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Experimental Design (experimental + design)
Kinds of Experimental Design Terms modified by Experimental Design Selected AbstractsBcl-2 mediated modulation of vascularization in prostate cancer xenografts,THE PROSTATE, Issue 5 2009Yoshihisa Sakai Abstract PURPOSE We previously demonstrated that Bcl-2 overexpression enhances the radiation resistance of PC-3 human prostate cancer cells and xenografts by inhibiting apoptosis, increasing proliferation, and promoting angiogenesis. To further elucidate the relationship between Bcl-2 expression and the angiogenic potential of PC-3-Bcl-2 cells, tumorigenicity, angiogenesis, and lymphangiogenesis were evaluated and compared in a Bcl-2 overexpressing clone in vitro and in vivo. EXPERIMENTAL DESIGN Human prostate cancer cells over expressing Bcl-2 were studied in vitro and in vivo to determine the angiogenic and lymphangiogenic properties of these cells. RESULTS Increased Bcl-2 expression enhanced the tumorigenicity of prostate cancer xenografts. It also enhanced the expression and secretion of key angiogenic and lymphangiogenic factors that stimulated the synthesis of CD31-positive blood vessels and LYVE-1 positive lymphatics. Specifically, the increased angiogenic and lymphangiogenic potential correlated with increased serum levels of basic fibroblast growth factor (bFGF), interleukin 8 (CXCL8), and matrix metalloproteinase (MMP 9). In vitro analysis demonstrated that Bcl-2 expressing tumor cells secreted bFGF and vascular endothelial growth factor (VEGF) into culture supernatants. Microarray analysis of Bcl-2 expressing PC-3 cells demonstrated increased transcription of genes involved in metabolism, such as interleukins, growth factors, tumor necrosis factors (TNF) family members, and peptidases. CONCLUSIONS Together, these results demonstrate that Bcl-2 can regulate tumoral angiogenesis and lymphangiogenesis and suggest that therapy targeted at Bcl-2 expression, angiogenesis, and lymphangiogenesis may synergistically modulate tumor growth and confirm that Bcl-2 is a pivotal target for cancer therapy. Prostate 69:459,470, 2009. © 2008 Wiley-Liss, Inc. [source] NF-,B2/p52 enhances androgen-independent growth of human LNCaP cells via protection from apoptotic cell death and cell cycle arrest induced by androgen-deprivationTHE PROSTATE, Issue 16 2008Nagalakshmi Nadiminty Abstract PURPOSE Androgen-deprivation therapy only causes a temporary regression of prostate cancer, as all tumors will eventually progress to refractory to hormonal therapy after 1,3 years of treatment. The underlying mechanisms of prostate cancer androgen refractory progression are incompletely understood. In this study, we employed in vitro as well as in vivo models to examine the role of NF-,B2/p52 in prostate cancer growth and androgen independent progression. EXPERIMENTAL DESIGN The effects of NF-,B2/p52 on cell growth, androgen responsiveness, cell cycle and apoptosis were examined in androgen sensitive LNCaP cells. The effect of NF-,B2/p52 on tumor growth was examined in intact and castrated male mice. RESULTS Overexpression of NF-,B2/p52 enhances androgen-sensitive LNCaP human prostate cancer cell growth and clonogenic ability in androgen-deprived condition in vitro. NF-,B2/p52 induced androgen-independent growth is through protecting LNCaP cells from apoptotic cell death and cell cycle arrest induced by androgen-deprivation. In addition, NF-,B2/p52 stimulates Cyclin D1 expression and knock down of Cyclin D1 expression by siRNA abolished NF-,B2/p52-induced cell growth in vitro. Adenoviral mediated NF-,B2/p52 expression in LNCaP cells enhances tumor growth in intact male nude mice and induces tumor growth in castrated male nude mice, suggesting that overexpression of NF-,B2/p52 induces androgen-independent growth of androgen-sensitive LNCaP cells. CONCLUSIONS Overexpression of NF-,B2/p52 protects androgen sensitive LNCaP cells from apoptotic cell death and cell cycle arrest induced by androgen-deprivation. NF-,B2/p52 activation induces androgen-independent growth in vitro and in vivo. Prostate © 2008 Wiley-Liss, Inc. [source] Magnetoencephalographic gamma power reduction in patients with schizophrenia during resting conditionHUMAN BRAIN MAPPING, Issue 10 2009Lindsay Rutter Abstract Objective: The "default network" represents a baseline condition of brain function and is of interest in schizophrenia research because its component brain regions are believed to be aberrant in the disorder. We hypothesized that magnetoencephalographic (MEG) source localization analysis would reveal abnormal resting activity within particular frequency bands in schizophrenia. Experimental Design: Eyes-closed resting state MEG signals were collected for two comparison groups. Patients with schizophrenia (N = 38) were age-gender matched with healthy control subjects (N = 38), and with a group of unmedicated unaffected siblings of patients with schizophrenia (N = 38). To localize 3D-brain regional differences, synthetic aperture magnetometry was calculated across established frequency bands as follows: delta (0.9,4 Hz), theta (4,8 Hz), alpha (8,14 Hz), beta (14,30 Hz), gamma (30,80 Hz), and super-gamma (80,150 Hz). Principle Observations: Patients with schizophrenia showed significantly reduced activation in the gamma frequency band in the posterior region of the medial parietal cortex. As a group, unaffected siblings of schizophrenia patients also showed significantly reduced activation in the gamma bandwidth across similar brain regions. Moreover, using the significant