Quantitative proteomic analysis by iTRAQ® for the identification of candidate biomarkers in ovarian cancer serum

<p>Abstract</p> <p>Background</p> <p>Ovarian cancer is the most lethal gynecologic malignancy, with the majority of cases diagnosed at an advanced stage when treatments are less successful. Novel serum protein markers are needed to detect ovarian cancer in its earliest stage; when detected early, survival rates are over 90%. The identification of new serum biomarkers is hindered by the presence of a small number of highly abundant proteins that comprise approximately 95% of serum total protein. In this study, we used pooled serum depleted of the most highly abundant proteins to reduce the dynamic range of proteins, and thereby enhance the identification of serum biomarkers using the quantitative proteomic method iTRAQ^®.</p> <p>Results</p> <p>Medium and low abundance proteins from 6 serum pools of 10 patients each from women with serous ovarian carcinoma, and 6 non-cancer control pools were labeled with isobaric tags using iTRAQ^®to determine the relative abundance of serum proteins identified by MS. A total of 220 unique proteins were identified and fourteen proteins were elevated in ovarian cancer compared to control serum pools, including several novel candidate ovarian cancer biomarkers: extracellular matrix protein-1, leucine-rich alpha-2 glycoprotein-1, lipopolysaccharide binding protein-1, and proteoglycan-4. Western immunoblotting validated the relative increases in serum protein levels for several of the proteins identified.</p> <p>Conclusions</p> <p>This study provides the first analysis of immunodepleted serum in combination with iTRAQ^®to measure relative protein expression in ovarian cancer patients for the pursuit of serum biomarkers. Several candidate biomarkers were identified which warrant further development.</p

The ratio of means method as an alternative to mean differences for analyzing continuous outcome variables in meta-analysis: A simulation study

<p>Abstract</p> <p>Background</p> <p>Meta-analysis of continuous outcomes traditionally uses mean difference (MD) or standardized mean difference (SMD; mean difference in pooled standard deviation (SD) units). We recently used an alternative ratio of mean values (RoM) method, calculating RoM for each study and estimating its variance by the delta method. SMD and RoM allow pooling of outcomes expressed in different units and comparisons of effect sizes across interventions, but RoM interpretation does not require knowledge of the pooled SD, a quantity generally unknown to clinicians.</p> <p>Objectives and methods</p> <p>To evaluate performance characteristics of MD, SMD and RoM using simulated data sets and representative parameters.</p> <p>Results</p> <p>MD was relatively bias-free. SMD exhibited bias (~5%) towards no effect in scenarios with few patients per trial (n = 10). RoM was bias-free except for some scenarios with broad distributions (SD 70% of mean value) and medium-to-large effect sizes (0.5–0.8 pooled SD units), for which bias ranged from -4 to 2% (negative sign denotes bias towards no effect). Coverage was as expected for all effect measures in all scenarios with minimal bias. RoM scenarios with bias towards no effect exceeding 1.5% demonstrated lower coverage of the 95% confidence interval than MD (89–92% vs. 92–94%). Statistical power was similar. Compared to MD, simulated heterogeneity estimates for SMD and RoM were lower in scenarios with bias because of decreased weighting of extreme values. Otherwise, heterogeneity was similar among methods.</p> <p>Conclusion</p> <p>Simulation suggests that RoM exhibits comparable performance characteristics to MD and SMD. Favourable statistical properties and potentially simplified clinical interpretation justify the ratio of means method as an option for pooling continuous outcomes.</p

FindFoci: a focus detection algorithm with automated parameter training that closely matches human assignments, reduces human inconsistencies and increases speed of analysis

Accurate and reproducible quantification of the accumulation of proteins into foci in cells is essential for data interpretation and for biological inferences. To improve reproducibility, much emphasis has been placed on the preparation of samples, but less attention has been given to reporting and standardizing the quantification of foci. The current standard to quantitate foci in open-source software is to manually determine a range of parameters based on the outcome of one or a few representative images and then apply the parameter combination to the analysis of a larger dataset. Here, we demonstrate the power and utility of using machine learning to train a new algorithm (FindFoci) to determine optimal parameters. FindFoci closely matches human assignments and allows rapid automated exploration of parameter space. Thus, individuals can train the algorithm to mirror their own assignments and then automate focus counting using the same parameters across a large number of images. Using the training algorithm to match human assignments of foci, we demonstrate that applying an optimal parameter combination from a single image is not broadly applicable to analysis of other images scored by the same experimenter or by other experimenters. Our analysis thus reveals wide variation in human assignment of foci and their quantification. To overcome this, we developed training on multiple images, which reduces the inconsistency of using a single or a few images to set parameters for focus detection. FindFoci is provided as an open-source plugin for ImageJ

