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Honeybees (Apis mellifera) are threatened by numerous pathogens and parasites. To prevent infections they apply cooperative behavioral defenses, such as allo-grooming and hygiene, or they use antimicrobial plant resin. Resin is a chemically complex and highly variable mixture of many bioactive compounds. Bees collect the sticky material from different plant species and use it for nest construction and protection. Despite its importance for colony health, comparatively little is known about the precise origins and variability in resin spectra collected by honeybees. To identify the botanical resin sources of A. mellifera in Western Europe we chemically compared resin loads of individual foragers and tree resins. We further examined the resin intake of 25 colonies from five different apiaries to assess the effect of location on variation in the spectra of collected resin. Across all colonies and apiaries, seven distinct resin types were categorized according to their color and chemical composition. Matches between bee-collected resin and tree resin indicated that bees used poplar (Populus balsamifera, P. x canadensis), birch (Betula alba), horse chestnut (Aesculus hippocastanum) and coniferous trees (either Picea abies or Pinus sylvestris) as resin sources. Our data reveal that honeybees collect a comparatively broad and variable spectrum of resin sources, thus assuring protection against a variety of antagonists sensitive to different resins and/or compounds. We further unravel distinct preferences for specific resins and resin chemotypes, indicating that honeybees selectively search for bioactive resin compounds.

Despite the abundance of recent publications about mind wandering (i.e., off-task thought), its interconnection with metacognition and cognitive control has not yet been examined. In the current study, we hypothesized that these three constructs would show clear interrelations. Metacognitive capacity was predicted to correlate positively with cognitive control ability, which in turn was predicted to be positively related to resistance to mind wandering during sustained attention. Moreover, it was expected that participants with good metacognitive capacity would be better at the subjective recognition of behaviorally present mind wandering. Three tasks were used: The Sustained Attention to Response Task (SART) to measure mind wandering, a perceptual decision task with confidence ratings to measure metacognitive efficiency, and a conflict task to measure cognitive control. Structural Equation Modelling was used to test the interrelations among the three constructs. As expected, metacognitive efficiency was positively related to cognitive control ability. Surprisingly, there was a negative relation between metacognitive efficiency and the degree to which subjective mind wandering reports tracked the behavioral index of mind wandering. No relation was found between cognitive control and behavioral mind wandering. The results of the current work are the first to shed light on the interrelations among these three constructs.

In several fields of research, like e.g. photosensitization, photovoltaics, organic electroluminescent devices, dynamic nuclear polarization, or pulsed dipolar electron paramagnetic resonance spectroscopy, triplet state kinetics play an important role. It is therefore desirable to tailor the kinetics of photoexcited triplet states, e.g. by exploiting the intramolecular heavy-atom effect, and to determine the respective kinetic parameters. In this work, we set out to systematically investigate the photoexcited triplet state kinetics of a series of haloanthracenes by time-resolved electron paramagnetic resonance spectroscopy in combination with synchronized laser excitation. For this purpose, a procedure to simulate time traces by solving the differential equation system governing the triplet kinetics numerically is developed. This way, spin lattice relaxation rates and zero-field triplet life times are obtained concurrently by a global fit to experimental data measured at three different cryogenic temperatures.

In many hyperthermophilic archaea the DNA binding protein TrmBL2 or one of its homologues is abundantly expressed. TrmBL2 is thought to play a significant role in modulating the chromatin architecture in combination with the archaeal histone proteins and Alba. However, its precise physiological role is poorly understood. It has been previously shown that upon binding TrmBL2 covers double-stranded DNA, which leads to the formation of a thick and fibrous filament. Here we investigated the filament formation process as well as the stabilization of DNA by TrmBL2 from Pyroccocus furiosus in detail. We used magnetic tweezers that allow to monitor changes of the DNA mechanical properties upon TrmBL2 binding on the single-molecule level. Extended filaments formed in a cooperative manner and were considerably stiffer than bare double-stranded DNA. Unlike Alba, TrmBL2 did not form DNA cross-bridges. The protein was found to bind double- and single-stranded DNA with similar affinities. In mechanical disruption experiments of DNA hairpins this led to stabilization of both, the double- (before disruption) and the single-stranded (after disruption) DNA forms. Combined, these findings suggest that the biological function of TrmBL2 is not limited to modulating genome architecture and acting as a global repressor but that the protein acts additionally as a stabilizer of DNA secondary structure.