region for the patients and examining the gamma band power gave an odds ratio of 6:1 for reductions of two standard deviations from the mean. This suggests that the measure might be the basis of an intermediate phenotype. Conclusions: MEG resting state analysis adds to the evidence that schizophrenic patients experience this condition very differently than healthy controls. Whether this baseline difference relates to network abnormalities remains to be seen. Hum Brain Mapp, 2009. © 2009 Wiley-Liss, Inc. [source] Virtual Experiments and Their Use in Teaching Experimental DesignINTERNATIONAL STATISTICAL REVIEW, Issue 3 2007Paul L. Darius Summary The ability to design experiments in an appropriate and efficient way is an important skill, but students typically have little opportunity to get that experience. Most textbooks introduce standard general-purpose designs, and then proceed with the analysis of data already collected. In this paper we explore a tool for gaining design experience: computer-based virtual experiments. These are software environments which mimic a real situation of interest and invite the user to collect data to answer a research question. Two prototype environments are described. The first one is suitable for a course that deals with screening or response surface designs, the second one allows experimenting with block and row-column designs. They are parts of a collection we developed called ENV2EXP, and can be freely used over the web. We also describe our experience in using them in several courses over the last few years. [source] Experimental Design Applied to Silicon Carbide SinteringJOURNAL OF THE AMERICAN CERAMIC SOCIETY, Issue 7 2003Juliana Marchi Silicon carbide is a promising structural ceramic used as abrasives and applied in metallurgical components, due to its low density, high hardness, and excellent mechanical properties. The composition and content of the additive can control liquid-phase sintering of SiC. Compositions based on the SiO2,Al2O3,RE2O3 system (RE = rare earth) have been largely used to promote silicon carbide densification, but most studies are not systematically presented. The aim of this work is to study the effect of several oxide additives in the SiO2,Al2O3,Y2O3 system on the densification of silicon carbide using experimental design. This technique seems to be effective in optimizing the values of maximum density with minimum weight loss. [source] Experimental Design and Analysis for Tree ImprovementJOURNAL OF THE ROYAL STATISTICAL SOCIETY: SERIES A (STATISTICS IN SOCIETY), Issue 2 2004Gillian M. Arnold No abstract is available for this article. [source] Consistency of a two clinical site sample collection: A proteomics studyPROTEOMICS - CLINICAL APPLICATIONS, Issue 8-9 2010Cedric Wiesner Abstract Purpose: We investigated the ability to perform a clinical proteomic study using samples collected at different times from two independent clinical sites. Experimental Design: Label-free 2-D-LC-MS proteomic analysis was used to differentially quantify tens of thousands of peptides from human plasma. We have asked whether samples collected from two sites, when analyzed by this type of peptide profiling, reproducibly contain detectable peptide markers that are differentially expressed in the plasma of disease (advanced renal cancer) patients relative to healthy normals. Results: We have demonstrated that plasma proteins enriched in disease patients are indeed detected reproducibly in both clinical collections. Regression analysis, unsupervised hierarchical clustering and PCA detected no systematic bias in the data related to site of sample collection and processing. Using a genetic algorithm, support vector machine classification method, we were able to correctly classify disease samples at 88% sensitivity and 94% specificity using the second site as an independent validation set. Conclusions and clinical relevance: We conclude that multiple site collection, when analyzed by label-free 2-D-LC-MS, generates data that are sufficiently reproducible to guide reliable biomarker discovery. [source] Experimental Design and Data Analysis for BiologistsAUSTRAL ECOLOGY, Issue 5 2003Julian R.W. Reid No abstract is available for this article. [source] DNA Methylation Microarrays: Experimental Design and Statistical Analysis by WANG, S.-C. and PETRONIS, A.BIOMETRICS, Issue 1 2009Kimberly D. Siegmund No abstract is available for this article. [source] Framework for the Rapid Optimization of Soluble Protein Expression in Escherichia coli Combining Microscale Experiments and Statistical Experimental DesignBIOTECHNOLOGY PROGRESS, Issue 4 2007R. S. Islam A major bottleneck in drug discovery is the production of soluble human recombinant protein in sufficient quantities for analysis. This problem is compounded by the complex relationship between protein yield and the large number of variables which affect it. Here, we describe a generic framework for the rapid identification and optimization of factors affecting soluble protein yield in microwell plate fermentations as a prelude to the predictive and reliable scaleup of optimized culture conditions. Recombinant expression of firefly luciferase in Escherichia coli was used as a model system. Two rounds of statistical design of experiments (DoE) were employed to first screen (D-optimal design) and then optimize (central composite face design) the yield of soluble protein. Biological variables from the initial screening experiments included medium type and growth and induction conditions. To provide insight into the impact of the engineering