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BACKGROUND: Molecular components in blood, such as proteins, are used as biomarkers to detect or predict disease states, guide clinical interventions and aid in the development of therapies. While multiplexing proteomics methods promote discovery of such biomarkers, their translation to clinical use is difficult due to the lack of substantial evidence regarding their reliability as quantifiable indicators of disease state or outcome. To overcome this challenge, a novel orthogonal strategy was developed and used to assess the reliability of biomarkers and analytically corroborate already identified serum biomarkers for Duchenne muscular dystrophy (DMD). DMD is a monogenic incurable disease characterized by progressive muscle damage that currently lacks reliable and specific disease monitoring tools. METHODS: Two technological platforms are used to detect and quantify the biomarkers in 72 longitudinally collected serum samples from DMD patients at 3 to 5 timepoints. Quantification of the biomarkers is achieved by detection of the same biomarker fragment either through interaction with validated antibodies in immuno-assays or through quantification of peptides by Parallel Reaction Monitoring Mass Spectrometry assay (PRM-MS). RESULTS: Five, out of ten biomarkers previously identified by affinity-based proteomics methods, were confirmed to be associated with DMD using the mass spectrometry-based method. Two biomarkers, carbonic anhydrase III and lactate dehydrogenase B, were quantified with two independent methods, sandwich immunoassays and PRM-MS, with Pearson correlations of 0.92 and 0.946 respectively. The median concentrations of CA3 and LDHB in DMD patients was elevated in comparison to those in healthy individuals by 35- and 3-fold, respectively. Levels of CA3 vary between 10.26 and 0.36 ng/ml in DMD patients whereas those of LDHB vary between 15.1 and 0.8 ng/ml. CONCLUSIONS: These results demonstrate that orthogonal assays can be used to assess the analytical reliability of biomarker quantification assays, providing a means to facilitate the translation of biomarkers to clinical practice. This strategy also warrants the development of the most relevant biomarkers, markers that can be reliably quantified with different proteomics methods.
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Longitudinal and high-dimensional measurements have become increasingly common in biomedical research. However, methods to predict survival outcomes using covariates that are both longitudinal and high-dimensional are currently missing. In this article, we propose penalized regression calibration (PRC), a method that can be employed to predict survival in such situations. PRC comprises three modeling steps: First, the trajectories described by the longitudinal predictors are flexibly modeled through the specification of multivariate mixed effects models. Second, subject-specific summaries of the longitudinal trajectories are derived from the fitted mixed models. Third, the time to event outcome is predicted using the subject-specific summaries as covariates in a penalized Cox model. To ensure a proper internal validation of the fitted PRC models, we furthermore develop a cluster bootstrap optimism correction procedure that allows to correct for the optimistic bias of apparent measures of predictiveness. PRC and the CBOCP are implemented in the R package pencal, available from CRAN. After studying the behavior of PRC via simulations, we conclude by illustrating an application of PRC to data from an observational study that involved patients affected by Duchenne muscular dystrophy, where the goal is predict time to loss of ambulation using longitudinal blood biomarkers.
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Calibragem , Viés , Biomarcadores , Humanos , Estudos Longitudinais , Modelos de Riscos ProporcionaisRESUMO
Rimeporide, a ï¬rst-in-class sodium/proton exchanger Type 1 inhibitor (NHE-1 inhibitor) is repositioned by EspeRare for patients with Duchenne Muscular Dystrophy (DMD). Historically, NHE-1 inhibitors were developed for cardiac therapeutic interventions. There is considerable overlap in the pathophysiological mechanisms in Congestive Heart Failure (CHF) and in cardiomyopathy in DMD, therefore NHE-1 inhibition could be a promising pharmacological approach to the cardiac dysfunctions observed in DMD. Extensive preclinical data was collected in various animal models including dystrophin-deficient (mdx) mice to characterise Rimeporide's anti-fibrotic and anti-inflammatory properties and there is evidence that NHE-1 inhibitors could play a significant role in modifying DMD cardiac and also skeletal pathologies, as the NHE-1 isoform is ubiquitous. We report here the first study with Rimeporide in DMD patients. This 4-week treatment, open label phase Ib, multiple oral ascending dose study, enrolled 20 ambulant boys with DMD (6-11 years), with outcomes including safety, pharmacokinetic (PK) and pharmacodynamic (PD) biomarkers. Rimeporide was safe and well-tolerated at all doses. PK evaluations showed that Rimeporide was well absorbed orally reaching pharmacological concentrations from the lowest dose, with exposure increasing linearly with dose and with no evidence of accumulation upon repeated dosing. Exploratory PD biomarkers showed positive effect upon a 4-week treatment, supporting its therapeutic potential in patients with DMD, primarily as a cardioprotective treatment, and provide rationale for further efficacy studies.