ObjectivesTreatment of diabetic subjects with cinnamon demonstrated an improvement in blood glucose concentrations and insulin sensitivity but the underlying mechanisms remained unclear. This work intends to elucidate the impact of cinnamon effects on the brain by using isolated astrocytes, and an obese and diabetic mouse model.MethodsCinnamon components (eugenol, cinnamaldehyde) were added to astrocytes and liver cells to measure insulin signaling and glycogen synthesis. Ob/ob mice were supplemented with extract from cinnamomum zeylanicum for 6 weeks and cortical brain activity, locomotion and energy expenditure were evaluated. Insulin action was determined in brain and liver tissues.ResultsTreatment of primary astrocytes with eugenol promoted glycogen synthesis, whereas the effect of cinnamaldehyde was attenuated. In terms of brain function in vivo, cinnamon extract improved insulin sensitivity and brain activity in ob/ob mice, and the insulin-stimulated locomotor activity was improved. In addition, fasting blood glucose levels and glucose tolerance were greatly improved in ob/ob mice due to cinnamon extracts, while insulin secretion was unaltered. This corresponded with lower triglyceride and increased liver glycogen content and improved insulin action in liver tissues. In vitro, Fao cells exposed to cinnamon exhibited no change in insulin action.ConclusionsTogether, cinnamon extract improved insulin action in the brain as well as brain activity and locomotion. This specific effect may represent an important central feature of cinnamon in improving insulin action in the brain, and mediates metabolic alterations in the periphery to decrease liver fat and improve glucose homeostasis.

Combat-related posttraumatic stress disorder (PTSD) can be a difficult condition to treat and has been associated with serious medical and economic issues among U.S. military veterans. Distinguishing between treatment responders vs. non-responders in this population has become an important public health priority. This study was conducted to identify pre-treatment characteristics of U.S. veterans with combat-related PTSD that might contribute to favorable and unfavorable responses to high value treatments for this condition.This study focused on 805 patients who completed a VHA PTSD residential program between 2000 and 2007. These patients completed the PTSD Clinical Checklist at pre-treatment, post-treatment, and a four-month follow-up assessment. Latent growth curve analysis (LCGA) was incorporated to determine trajectories of changes in PTSD across these assessments and whether several key clinical concerns for this population were associated with their treatment responses.LCGA indicated three distinct trajectories in PTSD outcomes and identified several clinical factors that were prospectively linked with changes in veterans' posttraumatic symptomatology. When compared to a group with high PTSD symptom severity that decreased over the program but relapsed at follow-up (41%), the near half (48.8%) of the sample with an improving trajectory had less combat exposure and superior physical/mental health. However, when compared to a minority (10.2%) with relatively low symptomatology that also remained somewhat stable, patients in the improving group were younger and also reported greater combat exposure, poorer physical/mental health status, and more problems with substance abuse before the start of treatment.Findings suggest that veterans are most likely to benefit from residential treatment in an intermediate range of symptoms and risk factors, including PTSD symptom severity, history of combat exposure, and comorbid issues with physical/mental health. Addressing these factors in an integrative manner could help to optimize the effectiveness of treatments of combat-related PTSD in many cases.

Glutathione (GSH) and GSH-dependent enzymes play a key role in cellular detoxification processes that enable organism to cope with various internal and environmental stressors. However, it is often not clear, which components of the complex GSH-metabolism are required for tolerance towards a certain stressor. To address this question, a small scale RNAi-screen was carried out in Caenorhabditis elegans where GSH-related genes were systematically knocked down and worms were subsequently analysed for their survival rate under sub-lethal concentrations of arsenite and the redox cycler juglone. While the knockdown of γ-glutamylcysteine synthetase led to a diminished survival rate under arsenite stress conditions, GSR-1 (glutathione reductase) was shown to be essential for survival under juglone stress conditions. gsr-1 is the sole GSR encoding gene found in C. elegans. Knockdown of GSR-1 hardly affected total glutathione levels nor reduced glutathione/glutathione disulphide (GSH/GSSG) ratio under normal laboratory conditions. Nevertheless, when GSSG recycling was impaired by gsr-1(RNAi), GSH synthesis was induced, but not vice versa. Moreover, the impact of GSSG recycling was potentiated under oxidative stress conditions, explaining the enormous effect gsr-1(RNAi) knockdown had on juglone tolerance. Accordingly, overexpression of GSR-1 was capable of increasing stress tolerance. Furthermore, expression levels of SKN-1-regulated GSR-1 also affected life span of C. elegans, emphasising the crucial role the GSH redox state plays in both processes.

