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Four nanostructured active semiconducting materials currently used in electronic inks have been structurally characterised using a combination of small angle scattering techniques and scanning electron microscopy. The percolation theory and scaling laws have been used to obtain quantitative correlations of the network topologies and the local micro-structures with the electronic and electrical properties of the printed, electronic devices. The small angle light scattering has been used to expand the lower q-range of the Ultra Small Angle x-ray Scattering curves of the 2503 metallurgical grade silicon (mSi), silicon dioxide (SiO2), aluminium dioxide (Al2O3) and titanium dioxide (TiO2) materials by close to an order of magnitude, thereby providing valuable clustering properties for each material. Each scattering curve presented a series of multiple structural levels, which are then quantified using the Unified power-law approach to provide valuable clustering characteristics such as the degree of aggregation, polydispersity and geometry standard deviation. Subsequently, a fully screen-printed field effect transistor that uses mSi as the active material is demonstrated. The transistor had an ON/OFF current-ratio of 104; an electron mobility of 0.7 cm2/V s; a leakage current in the order of 5 × 10-9 A, and no current saturation.
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Signaling pathways driven by protein and lipid kinases are altered in most human diseases. Therefore, pharmacological inhibitors of cell signaling are one of the most intensively pursued therapeutic approaches for the treatment of diseases such as cancer, neurodegeneration, and metabolic syndromes. Phosphoproteomics is a technique that measures the products of kinase activities and, with the appropriate bioinformatics techniques, the methodology can also provide measures of kinase pathway activation and network circuitry. Hence, due to recent technological advantages, LC-MS-based quantitative phosphoproteomics provides relevant information for the design and implementation of kinase inhibitor based therapies. Here, we review how phosphoproteome profiling is being used in translational research as a means to identify drug targets and biomarkers for personalizing therapies based on kinase inhibitors.
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Terapia de Alvo Molecular , Fosfoproteínas/metabolismo , Proteínas Quinases/metabolismo , Proteômica/métodos , Pesquisa Translacional Biomédica , Animais , Humanos , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM) is not only a proven tool for clinical chemistry, but also a versatile method to enhance the capability to quantify biomarkers for tumor biology research. As the treatment of cancer continues to evolve, the ability to assess multiple biomarkers to assign cancer phenotypes based on the genetic background and the signaling of the individual tumor becomes paramount to our ability to treat the patient. In breast cancer, the American Society of Clinical Oncology has defined biomarkers for patient assessment to guide selection of therapy: estrogen receptor, progesterone receptor, and the HER2/Neu receptor tyrosine kinase; therefore, these proteins were selected for LC-SRM assay development. Detailed molecular characterization of these proteins is necessary for patient treatment, so expression and phosphorylation assays have been developed and applied. In addition, other LC-SRM assays were developed to further evaluate tumor biology (e.g. Ki-67 for proliferation and vimentin for tumor aggressiveness related to the epithelial-to-mesenchymal transition). These measurements combined with biomarkers for tissue quality and histological content are implemented in a three-tier multiplexed assay platform, which is translated from cell line models into frozen tumor tissues banked from breast cancer patients.
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Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Proteômica/métodos , Linhagem Celular Tumoral , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Ligantes , Proteínas de Neoplasias/metabolismo , Fosforilação , Reprodutibilidade dos TestesRESUMO
The presence of native oxide on the surface of silicon nanoparticles is known to inhibit charge transport on the surfaces. Scanning electron microscopy (SEM) studies reveal that the particles in the printed silicon network have a wide range of sizes and shapes. High-resolution transmission electron microscopy reveals that the particle surfaces have mainly the (111)- and (100)-oriented planes which stabilizes against further oxidation of the particles. X-ray absorption spectroscopy (XANES) and X-ray photoelectron spectroscopy (XPS) measurements at the O 1s-edge have been utilized to study the oxidation and local atomic structure of printed layers of silicon nanoparticles which were milled for different times. XANES results reveal the presence of the +4 (SiO2) oxidation state which tends towards the +2 (SiO) state for higher milling times. Si 2p XPS results indicate that the surfaces of the silicon nanoparticles in the printed layers are only partially oxidized and that all three sub-oxide, +1 (Si2O), +2 (SiO) and +3 (Si2O3), states are present. The analysis of the change in the sub-oxide peaks of the silicon nanoparticles shows the dominance of the +4 state only for lower milling times.