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Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular de Duchenne/tratamento farmacológico , Fármacos Neuromusculares/administração & dosagem , Trocador 1 de Sódio-Hidrogênio/antagonistas & inibidores , Administração Oral , Criança , Esquema de Medicação , Europa (Continente) , Humanos , Masculino , Modelos Biológicos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatologia , Fármacos Neuromusculares/efeitos adversos , Fármacos Neuromusculares/farmacocinética , Trocador 1 de Sódio-Hidrogênio/metabolismo , Resultado do TratamentoRESUMO
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 â¼80% 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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Anticorpos/farmacologia , Expressão Gênica , Genômica/métodos , Especificidade de Órgãos/genética , Proteômica/métodos , Transcriptoma , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Masculino , Proteínas/genética , Proteínas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Integração de Sistemas , Análise Serial de TecidosRESUMO
Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.
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Quebras de DNA de Cadeia Simples , Reparo do DNA , Poli(ADP-Ribose) Polimerases/fisiologia , Alquilantes/toxicidade , Animais , Linhagem Celular , DNA Glicosilases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Cinética , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/genética , Ésteres do Ácido Sulfúrico/toxicidade , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to approximately 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.
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Anticorpos/imunologia , Bases de Dados Factuais , Perfilação da Expressão Gênica , Proteoma/metabolismo , Antígenos/análise , Antígenos/genética , Antígenos/imunologia , Atlas como Assunto , Humanos , Proteoma/análise , Proteoma/genética , Proteoma/imunologiaRESUMO
BACKGROUND: Duchenne Muscular Dystrophy is a severe, incurable disorder caused by mutations in the dystrophin gene. The disease is characterized by decreased muscle function, impaired muscle regeneration and increased inflammation. In a clinical context, muscle deterioration, is evaluated using physical tests and analysis of muscle biopsies, which fail to accurately monitor the disease progression. OBJECTIVES: This study aims to confirm and asses the value of blood protein biomarkers as disease progression markers using one of the largest longitudinal collection of samples. METHODS: A total of 560 samples, both serum and plasma, collected at three clinical sites are analyzed using a suspension bead array platform to assess 118 proteins targeted by 250 antibodies in microliter amount of samples. RESULTS: Nine proteins are confirmed as disease progression biomarkers in both plasma and serum. Abundance of these biomarkers decreases as the disease progresses but follows different trajectories. While carbonic anhydrase 3, microtubule associated protein 4 and collagen type I alpha 1 chain decline rather constantly over time, myosin light chain 3, electron transfer flavoprotein A, troponin T, malate dehydrogenase 2, lactate dehydrogenase B and nestin plateaus in early teens. Electron transfer flavoprotein A, correlates with the outcome of 6-minutes-walking-test whereas malate dehydrogenase 2 together with myosin light chain 3, carbonic anhydrase 3 and nestin correlate with respiratory capacity. CONCLUSIONS: Nine biomarkers have been identified that correlate with disease milestones, functional tests and respiratory capacity. Together these biomarkers recapitulate different stages of the disorder that, if validated can improve disease progression monitoring.
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Progressão da Doença , Distrofia Muscular de Duchenne/sangue , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/fisiopatologia , Proteômica , Adolescente , Adulto , Biomarcadores/sangue , Cadeia alfa 1 do Colágeno Tipo I , Humanos , Estudos Longitudinais , Masculino , Adulto JovemRESUMO
SUN-domain proteins form a novel and conserved family of inner nuclear membrane (INM) proteins, which establish physical connections between the nucleoplasm and the cytoskeleton. In the current study, we provide evidence that within the nuclear envelope (NE) Sun1 proteins form highly immobile oligomeric complexes in interphase cells. By performing inverse fluorescence recovery after photobleaching analysis, we demonstrate in vivo that both perinuclear and nucleoplasmic Sun1 segments are essential for maintenance of Sun1 immobility at the NE. Our data in particular underline the self-association properties of the C-terminal coiled-coil Sun1 segment, the ability of which to form dimers and tetramers is demonstrated. Furthermore, the Sun1 tertiary structure involves interchain disulfide bonds that might contribute to higher homo-oligomer formation, although the overall dynamics of the Sun1 C-terminus remains unaffected when the cysteins involved are mutated. While a major Sun1 pool colocalizes with nuclear pore complex proteins, a large fraction of the Sun1 protein assemblies colocalize with immunoreactive foci of Sun2, another SUN-domain paralogue at the NE. We demonstrate that the Sun1 coiled-coil domain permits these heterophilic associations with Sun2. Sun1 therefore provides a non-dynamic platform for the formation of different macromolecular assemblies at the INM. Our data support a model in which SUN-protein-containing multi-variate complexes may provide versatile outer nuclear membrane attachment sites for cytoskeletal filaments.