The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.

PURPOSE: To evaluate changes in nucleus pulposus volume as a potential parameter for the effects of disc decompression. METHODS: Fifty-two discs (T8 to L1) were extracted from 26 pigs and separated into thoracic (T8 to T11) and thoracolumbar discs (T12 to L1). The discs were imaged using 7.1 Tesla ultrahigh-field magnetic resonance imaging (MRI) with acquisition of axial T2-weighted turbo spin-echo sequences for determination of baseline and postinterventional nucleus pulposus volumes. Volumes were calculated using OsiriX® (http://www.osirix-viewer.com). After randomization, one group was treated with nucleoplasty, while the placebo group was treated with an identical procedure but without coblation current. The readers analyzing the MR images were blinded to the kind of procedure performed. Baseline and postinterventional volumes were compared between the nucleoplasty and placebo group. RESULTS: Average preinterventional nucleus volume was 0.799 (SD: 0.212) ml. Postinterventional volume reduction in the nucleoplasty group was significant at 0.052 (SD: 0.035) ml or 6.30% (p

The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.

OBJECTIVES: To assess the efficacy of a public health intervention to reduce blood mercury (Hg) concentration levels in pregnant Bermudian women. METHODS: In 2003, we conducted a study entitled "Prenatal exposure of the Bermudian Population to Environmental Contaminants" which provided Bermuda's first baseline data on prenatal exposure to several environmental contaminants, including Hg. The mean Hg concentration from 42 healthy newborns measured in umbilical cord blood was 41.3 nmol/L, ranging from 5-160 nmol/L. This concentration was much higher than expected, being approximately 8 times the general levels found in Canada and the U.S. Furthermore, we estimated that 85% of total Hg measured was in the form of methylmercury (MeHg), indicating that seafood consumption was the primary source of Hg exposure during pregnancy in Bermuda. Locally sourced seafood was identified as the most significant possible contributory source of Hg exposure. In 2005 the authors began a complementary research programme to study the levels of Hg in local commercial fish species. Coming out of this research were specific local fish consumption guidelines issued by the Department of Health advising pregnant women to avoid those local fish species found to be high in Hg while still encouraging consumption of fish species having lower Hg levels. RESULTS: In 2010, under another research initiative, we returned to Bermuda to carry out another evaluation of Hg in human blood. Hg was measured in the blood of 49 pregnant women. The arithmetic mean Hg blood concentration was 6.6 nmol/L and the geometric mean 4.2 nmol/L. The maximum concentration found was 24 nmol/L. CONCLUSIONS: Hg exposure of Bermudian pregnant women has dropped significantly by a factor of around 5 since the foetal cord blood study in 2003.

The signaling pathways associated with the Toll-like receptors (TLRs) and nuclear factor-kappaB (NF-κB) are essential to pro-inflammatory cytokine and chemokine expression, as well as initiating innate epithelial immune responses. The TLR/NF-κB signaling pathways must be stringently controlled through an intricate network of positive and negative regulatory elements. MicroRNAs (miRNAs) are non-coding small RNAs that regulate the stability and/or translation of protein-coding mRNAs. Herein we report that miR-16 promotes NF-κB-regulated transactivation of the IL-8 gene by suppression of the silencing mediator for retinoid and thyroid hormone receptor (SMRT). LPS stimulation activated miR-16 gene transcription in human monocytes (U937) and biliary epithelial cells (H69) through MAPK-dependent mechanisms. Transfection of cells with the miR-16 precursor promoted LPS-induced production of IL-8, IL-6, and IL-1α, without a significant effect on their RNA stability. Instead, an increase in NF-κB-regulated transactivation of the IL-8 gene was confirmed in cells following transfection of miR-16 precursor. Importantly, miR-16 targeted the 3'-untranslated region of SMRT and caused translational suppression of SMRT. LPS decreased SMRT expression via upregulation of miR-16. Moreover, functional manipulation of SMRT altered NF-κB-regulated transactivation of LPS-induced IL-8 expression. These data suggest that miR-16 targets SMRT and modulates NF-κB-regulated transactivation of the IL-8 gene.