environment on cell growth and expression, plate geometry, shaking speed, and liquid fill volume were included as factors since these strongly influence oxygen transfer into the wells. Compared to standard reference conditions, both the screening and optimization designs gave up to 3-fold increases in the soluble protein yield, i.e., a 9-fold increase overall. In general the highest protein yields were obtained when cells were induced at a relatively low biomass concentration and then allowed to grow slowly up to a high final biomass concentration, >8 g·L,1. Consideration and analysis of the model results showed 6 of the original 10 variables to be important at the screening stage and 3 after optimization. The latter included the microwell plate shaking speeds pre- and postinduction, indicating the importance of oxygen transfer into the microwells and identifying this as a critical parameter for subsequent scale translation studies. The optimization process, also known as response surface methodology (RSM), predicted there to be a distinct optimum set of conditions for protein expression which could be verified experimentally. This work provides a generic approach to protein expression optimization in which both biological and engineering variables are investigated from the initial screening stage. The application of DoE reduces the total number of experiments needed to be performed, while experimentation at the microwell scale increases experimental throughput and reduces cost. [source] Imaging of cell trafficking and metastases of paediatric rhabdomyosarcomaCELL PROLIFERATION, Issue 2 2008G. Seitz Objective:,The aim of this study was to establish a preclinical mouse model to study metastases of paediatric rhabdomyosarcoma at the macroscopic and cellular levels, with different imaging methods. Experimental Design:,The alveolar rhabdomyosarcoma cell line Rh30 was stably transfected with the red fluorescent protein (DsRed2) then was xenotransplanted (intravenous injection [n = 8], and footpad injection [n = 8]) into nude mice (NMRI nu/nu). Macroscopic imaging of metastases was performed using DsRed2-fluorescence and flat-panel volumetric computed tomography scan. In a further series of animals (n = 8), in vivo cell trafficking of rhabdomyosarcoma cells using cellular imaging with an Olympus OV100 variable-magnification small-animal imaging system was used. Results:,Metastases in the pelvis, thoracic wall and skin were visualized by fluorescence imaging. Pelvic metastases were found after tail vein injection and at other metastatic sites after footpad injection. Flat-panel volumetric computed tomography scan data allowed highly specific analysis of contrast between tumour and surrounding tissue. Correlation between fluorescence and flat-panel volumetric computed tomography scan imaging data was observed. Single-cell imaging visualized tumour cells in the vessels and demonstrated the arrest of tumour cells at vessel junctions followed by extravasation of the tumour cells. Conclusion:,We established a model for visualization of experimental metastatic invasion and describe relevant tools for imaging childhood rhabdomyosarcoma metastases at the macroscopic and cellular levels. Imaging of cell trafficking visualized the behaviour of tumour cells and development of metastases by accumulation and extravasation of rhabdomyosarcoma cells. [source] A Comparison of Three Experimental Designs for the Field Assessment of Resistance to Rice Blast Disease (Pyricularia oryzae)JOURNAL OF PHYTOPATHOLOGY, Issue 4 2007D. Katsantonis Abstract The reliability and the effectiveness of three field experimental designs for the assessment of varietal resistance to Pyricularia oryzae was studied for 2 years. Plants were arranged in the field using two sets of three designs: randomized compete block (RCB), adjacent control (AC) and honeycomb (HC). In the first set, plants were inoculated with the fungus and in the second one, plants were naturally infected. Four varieties were used, Maratelli (susceptible control), Roxani, Selenio and Senia (main varieties). Disease progress was recorded as leaf and neck blast. The number of plants of the main varieties required in each design was higher for RCB (90) and lower for AC and HC (30 and 32, respectively). Results showed that disease severity was significantly different in the varieties studied and it was consistent and not affected by the plant arrangement in the field. All experimental designs were reliable and effective for the assessment of leaf and neck blast under inoculation or natural infection. AC and HC designs had the advantage of requiring fewer plants over RCB one. This could be important in cases where seed availability is a limiting factor. [source] Optimum Experimental Designs, with SASJOURNAL OF THE ROYAL STATISTICAL SOCIETY: SERIES A (STATISTICS IN SOCIETY), Issue 2 2008Z. Q. John Lu No abstract is available for this article. [source] Searching and oviposition behavior of a mymarid egg parasitoid, Anagrus nigriventris, on five host plant species of its leafhopper host, Circulifer tenellusENTOMOLOGIA EXPERIMENTALIS ET APPLICATA, Issue 1 2000A.K. Al-Wahaibi Abstract Searching and oviposition behavior and parasitization ability of Anagrus nigriventris Girault (Hymenoptera: Mymaridae), an egg parasitoid of beet leafhopper, Circulifer tenellus (Baker) (Homoptera: Cicadellidae), were examined on five host plant species of beet leafhopper: sugar beet (Beta vulgaris L.), red stem filaree (Erodium cicutarium[L.]), peppergrass (Lepidium nitidum Nuttall), desert plantain (Plantago ovata