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Núcleo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Poro Nuclear/fisiologia , Sequência de Aminoácidos , Western Blotting , Núcleo Celular/ultraestrutura , Reagentes de Ligações Cruzadas , Dissulfetos/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Imunofluorescência , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Técnicas Imunoenzimáticas , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Membrana Nuclear/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-HíbridoRESUMO
The aim of this study was to investigate the gene and protein expression profiles of important drug-transporting proteins in human cell lines commonly used for studies of drug transport mechanisms. Human cell lines used to transiently or stably express single transporters [HeLa, human embryonic kidney (HEK) 293] and leukemia cell lines used to study drug resistance by ATP-binding cassette transporters (HL-60, K562) were investigated and compared with organotypic cell lines (HepG2, Saos-2, Caco-2, and Caco-2 TC7). For gene expression studies, real-time polymerase chain reaction was used, whereas monospecific polyclonal antibodies were generated and used to investigate protein expression by immunohistochemistry. Thirty-six transporters were studied for gene expression, and nine were studied for protein expression. The antibodies were validated using expression patterns in human tissues. Finally, the function of one ubiquitously expressed transporter, MCT1/SLC16A1, was investigated using [(14)C]lactic acid as a substrate. In general, the adherent cell lines (HeLa, HEK293) displayed low transporter expression, and the expression patterns were barely affected by transfection. The leukemia cell lines (K562, HL-60) and Saos-2 also had low endogenous transporter expression, whereas the organotypic cell lines (HepG2 and Caco-2) showed higher expression of some transporters. Comparison of gene and protein expression profiles gave poor correlations, but better agreement was obtained for antibodies with a good validation score, indicating that antibody quality was a significant variable. It is noteworthy that the monocarboxylic acid-transporting protein MCT1 was significantly expressed in all and was functional in most of the cell lines, indicating that MCT1 may be a confounding factor when the transport of small anionic drugs is investigated.
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Descoberta de Drogas/métodos , Perfilação da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Preparações Farmacêuticas/metabolismo , Proteômica , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico , Células CACO-2 , Adesão Celular , Células HL-60 , Células HeLa , Células Hep G2 , Humanos , Imuno-Histoquímica , Células K562 , Proteínas de Membrana Transportadoras/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simportadores/metabolismo , TransfecçãoRESUMO
Numerous biomarkers have been unveiled in the rapidly evolving biomarker discovery field, with an aim to improve the clinical management of disorders. In rare diseases, such as Duchenne muscular dystrophy, this endeavor has created a wealth of knowledge that, if effectively exploited, will benefit affected individuals, with respect to health care, therapy, improved quality of life and increased life expectancy. The most promising findings and molecular biomarkers are inspected in this review, with an aim to provide an overview of currently known biomarkers and the technological developments used. Biomarkers as cells, genetic variations, miRNAs, proteins, lipids and/or metabolites indicative of disease severity, progression and treatment response have the potential to improve development and approval of therapies, clinical management of DMD and patients' life quality. We highlight the complexity of translating research results to clinical use, emphasizing the need for biomarkers, fit for purpose and describe the challenges associated with qualifying biomarkers for clinical applications.
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There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.
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Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Ensaios de Triagem em Larga Escala/métodos , Estudos de Validação como Assunto , Animais , Western Blotting/métodos , Western Blotting/normas , Ensaios de Triagem em Larga Escala/normas , Humanos , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Yeast cells were grown in glucose-limited chemostat cultures and forced to switch to a new carbon source, the fatty acid oleate. Alterations in gene expression were monitored using DNA microarrays combined with bioinformatics tools, among which was included the recently developed algorithm REDUCE. Immediately after the switch to oleate, a transient and very specific stress response was observed, followed by the up-regulation of genes encoding peroxisomal enzymes required for fatty acid metabolism. The stress response included up-regulation of genes coding for enzymes to keep thioredoxin and glutathione reduced, as well as enzymes required for the detoxification of reactive oxygen species. Among the genes coding for various isoenzymes involved in these processes, only a specific subset was expressed. Not the general stress transcription factors Msn2 and Msn4, but rather the specific factor Yap1p seemed to be the main regulator of the stress response. We ascribe the initiation of the oxidative stress response to a combination of poor redox flux and fatty acid-induced uncoupling of the respiratory chain during the metabolic reprogramming phase.