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AIMS: This combined proteomic and histopathological study was aimed to compare tissue characteristics of immunoglobulin (Ig)G4-related sclerosing cholangitis (ISC) and primary sclerosing cholangitis (PSC) in a global, non-biased manner. METHODS AND RESULTS: Tissue proteomes and phosphorylomes of frozen large bile duct samples were analysed by a conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) protocol and additional phosphopeptide enrichment methods. The proteomic examination identified 23 373 peptides and 4870 proteins, including 4801 phosphopeptides and 1121 phosphoproteins. The expression profiles of phosphopeptides discriminated ISC from PSC more clearly than those of non-phosphopeptides. In the pathway analysis, ISC was found to have 11 more activated signal cascades, including three immunological pathways, all B cell- or immunoglobulin-related. On immunostaining, two immunological markers (FYN-binding protein and allograft inflammatory factor-1) up-regulated in ISC were expressed mainly in M2 macrophages, consistent with increased phagocytotic activity induced by the immunoglobulin (Ig)G-Fcγ receptor interaction. In contrast, PSC had two more activated signal pathways related to extracellular matrix (ECM) remodelling. Filamin-A involved in ECM remodelling was expressed aberrantly in injured bile ducts and associated cholangiocarcinomas in PSC, suggesting its possible roles in periductal fibrosis and carcinogenesis in PSC. CONCLUSIONS: This study suggested crucial roles of B cells and macrophages in ISC, and more dynamic ECM remodelling in PSC.
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Colangite Esclerosante/imunologia , Colangite Esclerosante/patologia , Matriz Extracelular/patologia , Imunoglobulina G/imunologia , Proteoma/análise , Idoso , Biomarcadores/análise , Colangite Esclerosante/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Análise por Conglomerados , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteômica/métodos , Espectrometria de Massas em TandemRESUMO
We present a novel tandem mass tag solid-phase amino labeling (TMT-SPAL) protocol using reversible immobilization of peptides onto octadecyl-derivatized (C18) solid supports. This method can reduce the number of steps required in complex protocols, saving time and potentially reducing sample loss. In our global phosphopeptide profiling workflow (SysQuant), we can cut 24 h from the protocol while increasing peptide identifications (20%) and reducing side reactions. Solid-phase labeling with TMTs does require some modification to typical labeling conditions, particularly pH. It has been found that complete labeling equivalent to standard basic pH solution-phase labeling for small and large samples can be achieved on C18 resins under slightly acidic buffer conditions. Improved labeling behavior on C18 compared to that with standard basic pH solution-phase labeling is demonstrated. We analyzed our samples for histidine, serine, threonine, and tyrosine labeling to determine the degree of overlabeling and observed higher than expected levels (25% of all peptide spectral matches (PSMs)) of overlabeling at all of these amino acids (predominantly at tyrosine and serine) in our standard solution-phase labeling protocol. Overlabeling at all of these sites is greatly reduced (4-fold, to 7% of all PSMs) by the low-pH conditions used in the TMT-SPAL protocol. Overlabeling seems to represent a so-far overlooked mechanism causing reductions in peptide identification rates with NHS-activated TMT labeling compared to that with label-free methods. Our results also highlight the importance of searching data for overlabeling when labeling methods are used.