Forsskal), and London rocket (Sisymbrium irio L.). Beet leafhopper embeds its eggs in the tissues of these plant species. For each plant species, A. nigriventris behavior was examined on plants with and without beet leafhopper eggs. Experimental design was a 5 (plant species) by 2 (host eggs present/absent) factorial. Additionally within each treatment, parasitoid behavior was observed over a 22-h period at five different observation periods: t=0, 3, 6, 9, and 22 h where t=0 h represents initial exposure of the insect with the plant. The behavioral events observed were: ,fast walking' (general searching), ,slow walking' (intensive searching), ovipositor probing, grooming, feeding, and resting. Significant differences (,=0.05) among plant species in time spent on the plant, percentage of host eggs parasitized, and behavioral variables associated with intensive searching and oviposition all indicated that the plant species fell into two groups: ,preferred' plants (sugar beet, London rocket, and peppergrass), and ,unpreferred' plants (filaree and plantago). These variables also indicated that the parasitoids spent more time on, searched more, probed more, and oviposited more in plants with host eggs than plants without host eggs. Consistent effects of time (over the observation periods from t=0 to t=22 h) generally were detected only in the preferred plant species that had host eggs present. In these cases, intensive searching and probing decreased as time advanced, while variables related to general searching (,fast walking') and abandoning host egg patches (leaving the plant) tended to increase over time. [source] Kinetics of intra- and intermolecular zymogen activation with formation of an enzyme,zymogen complexFEBS JOURNAL, Issue 1 2005Matilde Esther Fuentes A mathematical description was made of an autocatalytic zymogen activation mechanism involving both intra- and intermolecular routes. The reversible formation of an active intermediary enzyme,zymogen complex was included in the intermolecular activation route, thus allowing a Michaelis,Menten constant to be defined for the activation of the zymogen towards the active enzyme. Time,concentration equations describing the evolution of the species involved in the system were obtained. In addition, we have derived the corresponding kinetic equations for particular cases of the general model studied. Experimental design and kinetic data analysis procedures to evaluate the kinetic parameters, based on the derived kinetic equations, are suggested. The validity of the results obtained were checked by using simulated progress curves of the species involved. The model is generally good enough to be applied to the experimental kinetic study of the activation of different zymogens of physiological interest. The system is illustrated by following the transformation kinetics of pepsinogen into pepsin. [source] Effect of Evidence-Based Acute Pain Management Practices on Inpatient CostsHEALTH SERVICES RESEARCH, Issue 1 2009John M. Brooks Objectives. To estimate hospital cost changes associated with a behavioral intervention designed to increase the use of evidence-based acute pain management practices in an inpatient setting and to estimate the direct effect that changes in evidence-based acute pain management practices have on inpatient cost. Data Sources/Study Setting. Data from a randomized "translating research into practice" (TRIP) behavioral intervention designed to increase the use of evidence-based acute pain management practices for patients hospitalized with hip fractures. Study Design. Experimental design and observational "as-treated" and instrumental variable (IV) methods. Data Collection/Extraction Methods. Abstraction from medical records and Uniform Billing 1992 (UB92) discharge abstracts. Principal Findings. The TRIP intervention cost on average $17,714 to implement within a hospital but led to cost savings per inpatient stay of more than $1,500. The intervention increased the cost of nursing services, special operating rooms, and therapy services per inpatient stay, but these costs were more than offset by cost reductions within other cost categories. "As-treated" estimates of the effect of changes in evidence-based acute pain management practices on inpatient cost appear significantly underestimated, whereas IV estimates are statistically significant and are distinct from, but consistent with, estimates associated with the intervention. Conclusions. A hospital treating more that 12 patients with acute hip fractures can expect to lower overall cost by implementing the TRIP intervention. We also demonstrated the advantages of using IV methods over "as-treated" methods to assess the direct effect of practice changes on cost. [source] Translating Research into Practice Intervention Improves Management of Acute Pain in Older Hip Fracture PatientsHEALTH SERVICES RESEARCH, Issue 1 2009Marita G. Titler Objective. To test an interdisciplinary, multifaceted, translating research into practice (TRIP) intervention to (a) promote adoption, by physicians and nurses, of evidence-based (EB) acute pain management practices in hospitalized older adults, (b) decrease barriers to use of EB acute pain management practices, and (c) decrease pain intensity of older hospitalized adults. Study Design. Experimental design with the hospital as the unit of randomization. Study Setting. Twelve acute care hospitals in the Midwest. Data Sources. (a) Medical records (MRs) of patients ,65 years or older with a hip fracture admitted before and following implementation of the TRIP intervention