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Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , Saccharomyces cerevisiae/fisiologia , Transporte Ativo do Núcleo Celular , Algoritmos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Fúngicos , Ácido Oleico/metabolismo , Oxirredução , Peroxissomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarray-based immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level. Our tissue-based analysis detected more than 90% of the putative protein-coding genes. We used this approach to explore the human secretome, the membrane proteome, the druggable proteome, the cancer proteome, and the metabolic functions in 32 different tissues and organs. All the data are integrated in an interactive Web-based database that allows exploration of individual proteins, as well as navigation of global expression patterns, in all major tissues and organs in the human body.
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Bases de Dados de Proteínas , Proteoma/metabolismo , Processamento Alternativo , Linhagem Celular , Feminino , Genes , Código Genético , Humanos , Internet , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Análise Serial de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoma/genética , Distribuição Tecidual , Transcrição GênicaRESUMO
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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Neoplasias da Mama/metabolismo , Mama/patologia , Proteína Quinase CDC2/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Sequência de Aminoácidos , Mama/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Fosforilação , Prognóstico , Regiões Promotoras Genéticas , Proteólise , Proteínas Ligases SKP Culina F-Box/análise , Proteínas Ligases SKP Culina F-Box/genética , Transdução de Sinais , Análise de Sobrevida , Ativação Transcricional , UbiquitinaçãoRESUMO
Ovarian cancer is usually found at a late stage when the prognosis is often bad. Relative survival rates decrease with tumor stage or grade, and the 5-year survival rate for women with carcinoma is only 38%. Thus, there is a great need to find biomarkers that can be used to carry out routine screening, especially in high-risk patient groups. Here, we present a large-scale study of 64 tissue samples taken from patients at all stages and show that we can identify statistically valid markers using nonsupervised methods that distinguish between normal, benign, borderline, and malignant tissue. We have identified 217 of the significantly changing protein spots. We are expressing and raising antibodies to 35 of these. Currently, we have validated 5 of these antibodies for use in immunohistochemical analysis using tissue microarrays of healthy and diseased ovarian, as well as other, human tissues.
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Biomarcadores Tumorais/análise , Proteínas de Neoplasias/análise , Neoplasias Ovarianas/diagnóstico , Proteômica , Anticorpos/imunologia , Biomarcadores Tumorais/imunologia , Feminino , Humanos , Imuno-Histoquímica , Proteínas de Neoplasias/imunologia , Neoplasias Ovarianas/química , Neoplasias Ovarianas/patologiaRESUMO
The glomerular capillary tuft is a highly specialized microcapillary that is dedicated to function as a sophisticated molecular sieve. The glomerulus filter has a unique molecular composition, and several essential glomerular proteins are expressed in the kidney exclusively by glomerular podocytes. A catalog of >300 glomerulus-upregulated transcripts that were identified using expressed sequence tag profiling and microarray analysis was published recently. This study characterized the expression profile of five glomerulus-upregulated transcripts/proteins (ehd3, dendrin, sh2d4a, plekhh2, and 2310066E14Rik) in detail. The expression pattern of these novel glomerular transcripts in various mouse tissues was studied using reverse transcriptase-PCR, Northern blotting, and in situ hybridization. For studying the distribution of corresponding proteins, polyclonal antibodies were raised against the gene products, and Western blotting, immunofluorescence, and immunoelectron microscopic analyses were performed. Remarkably, it was discovered that all five transcripts/proteins were expressed in the kidney exclusively by glomerular cells. Ehd3 was expressed only by glomerular endothelial cells. Importantly, ehd3 is the first gene ever shown to be expressed exclusively by glomerular endothelial cells and not by other endothelial cells in the kidney. Dendrin, sh2d4a, plekhh2, and 2310066E14Rik, however, were transcribed solely by podocytes. With the use of polyclonal antibodies, dendrin, sh2d4a, and plekhh2 proteins were localized to the slit diaphragm and the foot process, whereas 2310066E14Rik protein was localized to the podocyte major processes and cell body. This study provides fresh insights into glomerular biology and uncovers new possibilities to explore the role of these novel proteins in the glomerular physiology and pathology.
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Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glomérulos Renais/metabolismo , Podócitos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Northern Blotting , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto , Glomérulos Renais/citologia , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.