Fundamental spray and combustion measurements of soy methyl-ester biodiesel

Although biodiesel has begun to penetrate the fuel market, its effect on injection processes, combustion and emission formation under diesel engine conditions remains somewhat unclear. Typical exhaust measurements from engines running biodiesel indicate that particulate matter, carbon monoxide and unburnt hydrocarbons are decreased, whereas nitrogen oxide emissions tend to be increased. However, these observations are the result of complex interactions between physical and chemical processes occurring in the combustion chamber, for which understanding is still needed. To characterize and decouple the physical and chemical influences of biodiesel on spray mixing, ignition, combustion and soot formation, a soy methyl-ester (SME) biodiesel is injected into a constant-volume combustion facility under diesel-like operating conditions. A range of optical diagnostics is performed, comparing biodiesel to a conventional #2 diesel at the same injection and ambient conditions. Schlieren high-speed imaging shows virtually the same vapour-phase penetration for the two fuels, while simultaneous Mie-scatter imaging shows that the maximum liquid-phase penetration of biodiesel is higher than diesel. Differences in the liquid-phase penetration are expected because of the different boiling-point temperatures of the two fuels. However, the different liquid-phase penetration does not affect overall mixing rate and downstream vapour-phase penetration because each fuel spray has similar momentum and spreading angle. For the biodiesel and diesel samples used in this study, the ignition delay and lift-off length are only slightly less for biodiesel compared to diesel, consistent with the fuel cetane number (51 for biodiesel, 46 for diesel). Because of the similarity in lift-off length, the differences in equivalence ratio distribution at the lift-off length are mainly affected by the oxygen content of the fuels. For biodiesel, the equivalence ratio is reduced, which, along with the fuel molecular structure and oxygen content, significantly affects soot formation downstream. Spatially resolved soot volume fraction measurements obtained by combining line-of-sight laser extinction measurements with planar laser-induced incandescence imaging show that the soot concentration can be reduced by an order of magnitude for biodiesel. These integrated measurements of spray mixing, combustion and quantitative soot concentration provide new validation data for the development of computational fluid dynamics spray, combustion and soot formation models suitable for the latest biofuels.This work was supported by the Spanish Ministry of Science and Innovation for Jean-Guillaume Nerva's visiting research, through the OPTICOMB project [TRA2007-67961-C03-01].Nerva, J.; Genzale, CL.; Kook, S.; García Oliver, JM.; Pickett, LM. (2013). Fundamental spray and combustion measurements of soy methyl-ester biodiesel. International Journal of Engine Research. 14(4):373-390. https://doi.org/10.1177/1468087412456688S373390144(2009). World Energy Outlook 2009. World Energy Outlook. doi:10.1787/weo-2009-enMonyem, A., & H. Van Gerpen, J. (2001). The effect of biodiesel oxidation on engine performance and emissions. Biomass and Bioenergy, 20(4), 317-325. doi:10.1016/s0961-9534(00)00095-7LAPUERTA, M., ARMAS, O., & RODRIGUEZFERNANDEZ, J. (2008). Effect of biodiesel fuels on diesel engine emissions. Progress in Energy and Combustion Science, 34(2), 198-223. doi:10.1016/j.pecs.2007.07.001Fisher, B. T., Knothe, G., & Mueller, C. J. (2010). Liquid-Phase Penetration under Unsteady In-Cylinder Conditions: Soy- and Cuphea-Derived Biodiesel Fuels Versus Conventional Diesel. Energy & Fuels, 24(9), 5163-5180. doi:10.1021/ef100594pFang, T., Lin, Y.