Integrative Proteomic Analysis of Serum and Peritoneal Fluids Helps Identify Proteins that Are Up-Regulated in Serum of Women with Ovarian Cancer

Background

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.

Methodology

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.

Findings

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.

Conclusion

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.     

Super-Resolution Imaging of Plasmodesmata Using Three-Dimensional Structured Illumination Microscopy

We used three-dimensional structured illumination microscopy (3D-SIM) to obtain subdiffraction (“super-resolution”) images of plasmodesmata (PD) expressing a green fluorescent protein-tagged viral movement protein (MP) in tobacco (Nicotiana tabacum). In leaf parenchyma cells, we were able to resolve individual components of PD (neck and central cavities) at twice the resolution of a confocal microscope. Within the phloem, MP-green fluorescent protein filaments extended outward from the specialized pore-PD that connect sieve elements (SEs) with their companion cells (CCs) along the tubular sieve element reticulum (SER). The SER was shown to interconnect individual pore-PD at the SE-CC interface. 3D-SIM resolved fine (less than 100 nm) endoplasmic reticulum threads running into individual pore-PD as well as strands that crossed sieve plate pores, structurally linking SEs within a file. Our data reveal that MP entering the SE from the CC may remain associated with the SER. Fluorescence recovery after photobleaching experiments revealed that this MP pool is relatively immobile compared with the membrane probe 3,3’-dihexyloxacarbocyanine iodide, suggesting that MP may become sequestered by the SER once it has entered the SE. The advent of 3D-SIM offers considerable potential in the subdiffraction imaging of plant cells, bridging an important gap between confocal and electron microscopy.Fluorescence-based imaging has revolutionized cell biology, allowing the localization of proteins to specific cells and organelles (Shaner et al., 2007; Frigault et al., 2009). However, conventional fluorescence microscopy is limited by the diffraction of light to approximately 200 nm in the lateral (x-y) plane and to about 500 nm in the axial (z) plane (Fernandez-Suarez and Ting, 2008; Huang et al., 2009). This is because light traveling through a lens cannot be focused to a point, only to an airy disc with a diameter of about half the wavelength of the visible emitted light (Huang et al., 2009). Confocal laser scanning microscopy has produced improvements in axial resolution due to the removal of out-of-focus flare, but it is also limited by diffraction (Huang et al., 2009). Thus, objects closer than about 200 nm cannot be resolved but appear merged into one. Many subcellular structures of interest to cell biologists lie below this resolution limit and have remained below the diffraction barrier. Such structures can be seen but not resolved.Recently, major innovations in biological imaging have broken the diffraction barrier. These include photoactivation localization microscopy (PALM) and stimulated emission and depletion (STED; for review, see Fernandez-Suarez and Ting, 2008; Huang et al., 2009). Most subdiffraction or “super-resolution” approaches have improved resolution in either the lateral (x-y) plane or the axial (z) plane, but usually not both (Schermelleh et al., 2008). Many of the structures of interest within plant cells lie some distance from the cell wall, posing problems for some super-resolution approaches (e.g. PALM) where the subject of interest must lie close to the coverslip (Huang et al., 2009). Recently, Schermelleh et al. (2008) described a subdiffraction multicolor imaging protocol using three-dimensional structured illumination microscopy (3D-SIM). In this method, objects beyond the diffraction limit are illuminated with multiple interfering beams of light transmitted through a series of diffraction gratings, producing a resolution of 100 nm in x-y and 200 nm in z (Schermelleh et al., 2008; Huang et al., 2009). These substantial increases in resolution are significant for plant cell