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Concentração de Íons de Hidrogênio , Fosfopeptídeos/química , Aminas/química , Linhagem Celular Tumoral , Humanos , Espectrometria de Massas em TandemRESUMO
BACKGROUND & AIMS: There has not been a broad analysis of the combined effects of altered activities of microRNAs (miRNAs) in pancreatic ductal adenocarcinoma (PDAC) cells, and it is unclear how these might affect tumor progression or patient outcomes. METHODS: We combined data from miRNA and messenger RNA (mRNA) expression profiles and bioinformatic analyses to identify an miRNA-mRNA regulatory network in PDAC cell lines (PANC-1 and MIA PaCa-2) and in PDAC samples from patients. We used this information to identify miRNAs that contribute most to tumorigenesis. RESULTS: We identified 3 miRNAs (MIR21, MIR23A, and MIR27A) that acted as cooperative repressors of a network of tumor suppressor genes that included PDCD4, BTG2, and NEDD4L. Inhibition of MIR21, MIR23A, and MIR27A had synergistic effects in reducing proliferation of PDAC cells in culture and growth of xenograft tumors in mice. The level of inhibition was greater than that of inhibition of MIR21 alone. In 91 PDAC samples from patients, high levels of a combination of MIR21, MIR23A, and MIR27A were associated with shorter survival times after surgical resection. CONCLUSIONS: In an integrated data analysis, we identified functional miRNA-mRNA interactions that contribute to growth of PDACs. These findings indicate that miRNAs act together to promote tumor progression; therapeutic strategies might require inhibition of several miRNAs.
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Carcinoma Ductal Pancreático/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor/fisiologia , MicroRNAs/fisiologia , Neoplasias Pancreáticas/genética , RNA Mensageiro/genética , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Complexos Endossomais de Distribuição Requeridos para Transporte/antagonistas & inibidores , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Perfilação da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/fisiologia , Camundongos , MicroRNAs/genética , Ubiquitina-Proteína Ligases Nedd4 , Prognóstico , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/fisiologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/fisiologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/fisiologiaRESUMO
A new small-angle scattering technique in reflection geometry is described which enables a topological study of rough surfaces. This is achieved by using long-wavelength soft X-rays which are scattered at wide angles but in the low-Q range normally associated with small-angle scattering. The use of nanometre-wavelength radiation restricts the penetration to a thin surface layer which follows the topology of the surface, while moving the scattered beam to wider angles preventing shadowing by the surface features. The technique is, however, only applicable to rough surfaces for which there is no specular reflection, so that only the scattered beam was detected by the detector. As an example, a study of the surfaces of rough layers of silicon produced by the deposition of nanoparticles by blade-coating is presented. The surfaces of the blade-coated layers have rough features of the order of several micrometers. Using 2 nm and 13 nm X-rays scattered at angular ranges of 5° ≤ θ ≤ 51° and 5° ≤ θ ≤ 45°, respectively, a combined range of scattering vector of 0.00842 Å(-1) ≤ Q ≤ 0.4883 Å(-1) was obtained. Comparison with previous transmission SAXS and USAXS studies of the same materials indicates that the new method does probe the surface topology rather than the internal microstructure.
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AIMS: Our recent proteomic study identified tubulin ß-III (TUBB3) as a potential tissue marker for intrahepatic cholangiocarcinomas (CCs). This validation study was conducted to see whether or not TUBB3 can serve as a novel immunohistochemical marker for peripheral CCs, using a large cohort (n = 197) covering various liver tumours and premalignant conditions. METHODS AND RESULTS: Immunostaining using a monoclonal antibody demonstrated TUBB3 expression in 14/28 cases of peripheral CCs (50%), while its expression was significantly less common in perihilar CCs (6/40, 15%) (P = 0.002). No significant difference was identified in clinicopathological features between TUBB3-positive and -negative cases. TUBB3 expression was entirely negative in hepatocellular carcinomas, biliary premalignant lesions (i.e., biliary intraepithelial neoplasias, intraductal papillary neoplasms), peribiliary gland hamartomas (bile duct adenomas), and non-neoplastic biliary epithelium. TUBB3 expression was only focally noted in 2/12 cases of mixed hepatocellular and cholangiocarcinomas (<10% of cancer cells). Compared with other biliary (CK7 and CK19) and malignant markers (p53 and MUC1), TUBB3 was less sensitive but more specific for peripheral CCs. TUBB3 was also expressed in 40% of metastatic colorectal or breast cancers. CONCLUSIONS: This study revealed that TUBB3 is a moderately sensitive and highly specific tissue marker for discriminating peripheral CCs from other primary liver tumours.