-C., Foong, T. M., & Lee, C. (2009). Biodiesel combustion in an optical HSDI diesel engine under low load premixed combustion conditions. Fuel, 88(11), 2154-2162. doi:10.1016/j.fuel.2009.02.033Pastor, J. V., García-Oliver, J. M., Nerva, J.-G., & Giménez, B. (2011). Fuel effect on the liquid-phase penetration of an evaporating spray under transient diesel-like conditions. Fuel, 90(11), 3369-3381. doi:10.1016/j.fuel.2011.05.006Fisher, B. T., & Mueller, C. J. (2010). Liquid penetration length of heptamethylnonane and trimethylpentane under unsteady in-cylinder conditions. Fuel, 89(10), 2673-2696. doi:10.1016/j.fuel.2010.04.024Kim, H. J., Park, S. H., Suh, H. K., & Lee, C. S. (2009). Atomization and Evaporation Characteristics of Biodiesel and Dimethyl Ether Compared to Diesel Fuel in a High-Pressure Injection System. Energy & Fuels, 23(3), 1734-1742. doi:10.1021/ef800811gSuh, H. K., Roh, H. G., & Lee, C. S. (2008). Spray and Combustion Characteristics of Biodiesel∕Diesel Blended Fuel in a Direct Injection Common-Rail Diesel Engine. Journal of Engineering for Gas Turbines and Power, 130(3). doi:10.1115/1.2835354Pickett, L. M., & Siebers, D. L. (2006). Soot Formation in Diesel Fuel Jets Near the Lift-Off Length. International Journal of Engine Research, 7(2), 103-130. doi:10.1243/146808705x57793Pickett, L. M., Kook, S., Persson, H., & Andersson, Ö. (2009). Diesel fuel jet lift-off stabilization in the presence of laser-induced plasma ignition. Proceedings of the Combustion Institute, 32(2), 2793-2800. doi:10.1016/j.proci.2008.06.082Yoo, C. S., Richardson, E. S., Sankaran, R., & Chen, J. H. (2011). A DNS study on the stabilization mechanism of a turbulent lifted ethylene jet flame in highly-heated coflow. Proceedings of the Combustion Institute, 33(1), 1619-1627. doi:10.1016/j.proci.2010.06.147Pastor, J. V., Payri, R., Gimeno, J., & Nerva, J. G. (2009). Experimental Study on RME Blends: Liquid-Phase Fuel Penetration, Chemiluminescence, and Soot Luminosity in Diesel-Like Conditions. Energy & Fuels, 23(12), 5899-5915. doi:10.1021/ef9007328Benajes, J., Molina, S., Novella, R., & Amorim, R. (2010). Study on Low Temperature Combustion for Light-Duty Diesel Engines. Energy & Fuels, 24(1), 355-364. doi:10.1021/ef900832cPickett, L. M., & Siebers, D. L. (2002). An investigation of diesel soot formation processes using micro-orifices. Proceedings of the Combustion Institute, 29(1), 655-662. doi:10.1016/s1540-7489(02)80084-0Siebers, D. L., & Pickett, L. M. (2004). Injection Pressure and Orifice Diameter Effects on Soot in DI Diesel Fuel Jets. Thermo- and Fluid Dynamic Processes in Diesel Engines 2, 109-132. doi:10.1007/978-3-662-10502-3_7Pickett, L. M., & Siebers, D. L. (2004). Soot in diesel fuel jets: effects of ambient temperature, ambient density, and injection pressure. Combustion and Flame, 138(1-2), 114-135. doi:10.1016/j.combustflame.2004.04.006Cheng, A. S., Upatnieks, A., & Mueller, C. J. (2006). Investigation of the impact of biodiesel fuelling on NOx emissions using an optical direct injection diesel engine. International Journal of Engine Research, 7(4), 297-318. doi:10.1243/14680874jer05005Cheng, A. S. (Ed), Upatnieks, A., & Mueller, C. J. (2007). Investigation of Fuel Effects on Dilute, Mixing-Controlled Combustion in an Optical Direct-Injection Diesel Engine. Energy & Fuels, 21(4), 1989-2002. doi:10.1021/ef0606456Klein-Douwel, R. J. H., Donkerbroek, A. J., van