imaging. The thickness of the plant cell wall is typically in the region of about 700 nm, allowing limited optical sectioning capacity with a confocal microscope (about 500 nm in z). A further advantage of 3D-SIM is that it permits the imaging of conventional fluorescent reporters and dyes that are compatible with confocal imaging, allowing a direct correlation of 3D-SIM and confocal images (Schermelleh et al., 2008).The phloem of higher plants is a major conduit for the long-distance transport of solutes (Oparka and Turgeon, 1999) and also functions as a “superhighway” for macromolecular trafficking (Lucas and Lee, 2004; Kehr and Buhtz, 2008; Lee and Cui, 2009). However, the phloem is difficult to image with conventional optical microscopy (Knoblauch and van Bel, 1998; Oparka and Turgeon, 1999; van Bel et al., 2002). Sieve elements (SEs), the conducting cells of the phloem, are enucleate yet contain a plethora of proteins and RNAs associated with long-distance signaling and defense (van Bel and Gaupels, 2004; Lee and Cui, 2009). Many of these macromolecules are synthesized in the companion cell (CC) and passed into the SE via the specialized pore-plasmodesmata (PD) that connect the two cell types (Oparka and Turgeon, 1999; van Bel et al., 2002). Pore-PD have been suggested to be a major “lifeline” from CC to SE (van Bel et al., 2002), but the exact nature of this pathway remains unresolved.Our current understanding of PD substructure is derived largely from electron microscope studies (Roberts, 2005). Such methods are time-consuming and do not permit facile protein localization within PD. Recent proteomics approaches have been successful in identifying new proteins associated with PD (Maule, 2008). Localization of these proteins with confocal microscopy results in the appearance of discrete punctae at the cell wall, consistent with the location of pit fields (Faulkner et al., 2008), but does not pinpoint specific protein locations within PD. In general, there is a growing gap between proteomics studies of plant organelles, including PD, and the ability to ascribe accurate addresses to these proteins (Millar et al., 2009; Moore and Murphy, 2009). The advent of 3D-SIM prompted us to explore the potential of subdiffraction imaging in plant cells, with a view to obtaining improved florescence resolution of PD. We used 3D-SIM to examine PD in a transgenic tobacco (Nicotiana tabacum) line expressing the viral movement protein (MP) of Tobacco mosaic virus (TMV) fused to GFP. Using a specific antibody to callose, a wall constituent located at the PD collar, we were able to resolve clearly the structure of single, simple PD in epidermal cells at 100-nm resolution, discriminating between the neck region of the pore and the central cavity to which it connects (Roberts and Oparka, 2003; Faulkner et al., 2008). 3D-SIM also revealed details of the central cavities of complex PD seen previously only with the electron microscope (Ding et al., 1992; Ehlers and Kollmann, 2001; Faulkner et al., 2008).Using 3D-SIM, we were able to image PD sequentially from the epidermis to the phloem within vascular bundles, producing unparalleled images of sieve plate pores and the specialized pore-PD that connect SEs with their CCs. In the SEs, MP was no longer restricted to the central cavities of PD but became distributed along the SE parietal layer, connecting all the pore-PD along the SE-CC interface. We were able to detect fine threads of MP-GFP that extended for up to 40 μm along the SE and also crossed individual sieve plate pores. Fluorescence recovery after photobleaching (FRAP) experiments revealed that this MP-GFP pool was relatively immobile within the SE parietal layer, suggesting that the SE may sequester TMV MP on or within the sieve element reticulum (SER).Our data reveal that 3D-SIM is especially suited to the subdiffraction imaging of plant cells and yields spatial information not previously possible with conventional fluorescence-based imaging. The unique optical sectioning capacity of 3D-SIM and the ability to produce multicolor imaging with conventional fluorophores offer enormous potential in plant cell biology.     