Elevated homocysteine concentrations have been associated with methotrexate-induced neurotoxicity. Based on methotrexate and homocysteine plasma concentrations of 494 children with acute lymphoblastic leukemia treated with high-dose methotrexate in the TOTAL XV study, a pharmacokinetic/pharmacodynamic (PK/PD) model was built with NONMEM. Several compartment and indirect response models were investigated. The pharmacokinetic disposition of methotrexate was best described by a two-compartment model. Homocysteine concentrations were included by an indirect response model where methotrexate inhibition of the homocysteine elimination rate was described by an E(max) model. The homocysteine baseline level was found to be age-dependent. Simulations revealed that folinate rescue therapy does not affect peak concentrations of homocysteine but leads to a modestly reduced homocysteine exposure. In conclusion, our PK/PD model describes the increase of methotrexate-induced HCY concentrations with satisfactory precision and can be applied to assess the effect of folinate regimens on the HCY concentration-time course.

In our studies of ovarian cancer cells we have identified subpopulations of cells that are in a transitory E/M hybrid stage, i.e. cells that simultaneously express epithelial and mesenchymal markers. E/M cells are not homogenous but, in vitro and in vivo, contain subsets that can be distinguished based on a number of phenotypic features, including the subcellular localization of E-cadherin, and the expression levels of Tie2, CD133, and CD44. A cellular subset (E/M-MP) (membrane E-cadherin(low)/cytoplasmic E-cadherin(high)/CD133(high), CD44(high), Tie2(low)) is highly enriched for tumor-forming cells and displays features which are generally associated with cancer stem cells. Our data suggest that E/M-MP cells are able to differentiate into different lineages under certain conditions, and have the capacity for self-renewal, i.e. to maintain a subset of undifferentiated E/M-MP cells during differentiation. Trans-differentiation of E/M-MP cells into mesenchymal or epithelial cells is associated with a loss of stem cell markers and tumorigenicity. In vivo xenograft tumor growth is driven by E/M-MP cells, which give rise to epithelial ovarian cancer cells. In contrast, in vitro, we found that E/M-MP cells differentiate into mesenchymal cells, in a process that involves pathways associated with an epithelial-to-mesenchymal transition. We also detected phenotypic plasticity that was dependent on external factors such as stress created by starvation or contact with either epithelial or mesenchymal cells in co-cultures. Our study provides a better understanding of the phenotypic complexity of ovarian cancer and has implications for ovarian cancer therapy.

Regeneration after hematopoietic stem cell transplantation (HSCT) depends on enormous activation of the stem cell pool. So far, it is hardly understood how these cells are recruited into proliferation and self-renewal. In this study, we have addressed the question if systemically released factors are involved in activation of hematopoietic stem and progenitor cells (HPC) after autologous HSCT. Serum was taken from patients before chemotherapy, during neutropenia and after hematopoietic recovery. Subsequently, it was used as supplement for in vitro culture of CD34(+) cord blood HPC. Serum taken under hematopoietic stress (4 to 11 days after HSCT) significantly enhanced proliferation, maintained primitive immunophenotype (CD34(+), CD133(+), CD45(-)) for more cell divisions and increased colony forming units (CFU) as well as the number of cobblestone area-forming cells (CAFC). The stimulatory effect decays to normal levels after hematopoietic recovery (more than 2 weeks after HSCT). Chemokine profiling revealed a decline of several growth-factors during neutropenia, including platelet-derived growth factors PDGF-AA, PDGF-AB and PDGF-BB, whereas expression of monocyte chemotactic protein-1 (MCP-1) increased. These results demonstrate that systemically released factors play an important role for stimulation of hematopoietic regeneration after autologous HSCT. This feedback mechanism opens new perspectives for in vivo stimulation of the stem cell pool.

