfunction or kidney damage.
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Glcci1 deficiency leads to proteinuria.
J. Am. Soc. Nephrol.
PUBLISHED: 09-23-2011
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Unbiased transcriptome profiling and functional genomics approaches identified glucocorticoid-induced transcript 1 (GLCCI1) as being a transcript highly specific for the glomerulus, but its role in glomerular development and disease is unknown. Here, we report that mouse glomeruli express far greater amounts of Glcci1 protein compared with the rest of the kidney. RT-PCR and Western blotting demonstrated that mouse glomerular Glcci1 is approximately 60 kD and localizes to the cytoplasm of podocytes in mature glomeruli. In the fetal kidney, intense Glcci1 expression occurs at the capillary-loop stage of glomerular development. Using gene knockdown in zebrafish with morpholinos, morphants lacking Glcci1 function had collapsed glomeruli with foot-process effacement. Permeability studies of the glomerular filtration barrier in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. Taken together, these data suggest that Glcci1 promotes the normal development and maintenance of podocyte structure and function.
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High RBM3 expression in prostate cancer independently predicts a reduced risk of biochemical recurrence and disease progression.
Diagn Pathol
PUBLISHED: 08-31-2011
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High expression of the RNA-binding protein RBM3 has previously been found to be associated with good prognosis in breast cancer, ovarian cancer, malignant melanoma and colorectal cancer. The aim of this study was to examine the prognostic impact of immunohistochemical RBM3 expression in prostate cancer.
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Variance decomposition of protein profiles from antibody arrays using a longitudinal twin model.
Proteome Sci
PUBLISHED: 08-08-2011
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The advent of affinity-based proteomics technologies for global protein profiling provides the prospect of finding new molecular biomarkers for common, multifactorial disorders. The molecular phenotypes obtained from studies on such platforms are driven by multiple sources, including genetic, environmental, and experimental components. In characterizing the contribution of different sources of variation to the measured phenotypes, the aim is to facilitate the design and interpretation of future biomedical studies employing exploratory and multiplexed technologies. Thus, biometrical genetic modelling of twin or other family data can be used to decompose the variation underlying a phenotype into biological and experimental components.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.