High-resolution melting analysis for rapid detection of linezolid resistance (mediated by G2576T mutation) in Staphylococcus epidermidis

We describe a novel HRM-PCR (high-resolution melting) assay capable of the accurate identification of the G2576T point mutation in domain V of the 23S rRNA genes attributed to linezolid resistance in Staphylococcus epidermidis. This rapid method demonstrated 100% correlation with the previously established NheI restriction digest assay.     

Expression of IL-20 in synovium and lesional skin of patients with psoriatic arthritis: differential response to alefacept treatment

Introduction

Psoriatic arthritis (PsA) is an inflammatory joint disease associated with psoriasis. Alefacept (a lymphocyte function-associated antigen (LFA)-3 Ig fusion protein that binds to CD2 and functions as an antagonist to T-cell activation) has been shown to result in improvement in psoriasis but has limited effectiveness in PsA. Interleukin-20 (IL-20) is a key proinflammatory cytokine involved in the pathogenesis of psoriasis. The effects of alefacept treatment on IL-20 expression in the synovium of patients with psoriasis and PsA are currently unknown.

Methods

Eleven patients with active PsA and chronic plaque psoriasis were treated with alefacept (7.5 mg per week for 12 weeks) in an open-label study. Skin biopsies were taken before and after 1 and 6 weeks, whereas synovial biopsies were obtained before and 4 and 12 weeks after treatment. Synovial biopsies from patients with rheumatoid arthritis (RA) (n = 10) were used as disease controls. Immunohistochemical analysis was performed to detect IL-20 expression, and stained synovial tissue sections were evaluated with digital image analysis. Double staining was performed with IL-20 and CD68 (macrophages), and conversely with CD55 (fibroblast-like synoviocytes, FLSs) to determine the phenotype of IL-20-positive cells in PsA synovium. IL-20 expression in skin sections (n = 6) was analyzed semiquantitatively.

Results

IL-20 was abundantly expressed in both PsA and RA synovial tissues. In inflamed PsA synovium, CD68⁺ macrophages and CD55⁺ FLSs coexpressed IL-20, and its expression correlated with the numbers of FLSs. IL-20 expression in lesional skin of PsA patients decreased significantly (P = 0.04) 6 weeks after treatment and correlated positively with the Psoriasis Area and Severity Index (PASI). IL-20 expression in PsA synovium was not affected by alefacept.

Conclusions

Conceivably, the relatively limited effectiveness of alefacept in PsA patients (compared with anti-tumor necrosis factor (TNF) therapy) might be explained in part by persistent FLS-derived IL-20 expression.     

Development of a Culturally Relevant Body Image Instrument among Urban African Americans

Objective: To validate a culturally relevant body image instrument among urban African Americans through three distinct studies. Research Methods and Procedures: In Study 1, 38 medical practitioners performed content validity tests on the instrument. In Study 2, three research staff rated the body image of 283 African‐American public housing residents (75% women, mean age = 44 years), with the residents completing body image, BMI, and percentage body fat measures. In Study 3, 35 African Americans (57% men, mean age = 42) completed body image measures and evaluated their cultural relevance. Results: In Study 1, 97% to 100% of practitioners sorted the jumbled figures into the correct ascending order. The correlation between the body image figures and the practitioners’ weight classifications of the figures was high (r = 0.91). In Study 2, observers arrived at similar ratings of body size with excellent consistency (α = 0.95). Ratings of body image were strongly correlated with participant BMI (r = 0.89 to 0.93 across observers and 0.81 for all participants) and percentage of body fat (r = 0.77 to 0.89 across observers and 0.76 for all participants). In Study 3, body image ratings with the new scale were positively correlated with other validated figural scales. The majority of participants reported that figures in the new body image scale looked most like themselves and other African Americans and were easiest to identify themselves with. Discussion: The instrument displayed strong psychometric performance and cultural relevance, suggesting that the scale is a promising tool for examining body image and obesity among African Americans.     

Structure of mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 suggests a bifunctional switch that couples kinase activation with nuclear export

MAPK-activated protein kinase 2 (MAPKAPK2), one of several kinases directly phosphorylated and activated by p38 MAPK, plays a central role in the inflammatory response. The activated MAPKAPK2 phosphorylates its nuclear targets CREB/ATF1, serum response factor, and E2A protein E47 and its cytoplasmic targets HSP25/27, LSP-1, 5-lipoxygenase, glycogen synthase, and tyrosine hydroxylase. The crystal structure of unphosphorylated MAPKAPK2, determined at 2.8 A resolution, includes the kinase domain and the C-terminal regulatory domain. Although the protein is inactive, the kinase domain adopts an active conformation with aspartate 366 mimicking the missing phosphorylated threonine 222 in the activation loop. The C-terminal regulatory domain forms a helix-turn-helix plus a long strand. Phosphorylation of threonine 334, which is located between the kinase domain and the C-terminal regulatory domain, may serve as a switch for MAPKAPK2 nuclear import and export. Phosphorylated MAPKAPK2 masks the nuclear localization signal at its C terminus by binding to p38. It unmasks the nuclear export signal, which is part of the second C-terminal helix packed along the surface of kinase domain C-lobe, and thereby carries p38 to the cytoplasm.     