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Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Biomarcadores Tumorais/análise , Colangiocarcinoma/patologia , Tubulina (Proteína)/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/metabolismo , Colangiocarcinoma/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Tubulina (Proteína)/análiseRESUMO
Release from natural enemies is considered to potentially play an important role in the initial establishment and success of introduced plants. With time, the species richness of herbivores using non-native plants may increase [species-time relationship (STR)]. We investigated whether enemy release may be limited to the early stages of invasion. Substituting space for time, we sampled invertebrates and measured leaf damage on the invasive species Senecio madagascariensis Poir. at multiple sites, north and south of the introduction site. Invertebrate communities were collected from plants in the field, and reared from collected plant tissue. We also sampled invertebrates and damage on the native congener Senecio pinnatifolius var. pinnatifolius A. Rich. This species served as a control to account for environmental factors that may vary along the latitudinal gradient and as a comparison for evaluating the enemy release hypothesis (ERH). In contrast to predictions of the ERH, greater damage and herbivore abundances and richness were found on the introduced species S. madagascariensis than on the native S. pinnatifolius. Supporting the STR, total invertebrates (including herbivores) decreased in abundance, richness and Shannon diversity from the point of introduction to the invasion fronts of S. madagascariensis. Leaf damage showed the opposite trend, with highest damage levels at the invasion fronts. Reared herbivore loads (as opposed to external collections) were greater on the invader at the point of introduction than on sites further from this region. These results suggest there is a complex relationship between the invader and invertebrate community response over time. S. madagascariensis may be undergoing rapid changes at its invasion fronts in response to environmental and herbivore pressure.
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Herbivoria , Espécies Introduzidas , Invertebrados/fisiologia , Senécio/fisiologia , Animais , Conservação dos Recursos Naturais , Cadeia Alimentar , New South Wales , Folhas de Planta/fisiologia , Plantas Daninhas/fisiologia , Queensland , Especificidade da Espécie , Fatores de TempoRESUMO
Reactive oxygen species (ROS) are both physiological intermediates in cellular signaling and mediators of oxidative stress. The cysteine-specific redox-sensitivity of proteins can shed light on how ROS are regulated and function, but low sensitivity has limited quantification of the redox state of many fundamental cellular regulators in a cellular context. Here we describe a highly sensitive and reproducible oxidation analysis approach (OxMRM) that combines protein purification, differential alkylation with stable isotopes, and multiple reaction monitoring mass spectrometry that can be applied in a targeted manner to virtually any cysteine or protein. Using this approach, we quantified the site-specific cysteine oxidation status of endogenous p53 for the first time and found that Cys182 at the dimerization interface of the DNA binding domain is particularly susceptible to diamide oxidation intracellularly. OxMRM enables analysis of sulfinic and sulfonic acid oxidation levels, which we validate by assessing the oxidation of the catalytic Cys215 of protein tyrosine phosphatase-1B under numerous oxidant conditions. OxMRM also complements unbiased redox proteomics discovery studies as a verification tool through its high sensitivity, accuracy, precision, and throughput.
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Cisteína/química , Espectrometria de Massas/métodos , Alquilação , Sequência de Aminoácidos , Linhagem Celular , Cisteína/metabolismo , Humanos , Dados de Sequência Molecular , Oxirredução , Processamento de Proteína Pós-Traducional , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
A systematic study of posttranslational modifications of the estrogen receptor isolated from the MCF-7 human breast cancer cell line is reported. Proteolysis with multiple enzymes, mass spectrometry, and tandem mass spectrometry achieved very high sequence coverage for the full-length 66-kDa endogenous protein from estradiol-treated cell cultures. Nine phosphorylated serine residues were identified, three of which were previously unreported and none of which were previously observed by mass spectrometry by any other laboratory. Two additional modified serine residues were identified in recombinant protein, one previously reported but not observed here in endogenous protein and the other previously unknown. Although major emphasis was placed on identifying new phosphorylation sites, N-terminal loss of methionine accompanied by amino acetylation and a lysine side chain acetylation (or possibly trimethylation) were also detected. The use of both HPLC-ESI and MALDI interfaced to different mass analyzers gave higher sequence coverage and identified more sites than could be achieved by either method alone. The estrogen receptor is critical in the development and progression of breast cancer. One previously unreported phosphorylation site identified here was shown to be strongly dependent on estradiol, confirming its potential significance to breast cancer. Greater knowledge of this array of posttranslational modifications of estrogen receptor, particularly phosphorylation, will increase our understanding of the processes that lead to estradiol-induced activation of this protein and may aid the development of therapeutic strategies for management of hormone-dependent breast cancer.