and (b) physicians and nurses who care for those patients. Data Collection. Data were abstracted from MRs and questions distributed to nurses and physicians. Principal Findings. The Summative Index for Quality of Acute Pain Care (0,18 scale) was significantly higher for the experimental (10.1) than comparison group (8.4) at the end of the TRIP implementation phase. At the end of the TRIP implementation phase, patients in the experimental group had a lower mean pain intensity rating than those in the comparison group ( p<.0001). Conclusion. The TRIP intervention improved quality of acute pain management of older adults hospitalized with a hip fracture. [source] A Process Analytical Technology approach to near-infrared process control of pharmaceutical powder blending.JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2006Part I: D-optimal design for characterization of powder mixing, preliminary spectral data evaluation Abstract Experimental design, multivariate data acquisition, and analysis in addition to real time monitoring and control through process analyzers, represent an integrated approach for implementation of Process Analytical Technology (PAT) in the pharmaceutical industry. This study, which is the first in a series of three parts, uses an experimental design approach to identify critical factors affecting powder blending. Powder mixtures composed of salicylic acid and lactose were mixed in an 8 qt. V-blender. D-optimal design was employed to characterize the blending process, by studying the effect of humidity, component concentration, and blender speed on mixing end point. Additionally, changes in particle size and density of powder mixtures were examined. A near-infrared (NIR) fiber-optic probe was used to monitor mixing, through multiple optical ports on the blender. Humidity, component concentration, and blender speed were shown to have a significant impact on the blending process. Furthermore, humidity and concentration had a significant effect on particle size and density of powder mixtures. NIRS was sensitive to changes in physicochemical properties of the mixtures, resulting from process variables. Proper selection of NIR spectral preprocessing is of ultimate importance for successful implementation of this technology in the monitoring and control of powder blending and is discussed. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:392,406, 2006 [source] Dynamic Compressive Failure of AlON Under Controlled Planar ConfinementJOURNAL OF THE AMERICAN CERAMIC SOCIETY, Issue 11 2008Bhasker Paliwal An experimental technique is developed to impose a planar lateral confinement in a prismatic specimen (with a rectangular cross section); the setup enabled a controlled and homogeneous stress state with high lateral compressive stresses. A transparent polycrystalline aluminum oxynitride (AlON) specimen was used for the study. The statically precompressed specimen was then subjected to axial dynamic compressive loading using a modified compression Kolsky bar setup. Experimental design was performed using 3D computational modeling. Initial exploratory experiments were conducted on AlON at an average planar confinement of 400,410 MPa; the results suggested a higher compressive strength and a nonlinear stress evolution in AlON due to the confinement. A high-speed camera was used to observe the damage evolution in the specimen during the course of loading. The photographs and stress evolution are suggestive of an additional inelastic deformation mechanism, whose evolution is slower than the typical brittle-cracking type of damage apparent in the unconfined case. The TEM and high-resolution electron microscope analysis indicated dislocation plasticity in some fragments; dislocations in the slip bands were characterized to be dissociated ,110, dislocations on {111} planes. The width between two partial dislocations was about 15 nm, suggesting low stacking fault energy of AlON. Microscopic characterization also shows that the eventual fragmentation of AlON is by cleavage mainly along low-index {111} planes. [source] Experimental design and taxonomic scope of fragmentation studies on European mammals: current status and future prioritiesMAMMAL REVIEW, Issue 2 2010Alessio MORTELLITI ABSTRACT 1Habitat loss, habitat fragmentation and habitat degradation are the greatest threats to mammals in Europe and the rest of the world. Despite the fact that extensive literature exists, no comprehensive review or synthesis is available to date and this may slow down scientific progress and hamper conservation efforts. 2The goal of this study is to understand if and in what direction progress has been made in the study of the effects of habitat loss and fragmentation on the spatial distribution of European terrestrial mammals. Firstly, we carry out a general synthesis which is structured around 11 key points. The aim of this point-by-point analysis is to identify trends, knowledge gaps and any significant bias in the available literature, and to highlight strengths and shortfalls of the different approaches which have to date been applied. Secondly, we follow a species-specific systematic approach: for each species, we synthesise key results. 3Our results show how substantial progress has been hampered for several reasons including: a large predominance of small-scale field studies of short duration, and a generalised lack of control of: (i) confounding variables; (ii) spatial autocorrelation; and (iii) false absences. Also, despite the relatively high number of studies, few were theoretical studies and even fewer were meta-analyses. The lack of meta-analyses is likely to be due to the small amount of crucial details included in the publications, such as model parameters or information on the landscape context (such as the amount of residual forest cover). 