In populations of most social insects, gene flow is maintained through mating between reproductive individuals from different colonies in periodic nuptial flights followed by dispersal of the fertilized foundresses. Some ant species, however, form large polygynous supercolonies, in which mating takes place within the maternal nest (intranidal mating) and fertilized queens disperse within or along the boundary of the supercolony, leading to supercolony growth (colony budding). As a consequence, gene flow is largely confined within supercolonies. Over time, such supercolonies may diverge genetically and, thus, also in recognition cues (cuticular hydrocarbons, CHC's) by a combination of genetic drift and accumulation of colony-specific, neutral mutations.We tested this hypothesis for six supercolonies of the invasive ant Anoplolepis gracilipes in north-east Borneo. Within supercolonies, workers from different nests tolerated each other, were closely related and showed highly similar CHC profiles. Between supercolonies, aggression ranged from tolerance to mortal encounters and was negatively correlated with relatedness and CHC profile similarity. Supercolonies were genetically and chemically distinct, with mutually aggressive supercolony pairs sharing only 33.1%±17.5% (mean ± SD) of their alleles across six microsatellite loci and 73.8%±11.6% of the compounds in their CHC profile. Moreover, the proportion of alleles that differed between supercolony pairs was positively correlated to the proportion of qualitatively different CHC compounds. These qualitatively differing CHC compounds were found across various substance classes including alkanes, alkenes and mono-, di- and trimethyl-branched alkanes.We conclude that positive feedback between genetic, chemical and behavioural traits may further enhance supercolony differentiation through genetic drift and neutral evolution, and may drive colonies towards different evolutionary pathways, possibly including speciation.

We used intensive modern proteomics approaches to identify predictive proteins in ovary cancer. We identify up-regulated proteins in both serum and peritoneal fluid. To evaluate the overall performance of the approach we track the behavior of 20 validated markers across these experiments.Mass spectrometry based quantitative proteomics following extensive protein fractionation was used to compare serum of women with serous ovarian cancer to healthy women and women with benign ovarian tumors. Quantitation was achieved by isotopically labeling cysteine amino acids. Label-free mass spectrometry was used to compare peritoneal fluid taken from women with serous ovarian cancer and those with benign tumors. All data were integrated and annotated based on whether the proteins have been previously validated using antibody-based assays.We selected 54 quantified serum proteins and 358 peritoneal fluid proteins whose case-control differences exceeded a predefined threshold. Seventeen proteins were quantified in both materials and 14 are extracellular. Of 19 validated markers that were identified all were found in cancer peritoneal fluid and a subset of 7 were quantified in serum, with one of these proteins, IGFBP1, newly validated here.Proteome profiling applied to symptomatic ovarian cancer cases identifies a large number of up-regulated serum proteins, many of which are or have been confirmed by immunoassays. The number of currently known validated markers is highest in peritoneal fluid, but they make up a higher percentage of the proteins observed in both serum and peritoneal fluid, suggesting that the 10 additional markers in this group may be high quality candidates.

BACKGROUND:Epithelial ovarian carcinoma is a significant cause of cancer mortality in women worldwide and in the United States. Epithelial ovarian cancer comprises several histological subtypes, each with distinct clinical and molecular characteristics. The natural history of this heterogeneous disease, including the cell types of origin, is poorly understood. This study applied recently developed methods for high-throughput DNA methylation profiling to characterize ovarian cancer cell lines and tumors, including representatives of three major histologies. METHODOLOGY/PRINCIPAL FINDINGS:We obtained DNA methylation profiles of 1,505 CpG sites (808 genes) in 27 primary epithelial ovarian tumors and 15 ovarian cancer cell lines. We found that the DNA methylation profiles of ovarian cancer cell lines were markedly different from those of primary ovarian tumors. Aggregate DNA methylation levels of the assayed CpG sites tended to be higher in ovarian cancer cell lines relative to ovarian tumors. Within the primary tumors, those of the same histological type were more alike in their methylation profiles than those of different subtypes. Supervised analyses identified 90 CpG sites (68 genes) that exhibited 'subtype-specific' DNA methylation patterns (FDR