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Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Sequência de Aminoácidos , Linhagem Celular Tumoral , Cromatografia de Afinidade , Cromatografia Líquida , Cisteína/metabolismo , Humanos , Dados de Sequência Molecular , Mapeamento de Peptídeos , Fosfopeptídeos/análise , Fosfopeptídeos/química , Fosforilação , Fosfosserina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Cholangiocarcinoma is a form of hepatobiliary cancer with an abysmal prognosis. Despite advances in our understanding of cholangiocarcinoma pathophysiology and its genomic landscape, targeted therapies have not yet made a significant impact on its clinical management. The low response rates of targeted therapies in cholangiocarcinoma suggest that patient heterogeneity contributes to poor clinical outcome. Here we used mass spectrometry-based phosphoproteomics and computational methods to identify patient-specific drug targets in patient tumors and cholangiocarcinoma-derived cell lines. We analyzed 13 primary tumors of patients with cholangiocarcinoma with matched nonmalignant tissue and 7 different cholangiocarcinoma cell lines, leading to the identification and quantification of more than 13,000 phosphorylation sites. The phosphoproteomes of cholangiocarcinoma cell lines and patient tumors were significantly correlated. MEK1, KIT, ERK1/2, and several cyclin-dependent kinases were among the protein kinases most frequently showing increased activity in cholangiocarcinoma relative to nonmalignant tissue. Application of the Drug Ranking Using Machine Learning (DRUML) algorithm selected inhibitors of histone deacetylase (HDAC; belinostat and CAY10603) and PI3K pathway members as high-ranking therapies to use in primary cholangiocarcinoma. The accuracy of the computational drug rankings based on predicted responses was confirmed in cell-line models of cholangiocarcinoma. Together, this study uncovers frequently activated biochemical pathways in cholangiocarcinoma and provides a proof of concept for the application of computational methodology to rank drugs based on efficacy in individual patients. SIGNIFICANCE: Phosphoproteomic and computational analyses identify patient-specific drug targets in cholangiocarcinoma, supporting the potential of a machine learning method to predict personalized therapies.
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Antineoplásicos/farmacologia , Colangiocarcinoma/metabolismo , Biologia Computacional/métodos , Fosfoproteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Proteoma/metabolismo , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/patologia , Descoberta de Drogas , Humanos , Fosfoproteínas/análise , Fosfoproteínas/antagonistas & inibidores , Proteoma/análise , Células Tumorais CultivadasRESUMO
The anti-CD20 monoclonal antibodies rituximab and obinutuzumab differ in their mechanisms of action, with obinutuzumab evoking greater direct B cell death. To characterize the signaling processes responsible for improved B cell killing by obinutuzumab, we undertook a phosphoproteomics approach and demonstrate that rituximab and obinutuzumab differentially activate pathways downstream of the B cell receptor. Although both antibodies induce strong ERK and MYC activation sufficient to promote cell-cycle arrest and B cell death, obinutuzumab exceeds rituximab in supporting apoptosis induction by means of aberrant SYK phosphorylation. In contrast, rituximab elicits stronger anti-apoptotic signals by activating AKT, by impairing pro-apoptotic BAD, and by releasing membrane-bound NOTCH1 to up-regulate pro-survival target genes. As a consequence, rituximab appears to reinforce BCL2-mediated apoptosis resistance. The unexpected complexity and differences by which rituximab and obinutuzumab interfere with signaling pathways essential for lymphoma pathogenesis and treatment provide important impetus to optimize and personalize the application of different anti-CD20 treatments.