4We synthesise the main results for 14 species. The level of progress is highly variable: for some species, such as the red squirrel Sciurus vulgaris, a series of long-term, large-scale process-oriented studies has allowed an in-depth understanding of its ecology in fragmented landscapes. On the other hand, with other species such as the bank vole Myodes glareolus, despite a relatively large number of field studies, little progress has been made. [source] Impact of the storage temperature on human plasma proteomic analysis: Implications for the use of human plasma collections in researchPROTEOMICS - CLINICAL APPLICATIONS, Issue 8-9 2010María Insenser Abstract Purpose: To analyze the impact of storage temperature on the 2D-DIGE profile of human plasma. Experimental design: 2D-DIGE and MS were used to identify the differences in the proteomic profiles of aliquots of eight human plasma samples stored at either ,30 or ,80°C for 18 months. Results: After the depletion of albumin and IgG, 2D-DIGE identified four spots significantly and reproducibly increased, and five spots that were decreased, in samples stored at ,30°C compared with those stored at ,80°C. These nine spots were manually excised, digested in-gel and analyzed by MALDI-TOF/TOF. MASCOT database search using the PMF spectra allowed the identification of the proteins present in eight of the nine spots. All the spots corresponded to the complement C3 precursor protein. Conclusions and clinical relevance: Our present results indicate that plasma collections stored at ,30°C, and not only those stored at ,80°C, may be used for 2D-DIGE analyses of albumin- and IgG-depleted human plasma without loosing the essential information about highly abundant proteins. This finding expands the applicability of 2D-DIGE to the study of human disease by permitting the analysis of human plasma samples stored at temperatures between ,30 and ,80°C. [source] Identification of breast cancer biomarkers in transgenic mouse models: A proteomics approachPROTEOMICS - CLINICAL APPLICATIONS, Issue 6-7 2010Wendy Rodenburg Abstract Purpose: Transgenic mouse models for cancer circumvent many challenges that hamper human studies aimed at biomarker discovery. Lower biological variances among mice combined with controllable factors such as food uptake and health status may enable the detection of more subtle protein expression differences. This is envisioned to result in the identification of biomarkers better discriminating cancer cases from controls. Experimental design: The current study used two innovative mouse models for breast-cancer to identify new serum biomarkers. Multi-analyte profiling technique was used to analyze 70 proteins in individual serum samples of non-tumor and mammary tumor-bearing Tg.NK (MMTV/c-neu) mice. Results: A small set of proteins fully differentiated tumor samples from controls. These comprised osteopontin, interleukin-18, cystatin C and CD40 antigen. Comparison of protein expression in another breast-cancer mouse model, the humanized p53.R270H mice, showed common discriminatory expression of osteopontin. However, other biomarkers showed distinct expression in the two different breast-cancer models, indicating that different mammary tumor sub-types with respect to molecular and estrogen receptor status reveal divergent serum biomarker sets. Conclusions and clinical relevance: The current study supports the concept that serum proteins can discriminate mammary tumor cases from controls, and yielded interesting biomarkers that need further testing and validation in human studies. [source] A non-invasive method based on saliva to characterize transthyretin in familial amyloidotic polyneuropathy patients using FT-ICR high-resolution MSPROTEOMICS - CLINICAL APPLICATIONS, Issue 6-7 2010Gonçalo da Costa Abstract Purpose: To identify, characterize and perform a relative quantification of human transthyretin (TTR) variants in human saliva. Experimental design: Serum and saliva samples were collected from healthy and familial amyloidotic polyneuropathy (FAP) patients, proteins separated by SDS-PAGE, TTR bands excised, in-gel digested and analyzed by MALDI-FTICR. Results: We identified and performed a relative quantification of mutated and native TTR forms in human saliva, based on FTICR-MS. The results are quantitatively identical to the ones obtained with human serum. In FAP patients subjected to cadaveric liver transplant, the TTR mutant form is no longer detected in saliva, while in patients receiving a domino liver from a FAP donor the mutant form of TTR becomes detectable in saliva, thus demonstrating the serum origin of TTR in saliva. Conclusions and clinical relevance: Saliva TTR originates in serum and the ratio of mutant to native TTR is preserved. The method provides a non-invasive detection of mutated TTR and a relative quantification of TTR forms. Diagnostic and disease prognosis of FAP is crucial at early stages of the disease and after liver transplantation, the only curative therapy. A suitable non-invasive method was developed for monitoring the most important FAP biomarker in human saliva. [source] MS analysis of rheumatoid arthritic synovial tissue identifies specific citrullination sites on fibrinogenPROTEOMICS - CLINICAL APPLICATIONS, Issue 5 2010Monika Hermansson Abstract Purpose: Citrullination is a post-translational