Vliet, A. P., Boot, M. D., Somers, L. M. T., Baert, R. S. G., … ter Meulen, J. J. (2009). Soot and chemiluminescence in diesel combustion of bio-derived, oxygenated and reference fuels. Proceedings of the Combustion Institute, 32(2), 2817-2825. doi:10.1016/j.proci.2008.06.140Fang, T., & Lee, C. F. (2009). Bio-diesel effects on combustion processes in an HSDI diesel engine using advanced injection strategies. Proceedings of the Combustion Institute, 32(2), 2785-2792. doi:10.1016/j.proci.2008.07.031Payri, F., Pastor, J. V., Nerva, J.-G., & Garcia-Oliver, J. M. (2011). Lift-Off Length and KL Extinction Measurements of Biodiesel and Fischer-Tropsch Fuels under Quasi-Steady Diesel Engine Conditions. SAE International Journal of Engines, 4(2), 2278-2297. doi:10.4271/2011-24-0037Kook, S., & Pickett, L. M. (2012). Liquid length and vapor penetration of conventional, Fischer–Tropsch, coal-derived, and surrogate fuel sprays at high-temperature and high-pressure ambient conditions. Fuel, 93, 539-548. doi:10.1016/j.fuel.2011.10.004Settles, G. S. (2001). Schlieren and Shadowgraph Techniques. doi:10.1007/978-3-642-56640-0Pickett, L. M., Manin, J., Genzale, C. L., Siebers, D. L., Musculus, M. P. B., & Idicheria, C. A. (2011). Relationship Between Diesel Fuel Spray Vapor Penetration/Dispersion and Local Fuel Mixture Fraction. SAE International Journal of Engines, 4(1), 764-799. doi:10.4271/2011-01-0686MUSCULUS, M., & PICKETT, L. (2005). Diagnostic considerations for optical laser-extinction measurements of soot in high-pressure transient combustion environments. Combustion and Flame, 141(4), 371-391. doi:10.1016/j.combustflame.2005.01.013Williams, T. C., Shaddix, C. R., Jensen, K. A., & Suo-Anttila, J. M. (2007). Measurement of the dimensionless extinction coefficient of soot within laminar diffusion flames. International Journal of Heat and Mass Transfer, 50(7-8), 1616-1630. doi:10.1016/j.ijheatmasstransfer.2006.08.024Kook, S., & Pickett, L. M. (2011). Soot volume fraction and morphology of conventional and surrogate jet fuel sprays at 1000-K and 6.7-MPa ambient conditions. Proceedings of the Combustion Institute, 33(2), 2911-2918. doi:10.1016/j.proci.2010.05.073De Francqueville, L., Bruneaux, G., & Thirouard, B. (2010). Soot Volume Fraction Measurements in a Gasoline Direct Injection Engine by Combined Laser Induced Incandescence and Laser Extinction Method. SAE International Journal of Engines, 3(1), 163-182. doi:10.4271/2010-01-0346Musculus, M. P. B., & Kattke, K. (2009). Entrainment Waves in Diesel Jets. SAE International Journal of Engines, 2(1), 1170-1193. doi:10.4271/2009-01-1355Desantes, J. M., Pastor, J. V., García-Oliver, J. M., & Pastor, J. M. (2009). A 1D model for the description of mixing-controlled reacting diesel sprays. Combustion and Flame, 156(1), 234-249. doi:10.1016/j.combustflame.2008.10.008Idicheria, C. A., & Pickett, L. M. (2011). Ignition, soot formation, and end-of-combustion transients in diesel combustion under high-EGR conditions. International Journal of Engine Research, 12(4), 376-392. doi:10.1177/1468087411399505Aizawa, T., & Kosaka, H. (2008). Investigation of early soot formation process in a diesel spray flame via excitation—emission matrix using a multi-wavelength laser source. International Journal of Engine Research, 9(1), 79-97. doi:10.1243/14680874jer01407Bruneaux, G. (2008). Combustion structure of free and wall-impinging diesel jets by simultaneous laser-induced fluorescence of formaldehyde, poly-aromatic hydrocarbons, and hydroxides. International Journal of Engine Research, 9(3), 249-265. doi:10.1243/14680874jer0010