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Artificial intelligence and machine learning (ML) promise to transform cancer therapies by accurately predicting the most appropriate therapies to treat individual patients. Here, we present an approach, named Drug Ranking Using ML (DRUML), which uses omics data to produce ordered lists of >400 drugs based on their anti-proliferative efficacy in cancer cells. To reduce noise and increase predictive robustness, instead of individual features, DRUML uses internally normalized distance metrics of drug response as features for ML model generation. DRUML is trained using in-house proteomics and phosphoproteomics data derived from 48 cell lines, and it is verified with data comprised of 53 cellular models from 12 independent laboratories. We show that DRUML predicts drug responses in independent verification datasets with low error (mean squared error < 0.1 and mean Spearman's rank 0.7). In addition, we demonstrate that DRUML predictions of cytarabine sensitivity in clinical leukemia samples are prognostic of patient survival (Log rank p < 0.005). Our results indicate that DRUML accurately ranks anti-cancer drugs by their efficacy across a wide range of pathologies.
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Antineoplásicos/uso terapêutico , Biologia Computacional/métodos , Citarabina/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Leucemia/tratamento farmacológico , Aprendizado de Máquina , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Hep G2 , Humanos , Leucemia/mortalidade , Neoplasias/tratamento farmacológico , Prognóstico , Proteômica/métodosRESUMO
LAMP assays are targeted molecular tests for the rapid detection of species in the laboratory and field. We developed a LAMP assay for an economically important fruit fly species, Queensland fruit fly, Bactrocera tryoni. This assay was assessed against a broad panel of target and non-target species and found to be specific, only amplifying the target species and closest relatives, in a portable real-time fluorometer (Genie III) in under 15 minutes with an anneal derivative temperature of 82.5 oC. The assay is sensitive to low levels of target DNA (>0.016 ng/µl), performing equally to the existing qPCR test. To enable retention of a physical voucher specimen, for potential morphological confirmation of LAMP results, a novel whole-specimen non-destructive DNA extraction method was developed, suitable for LAMP in the field. The stability of DNA extraction and LAMP reagents was tested under simulated and actual field conditions and shown to be robust. Our new assay now provides a portable molecular tool for the detection of this significant tephritid fruit fly pest species of biosecurity/quarantine concern. This has already proven invaluable for in-field diagnostics, providing real-time support influencing immediate actions, with negative results allowing the release of fruit produce, and positive results initiating fruit fly control measures.
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Bioensaio/métodos , Tephritidae/genética , Animais , Quarentena/métodos , Especificidade da EspécieRESUMO
Activated estrogen receptor (ERalpha) plays a critical role in breast cancer development and is a major target for drug treatment. Serine phosphorylation within the N-terminal domain (NTD) contributes to ERalpha activation and may also cause drug resistance. Previous biochemical identification of phosphorylated ERalpha residues was limited to protein artificially overexpressed in transfected cell lines. We report mass spectrometric methods that have allowed the identification of a new site within the NTD of ERalpha isolated from cultured human breast cancer cells. Immunoprecipitation, trypsin digestion, and analysis by nano-LC-ESI-MS/MS (Q-STAR, MDS Sciex) and vMALDI-MS(n) (Finnigan LTQ, Thermo-Electron) identified peptides containing 8 of 14 serine residues within the NTD, one being partially phosphorylated Ser-167, known but not previously reported by MS. Chymotrypsin digestion revealed other known sites at Ser-102/104/106 and 118. Tandem methods developed for the peptide containing Ser-118 and the use of hypothesis-driven experiments--i.e., the assumption that an intact phosphopeptide showing no molecular ion might yield fragment ions including loss of phosphoric acid in vMALDI-MS/MS--allowed the identification of a novel site at Ser-154. Quantitation by selected reaction monitoring demonstrated 6-fold and 2.5-fold increases in Ser-154 phosphorylation in estradiol- and EGF-treated cells, respectively, compared to controls, confirmed by immunoblotting with a novel rabbit polyclonal antibody. Thus, the protein isolation and MS strategies described here can facilitate discovery of novel phosphorylation sites within low abundance, clinically important cancer targets like ERalpha, and may thereby contribute to our understanding of the role of phosphorylation in the development of breast cancer.