modification of arginine residues to citrulline catalyzed by peptidyl arginine deiminases. Induced expression of citrullinated proteins are frequently detected in various inflammatory states including arthritis; however, direct detection of citrullination in arthritic samples has not been successfully performed in the past. Experimental design: Citrullination of human fibrinogen, a candidate autoantigen in arthritis, was studied. Accurate identification of citrullinated fibrinogen peptides from rheumatoid arthritis synovial tissue specimens was performed using accurate mass and retention time analysis. Results: A peptide with the sequence ESSSHHPGIAEFPSRGK corresponding to amino acids 559,575 of fibrinogen ,-chain was identified to be citrullinated with an occupancy rate between 1.4 and 2.5%. Citrullination of the peptide KREEAPSLRPAPPPISGGGYRARPAK corresponding to amino acids 52,77 of the fibrinogen ,-chain was identified with an occupancy rate of 1.2%. Conclusions and clinical relevance: We report a proof of principle study for the identification of citrullinated proteins and within them, identification of citrullination sites and quantification of their occupancies in synovial tissue from rheumatoid arthritis patients using high-resolution MS. Detailed studies on which molecules are citrullinated in arthritis can provide information about their role in immune regulation and serve as novel biomarkers and potentially even as therapeutic targets. [source] Protein profiling in pathology: Analysis and evaluation of 239 frozen tissue biopsies for diagnosis of B-cell lymphomasPROTEOMICS - CLINICAL APPLICATIONS, Issue 5 2010Corine Jansen Abstract Purpose: We determined the potential value of protein profiling of tissue samples by assessing how precise this approach enables discrimination of B-cell lymphoma from reactive lymph nodes, and how well the profiles can be used for lymphoma classification. Experimental design: Protein lysates from lymph nodes (n=239) from patients with the diagnosis of reactive hyperplasia (n=44), follicular lymphoma (n=63), diffuse large B-cell lymphoma (n=43), mantle cell lymphoma (n=47), and chronic lymphocytic leukemia/small lymphocytic B-cell lymphoma (n=42) were analysed by SELDI-TOF MS. Data analysis was performed by (i) classification and regression tree-based analysis and (ii) binary and polytomous logistic regression analysis. Results: After internal validation by the leave-one-out principle, both the classification and regression tree and logistic regression classification correctly identified the majority of the malignant (87 and 96%, respectively) and benign cases (73 and 75%, respectively). Classification was less successful since approximately one-third of the cases of each group were misclassified according to the histological classification. However, an additional mantle cell lymphoma case that was misclassified as chronic lymphocytic leukemia/small lymphocytic B-cell lymphoma initially was identified based on the protein profile. Conclusions and clinical relevance: SELDI-TOF MS protein profiling allows for reliable identification of the majority of malignant lymphoma cases; however, further validation and testing robustness in a diagnostic setting is needed. [source] Glycosylation of liver acute-phase proteins in pancreatic cancer and chronic pancreatitisPROTEOMICS - CLINICAL APPLICATIONS, Issue 4 2010Ariadna Sarrats Abstract Purpose: Glycosylation of acute-phase proteins (APP), which is partially regulated by cytokines, may be distinct in disease and provide useful tumour markers. Thus, we have examined the glycosylation of major serum APP in pancreatic cancer (PaC), chronic pancreatitis (CP) and control patients. Experimental design: Using a specific anti-sialyl Lewis X antibody and N -glycan sequencing, we have determined glycosylation changes on ,-1-acid glycoprotein (AGP), haptoglobin (HPT), fetuin (FET), ,-1-antitrypsin (AT) and transferrin (TRF). Results: Increased levels of sialyl Lewis X (SLex) were detected on AGP in advanced PaC and CP and on HPT, FET, AT and TRF in CP. An increase in N -glycan branching was detected on AGP and HPT in the advanced stage of PaC and CP and on FET and TRF in the CP. A core fucosylated structure was increased on AGP and HPT only in the advanced PaC patients. Conclusions and clinical relevance: Changes in APP SLex and branching are probably associated with an inflammatory response because they were detected in both advanced PaC and CP patients and these conditions give rise to inflammation. On the contrary, the increase in APP core fucosylation could be cancer associated and the presence of this glycoform may give an advantage to the tumour. [source] Comparative proteomic analysis of differentially expressed proteins in primary retinoblastoma tumorsPROTEOMICS - CLINICAL APPLICATIONS, Issue 4 2010Kandalam Mallikarjuna Abstract Purpose: To understand the disease mechanism and to identify the potential tumor markers that would help in therapeutics, comparative proteomic analysis of 29 retinoblastoma (RB) tumors was performed using 14 non-neoplastic retinas (age ranged from 45 to 89 years) as control tissues. Experimental design: 2-DE and MALDI-TOF-TOF MS/MS were used to identify differentially expressed proteins. Results: Twenty-seven distinct differentially expressed proteins were identified, including 16 upregulated 11 downregulated proteins. Significantly, higher mRNA levels of apolipoprotein A1 (p<0.001), transferrin (TF; p<0.001), CRABP2 (p<0.001), ,-crystallin A (CRYAA; p<0.001) were observed in RBs when compared with normal