MicroRNAs (miRNAs) are small regulatory RNAs that are implicated in cancer pathogenesis and have recently shown promise as blood-based biomarkers for cancer detection. Epithelial ovarian cancer is a deadly disease for which improved outcomes could be achieved by successful early detection and enhanced understanding of molecular pathogenesis that leads to improved therapies. A critical step toward these goals is to establish a comprehensive view of miRNAs expressed in epithelial ovarian cancer tissues as well as in normal ovarian surface epithelial cells.We used massively parallel pyrosequencing (i.e., "454 sequencing") to discover and characterize novel and known miRNAs expressed in primary cultures of normal human ovarian surface epithelium (HOSE) and in tissue from three of the most common histotypes of ovarian cancer. Deep sequencing of small RNA cDNA libraries derived from normal HOSE and ovarian cancer samples yielded a total of 738,710 high-quality sequence reads, generating comprehensive digital profiles of miRNA expression. Expression profiles for 498 previously annotated miRNAs were delineated and we discovered six novel miRNAs and 39 candidate miRNAs. A set of 124 miRNAs was differentially expressed in normal versus cancer samples and 38 miRNAs were differentially expressed across histologic subtypes of ovarian cancer. Taqman qRT-PCR performed on a subset of miRNAs confirmed results of the sequencing-based study.This report expands the body of miRNAs known to be expressed in epithelial ovarian cancer and provides a useful resource for future studies of the role of miRNAs in the pathogenesis and early detection of ovarian cancer.

Epithelial ovarian cancer is a significant cause of mortality both in the United States and worldwide, due largely to the high proportion of cases that present at a late stage, when survival is extremely poor. Early detection of epithelial ovarian cancer, and of the serous subtype in particular, is a promising strategy for saving lives. The low prevalence of ovarian cancer makes the development of an adequately sensitive and specific test based on blood markers very challenging. We evaluated the performance of a set of candidate blood markers and combinations of these markers in detecting serous ovarian cancer.We selected 14 candidate blood markers of serous ovarian cancer for which assays were available to measure their levels in serum or plasma, based on our analysis of global gene expression data and on literature searches. We evaluated the performance of these candidate markers individually and in combination by measuring them in overlapping sets of serum (or plasma) samples from women with clinically detectable ovarian cancer and women without ovarian cancer. Based on sensitivity at high specificity, we determined that 4 of the 14 candidate markers--MUC16, WFDC2, MSLN and MMP7--warrant further evaluation in precious serum specimens collected months to years prior to clinical diagnosis to assess their utility in early detection. We also reported differences in the performance of these candidate blood markers across histological types of epithelial ovarian cancer.By systematically analyzing the performance of candidate blood markers of ovarian cancer in distinguishing women with clinically apparent ovarian cancer from women without ovarian cancer, we identified a set of serum markers with adequate performance to warrant testing for their ability to identify ovarian cancer months to years prior to clinical diagnosis. We argued for the importance of sensitivity at high specificity and of magnitude of difference in marker levels between cases and controls as performance metrics and demonstrated the importance of stratifying analyses by histological type of ovarian cancer. Also, we discussed the limitations of studies (like this one) that use samples obtained from symptomatic women to assess potential utility in detection of disease months to years prior to clinical detection.

BACKGROUND: Elucidation of the repertoire of secreted and cell surface proteins of tumor cells is relevant to molecular diagnostics, tumor imaging and targeted therapies. We have characterized the cell surface proteome and the proteins released into the extra-cellular milieu of three ovarian cancer cell lines, CaOV3, OVCAR3 and ES2 and of ovarian tumor cells enriched from ascites fluid. METHODOLOGY AND FINDINGS: To differentiate proteins released into the media from protein constituents of media utilized for culture, cells were grown in the presence of [(13)C]-labeled lysine. A biotinylation-based approach was used to capture cell surface associated proteins. Our general experimental strategy consisted of fractionation of proteins from individual compartments followed by proteolytic digestion and LC-MS/MS analysis. In total, some 6,400 proteins were identified with high confidence across all specimens and fractions. CONCLUSIONS AND SIGNIFICANCE: Protein profiles of the cell lines had substantial similarity to the profiles of human ovarian cancer cells from ascites fluid and included protein markers known to be associated with ovarian cancer. Proteomic analysis indicated extensive shedding from extra-cellular domains of proteins expressed on the cell surface, and remarkably high secretion rates for some proteins (nanograms per million cells per hour). Cell surface and secreted proteins identified by in-depth proteomic profiling of ovarian cancer cells may provide new targets for diagnosis and therapy.