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Neoplasias da Mama/química , Cromatografia Líquida de Alta Pressão/métodos , Receptor alfa de Estrogênio/química , Serina/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sítios de Ligação , Feminino , Humanos , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Células Tumorais CultivadasRESUMO
Liquid chromatography-selected reaction monitoring (LC-SRM) mass spectrometry has developed into a versatile tool for quantification of proteins with a wide range of applications in basic science, translational research, and clinical patient assessment. This strategy uniquely complements traditional pathology approaches, like hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). The multiplexing capabilities offered by mass spectrometry are currently unmatched by other techniques. However, quantification of biomarkers in tissue specimens without the other data obtained from H&E-stained slides or IHC, including tumor cellularity or percentage of positively stained cells inter alia, may not provide as much information that is needed to fully understand tumor biology or properly assess the patient. Therefore, additional characterization of the tissue proteome is needed, which in turn requires the ability to assess protein markers across a wide range of expression levels from a single sample. This protocol provides an example of multiplexed analysis in breast tumor tissue quantifying specific biomarkers, specifically estrogen receptor, progesterone receptor, and the HER2 receptor tyrosine kinase, in combination with other proteins that can report on tissue content and other aspects of tumor biology.
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Neoplasias da Mama/patologia , Mama/patologia , Proteômica/métodos , Receptor ErbB-2/análise , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Sequência de Aminoácidos , Biomarcadores Tumorais/análise , Neoplasias da Mama/diagnóstico , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Feminino , Humanos , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Proteoma/análise , Espectrometria de Massas em Tandem/métodosRESUMO
Allocation of organic carbon (OC) to primary energetic pathways was estimated under seasonal and artificially elevated ambient temperatures for a field population of a freshwater pulmonate snail, Physella virgata. Allocation to respiration increased with temperature. Snails allocated most assimilated OC to reproduction within their natural temperature range (15 degrees -35 degrees C), where assimilation efficiencies remained relatively stable at 25%-35%. However, in artificially heated waters exceeding 35 degrees C, declining assimilation rates and increasing respiratory demands inhibited allocation to reproduction and growth. At the species' 40 degrees C upper thermal limit, assimilation efficiencies fell below 10%, while average consumption levels more than doubled relative to snails unaffected by the thermal effluent. Ambient temperature substantially influenced OC allocation over P. virgata's natural temperature range and negatively affected growth and reproduction at temperatures approaching or exceeding maximum natural levels.
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Metabolismo Energético/fisiologia , Estações do Ano , Caramujos/fisiologia , Temperatura , Animais , Carbono/fisiologia , Fezes/química , Água Doce , Consumo de Oxigênio/fisiologia , Reprodução/fisiologia , Caramujos/crescimento & desenvolvimento , TexasRESUMO
OBJECTIVE: LC-MS/MS phospho-proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed a global phospho-proteomic workflow to determine activity of signaling pathways and drug targets in pancreatic cancer tissue for clinical application. METHODS: Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (nâ=â12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using IMAC and TiO2, prior to analysis. In-house, commercial and freeware bioinformatic platforms were used to identify relevant biological events from the complex dataset. RESULTS: Of 2,101 proteins identified, 152 demonstrated significant difference in abundance between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as HIPK1 & MLCK. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho guanine nucleotide exchange factors & MRCKα) and formation of focal adhesions. Activator phosphorylation sites on FYN, AKT1, ERK2, HDAC1 and other drug targets were found to be highly modulated (≥2 fold) in different cases highlighting their predictive power. CONCLUSION: Here we provided critical information enabling us to identify the common and unique molecular events likely contributing to cancer in each case. Such information may be used to help predict more bespoke therapy suitable for an individual case.