A meta-analytic review of stand-alone interventions to improve body image

Objective Numerous stand-alone interventions to improve body image have been developed. The present review used meta-analysis to estimate the effectiveness of such interventions, and to identify the specific change techniques that lead to improvement in body image. Methods The inclusion criteria were that (a) the intervention was stand-alone (i.e., solely focused on improving body image), (b) a control group was used, (c) participants were randomly assigned to conditions, and (d) at least one pretest and one posttest measure of body image was taken. Effect sizes were meta-analysed and moderator analyses were conducted. A taxonomy of 48 change techniques used in interventions targeted at body image was developed; all interventions were coded using this taxonomy. Results The literature search identified 62 tests of interventions (N = 3,846). Interventions produced a small-to-medium improvement in body image (d+ = 0.38), a small-to-medium reduction in beauty ideal internalisation (d+ = -0.37), and a large reduction in social comparison tendencies (d+ = -0.72). However, the effect size for body image was inflated by bias both within and across studies, and was reliable but of small magnitude once corrections for bias were applied. Effect sizes for the other outcomes were no longer reliable once corrections for bias were applied. Several features of the sample, intervention, and methodology moderated intervention effects. Twelve change techniques were associated with improvements in body image, and three techniques were contra-indicated. Conclusions The findings show that interventions engender only small improvements in body image, and underline the need for large-scale, high-quality trials in this area. The review identifies effective techniques that could be deployed in future interventions

Sibling relationships and family functioning in siblings of early adolescents, adolescents and young adults with autism spectrum disorder

The purpose of the study was to investigate how family functioning (defined as the ability that family members hold to manage stressful events, and intimate and social relationships), the degree to which family members feel happy and fulfilled with each other (called family satisfaction), and the demographical characteristics of siblings (age and gender) impacted on sibling relationships. The Circumplex Model of Marital and Family Systems and Behavioral Systems constituted the theoretical frameworks that guided our study. Eighty-six typically developing adolescents and young adults having a sister or a brother with autism spectrum disorder were enrolled. Results indicated that the youngest age group (early adolescents) reported to engage more frequently in negative behaviors with their siblings with ASD than the two older age groups (middle adolescents and young adults). No significant differences were found among the three age groups regarding behaviors derived from attachment, caregiving and affiliative systems. Family satisfaction and age significantly predicted behaviors during sibling interactions. Suggestions on prevention and intervention programs were discussed in order to prevent parentification among typically developing siblings and decrease episodes of quarrels and overt conflicts between brothers and sisters with and without AS

Selection at a single locus leads to widespread expansion of toxoplasma gondii lineages that are virulent in mice

The determinants of virulence are rarely defined for eukaryotic parasites such as T. gondii, a widespread parasite of mammals that also infects humans, sometimes with serious consequences. Recent laboratory studies have established that variation in a single secreted protein, a serine/threonine kinase known as ROPO18, controls whether or not mice survive infection. Here, we establish the extent and nature of variation in ROP18among a collection of parasite strains from geographically diverse regions. Compared to other genes, ROP18 showed extremely high levels of diversification and changes in expression level, which correlated with severity of infection in mice. Comparison with an out-group demonstrated that changes in the upstream region that regulates expression of ROP18 led to an historical increase in the expression and exposed the protein to diversifying selective pressure. Surprisingly, only three atypically distinct protein variants exist despite marked genetic divergence elsewhere in the genome. These three forms of ROP18 are likely adaptations for different niches in nature, and they confer markedly different virulence to mice. The widespread distribution of a single mouse-virulent allele among geographically and genetically disparate parasites may have consequences for transmission and disease in other hosts, including humans

MTSS1 and SCAMP1 cooperate to prevent invasion in breast cancer

Cell–cell adhesions constitute the structural “glue” that retains cells together and contributes to tissue organisation and physiological function. The integrity of these structures is regulated by extracellular and intracellular signals and pathways that act on the functional units of cell adhesion such as the cell adhesion molecules/adhesion receptors, the extracellular matrix (ECM) proteins and the cytoplasmic plaque/peripheral membrane proteins. In advanced cancer, these regulatory pathways are dysregulated and lead to cell–cell adhesion disassembly, increased invasion and metastasis. The Metastasis suppressor protein 1 (MTSS1) plays a key role in the maintenance of cell–cell adhesions and its loss correlates with tumour progression in a variety of cancers. However, the mechanisms that regulate its function are not well-known. Using a system biology approach, we unravelled potential interacting partners of MTSS1. We found that the secretory carrier-associated membrane protein 1 (SCAMP1), a molecule involved in post-Golgi recycling pathways and in endosome cell membrane recycling, enhances Mtss1 anti-invasive function in HER2+/ER−/PR− breast cancer, by promoting its protein trafficking leading to elevated levels of RAC1-GTP and increased cell–cell adhesions. This was clinically tested in HER2 breast cancer tissue and shown that loss of MTSS1 and SCAMP1 correlates with reduced disease-specific survival. In summary, we provide evidence of the cooperative roles of MTSS1 and SCAMP1 in preventing HER2+/ER−/PR− breast cancer invasion and we show that the loss of Mtss1 and Scamp1 results in a more aggressive cancer cell phenotype