retinas and hence are consistent with the proteomic data. Immunohistochemistry was also performed for selected proteins on paraffin RB blocks to confirm protein expression. RB with invasion showed significantly higher expression by 2-DE-MS/MS analysis of CRABP2 (p<0.001), peroxiredoxin 6 (p=0.025), apolipoprotein A1 (p<0.001), recoverin (p<0.001). Conclusions and clinical relevance: Thus, this study provides a dynamic protein profile of RB tumors, which could provide clues to study the mechanisms of RB oncogenesis and possibly be developed as potential biomarkers for prognosis and therapy. [source] Comparison of proteomic biomarker panels in urine and serum for ovarian cancer diagnosisPROTEOMICS - CLINICAL APPLICATIONS, Issue 3 2010Anette Lykke Petri Abstract Purpose: The purposes of this study were to confirm previously found candidate epithelial ovarian cancer biomarkers in urine and to compare a paired serum biomarker panel and a urine biomarker panel from the same study cohort with regard to the receiver operating characteristic curve (ROC) area under the ROC curve (AUC) values. Experimental design: Four significant urine biomarkers were confirmed among 130 pelvic mass patients in the present study. The four biomarkers form a potential urine biomarker panel. From the same study cohort, the potential urine biomarker panel was compared to a serum biomarker panel, consisting of seven proteins/peptides, OvaRI. Results: Multivariate analysis of the urine panel demonstrated a significant differentiation (p<0.0001) between epithelial ovarian cancer patients and patients with benign ovarian pelvic masses. The ROC AUC of the urine panel was 0.84 and the ROC AUC of OvaRI was 0.83. Combining the urine panel with OvaRI demonstrated a significant contribution from both, for urine peaks, OR=2.12 and for OvaRI, OR=1.39; the ROC AUC of this model was 0.88. Conclusions and clinical relevance: We demonstrated that both urine and serum can be used individually or in combination to potentially aid in ovarian cancer diagnostics. Urine proteomic profiling could provide biomarkers for the non-invasive test required in clinical practice. [source] Is GRP78/BiP a potential salivary biomarker in patients with rheumatoid arthritis?PROTEOMICS - CLINICAL APPLICATIONS, Issue 3 2010Laura Giusti Abstract Purpose: In the last few years, serum and joint synovial fluid have been extensively analyzed for the proteomic research of rheumatoid arthritis (RA) biomarkers. Nonetheless, to date, there have been no studies investigating salivary biomarkers in this condition. Therefore, aim of this study is to investigate the presence of potential biomarkers of RA in human whole saliva. Experimental design: We combined 2-DE and MS to analyze the whole saliva protein profile of 20 RA patients in comparison with 20 sex- and age-matched healthy subjects. Results: Eight salivary proteins resulted differentially expressed, namely calgranulin A, calgranulin B, apolipoprotein A-1, 6-phosphogluconate dehydrogenase, peroxiredoxin 5, epidermal fatty acid-binding protein, 78,kDa glucose-regulated protein precursor (GRP78/BiP), and 14-3-3 proteins. It is particularly interesting that chaperone GRP78/BiP showed the greatest increase in RA patients. This finding was validated by Western Blot analysis and the over-expression of GRP78/BiP appear to be distinctive of RA and drugs treatment independent. Conclusions and clinical relevance: This study provides a rationale for further studies aimed at evaluating any correlation between GRP78/BiP and different clinical/serological aspects of the disease in order to improve the diagnostic algorithms of RA. [source] Proteomic examination of Leishmania chagasi plasma membrane proteins: Contrast between avirulent and virulent (metacyclic) parasite formsPROTEOMICS - CLINICAL APPLICATIONS, Issue 1 2010Chaoqun Yao Abstract Purpose: About two million new cases of leishmaniasis with 50 000 associated deaths occur worldwide each year. Promastigotes of the causative Leishmania spp. develop from the procyclic stage to the highly virulent metacyclic stage within the sand fly vector. We hypothesized that proteins important for promastigote virulence might be uniquely represented in the plasma membrane of metacyclic, but not procyclic, promastigotes. Experimental design: Procyclic (logarithmic) promastigotes and purified metacyclic promastigotes from stationary phase cultures of Leishmania chagasi were used to prepare membrane preparations either by surface biotinylation-streptavidin affinity separation or by octyl glucoside detergent extraction. Results: These membrane fractions were enriched over 130- and 250-fold, respectively, as estimated by Western blotting for the plasma membrane's major surface protease. Hundreds or dozens of proteins were identified by LC-MS/MS in the surface biotinylation or detergent extraction, respectively. Confocal microscopy suggested the difference between the lists was due to the fact that proteins localized both on the surface membrane and within the flagellar pocket were accessible to surface biotinylation, whereas only proteins on the membrane were obtained by detergent extraction. Using detergent extraction, we found different proteins were present in membranes of the procyclic stage compared to metacyclic stage promastigotes. Several dozen were stage specific. Conclusions and clinical relevance: These data provide a foundation for identifying virulence factors in the plasma membranes of Leishmania spp. promastigotes during metacyclogenesis. [source] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||