Biopsy confirmation of metastatic sites in breast cancer patients:clinical impact and future perspectives

Determination of hormone receptor (estrogen receptor and progesterone receptor) and human epidermal growth factor receptor 2 status in the primary tumor is clinically relevant to define breast cancer subtypes, clinical outcome,and the choice of therapy. Retrospective and prospective studies suggest that there is substantial discordance in receptor status between primary and recurrent breast cancer. Despite this evidence and current recommendations,the acquisition of tissue from metastatic deposits is not routine practice. As a consequence, therapeutic decisions for treatment in the metastatic setting are based on the features of the primary tumor. Reasons for this attitude include the invasiveness of the procedure and the unreliable outcome of biopsy, in particular for biopsies of lesions at complex visceral sites. Improvements in interventional radiology techniques mean that most metastatic sites are now accessible by minimally invasive methods, including surgery. In our opinion, since biopsies are diagnostic and changes in biological features between the primary and secondary tumors can occur, the routine biopsy of metastatic disease needs to be performed. In this review, we discuss the rationale for biopsy of suspected breast cancer metastases, review issues and caveats surrounding discordance of biomarker status between primary and metastatic tumors, and provide insights for deciding when to perform biopsy of suspected metastases and which one (s) to biopsy. We also speculate on the future translational implications for biopsy of suspected metastatic lesions in the context of clinical trials and the establishment of bio-banks of biopsy material taken from metastatic sites. We believe that such bio-banks will be important for exploring mechanisms of metastasis. In the future,advances in targeted therapy will depend on the availability of metastatic tissue

Evaluating the Quality of Research into a Single Prognostic Biomarker: A Systematic Review and Meta-analysis of 83 Studies of C-Reactive Protein in Stable Coronary Artery Disease

Background Systematic evaluations of the quality of research on a single prognostic biomarker are rare. We sought to evaluate the quality of prognostic research evidence for the association of C-reactive protein (CRP) with fatal and nonfatal events among patients with stable coronary disease. Methods and Findings We searched MEDLINE (1966 to 2009) and EMBASE (1980 to 2009) and selected prospective studies of patients with stable coronary disease, reporting a relative risk for the association of CRP with death and nonfatal cardiovascular events. We included 83 studies, reporting 61,684 patients and 6,485 outcome events. No study reported a prespecified statistical analysis protocol; only two studies reported the time elapsed (in months or years) between initial presentation of symptomatic coronary disease and inclusion in the study. Studies reported a median of seven items (of 17) from the REMARK reporting guidelines, with no evidence of change over time. The pooled relative risk for the top versus bottom third of CRP distribution was 1.97 (95% confidence interval [CI] 1.78–2.17), with substantial heterogeneity (I2 = 79.5). Only 13 studies adjusted for conventional risk factors (age, sex, smoking, obesity, diabetes, and low-density lipoprotein [LDL] cholesterol) and these had a relative risk of 1.65 (95% CI 1.39–1.96), I2 = 33.7. Studies reported ten different ways of comparing CRP values, with weaker relative risks for those based on continuous measures. Adjusting for publication bias (for which there was strong evidence, Egger's p<0.001) using a validated method reduced the relative risk to 1.19 (95% CI 1.13–1.25). Only two studies reported a measure of discrimination (c-statistic). In 20 studies the detection rate for subsequent events could be calculated and was 31% for a 10% false positive rate, and the calculated pooled c-statistic was 0.61 (0.57–0.66). Conclusion Multiple types of reporting bias, and publication bias, make the magnitude of any independent association between CRP and prognosis among patients with stable coronary disease sufficiently uncertain that no clinical practice recommendations can be made. Publication of prespecified statistical analytic protocols and prospective registration of studies, among other measures, might help improve the quality of prognostic biomarker research