Difference between the spore sizes of Bacillus anthracis and other Bacillus species

AIMS: To determine the size distribution of the spores of Bacillus anthracis, and compare its size with other Bacillus species grown and sporulated under similar conditions. METHODS AND RESULTS: Spores from several Bacillus species, including seven strains of B. anthracis and six close neighbours, were prepared and studied using identical media, protocols and instruments. Here, we report the spore length and diameter distributions, as determined by transmission electron microscopy (TEM). We calculated the aspect ratio and volume of each spore. All the studied strains of B. anthracis had similar diameter (mean range between 0.81 +/- 0.08 microm and 0.86 +/- 0.08 microm). The mean lengths of the spores from different B. anthracis strains fell into two significantly different groups: one with mean spore lengths 1.26 +/- 0.13 microm or shorter, and another group of strains with mean spore lengths between 1.49 and 1.67 microm. The strains of B. anthracis that were significantly shorter also sporulated with higher yield at relatively lower temperature. The grouping of B. anthracis strains by size and sporulation temperature did not correlate with their respective virulence. CONCLUSIONS: The spores of Bacillus subtilis and Bacillus atrophaeus (previously named Bacillus globigii), two commonly used simulants of B. anthracis, were considerably smaller in length, diameter and volume than all the B. anthracis spores studied. Although rarely used as simulants, the spores of Bacillus cereus and Bacillus thuringiensis had dimensions similar to those of B. anthracis. SIGNIFICANCE AND IMPACT OF THE STUDY: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefence against B. anthracis. The data presented here should help in the selection of simulants that better resemble the properties of B. anthracis, and thus, more accurately represent the performance of collectors, detectors and other countermeasures against this threat agent.     

A platform for accurate mass and time analyses of mass spectrometry data

We describe an integrated suite of algorithms and software for general accurate mass and time (AMT) tagging data analysis of mass spectrometry data. The AMT approach combines identifications from liquid chromatography (LC) tandem mass spectrometry (MS/MS) data with peptide accurate mass and retention time locations from high-resolution LC-MS data. Our workflow includes the traditional AMT approach, in which MS/MS identifications are located in external databases, as well as methods based on more recent hybrid instruments such as the LTQ-FT or Orbitrap, where MS/MS identifications are embedded with the MS data. We demonstrate our AMT workflow's utility for general data synthesis by combining data from two dissimilar biospecimens. Specifically, we demonstrate its use relevant to serum biomarker discovery by identifying which peptides sequenced by MS/MS analysis of tumor tissue may also be present in the plasma of tumor-bearing and control mice. The analysis workflow, referred to as msInspect/AMT, extends and combines existing open-source platforms for LC-MS/MS (CPAS) and LC-MS (msInspect) data analysis and is available in an unrestricted open-source distribution.     

The Lck inhibitor,AMG-47a,blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

Necroptosis is a form of caspase-independent programmed cell death that arises from disruption of cell membranes by the mixed lineage kinase domain-like (MLKL) pseudokinase after its activation by the upstream kinases, receptor interacting protein kinase (RIPK)-1 and RIPK3, within a complex known as the necrosome. Dysregulated necroptosis has been implicated in numerous inflammatory pathologies. As such, new small molecule necroptosis inhibitors are of great interest, particularly ones that operate downstream of MLKL activation, where the pathway is less well defined. To better understand the mechanisms involved in necroptosis downstream of MLKL activation, and potentially uncover new targets for inhibition, we screened known kinase inhibitors against an activated mouse MLKL mutant, leading us to identify the lymphocyte-specific protein tyrosine kinase (Lck) inhibitor AMG-47a as an inhibitor of necroptosis. We show that AMG-47a interacts with both RIPK1 and RIPK3, that its ability to protect from cell death is dependent on the strength of the necroptotic stimulus, and that it blocks necroptosis most effectively in human cells. Moreover, in human cell lines, we demonstrate that AMG-47a can protect against cell death caused by forced dimerisation of MLKL truncation mutants in the absence of any upstream signalling, validating that it targets a process downstream of MLKL activation. Surprisingly, however, we also found that the cell death driven by activated MLKL in this model was completely dependent on the presence of RIPK1, and to a lesser extent RIPK3, although it was not affected by known inhibitors of these kinases. Together, these results suggest an additional role for RIPK1, or the necrosome, in mediating human necroptosis after MLKL is phosphorylated by RIPK3 and provide further insight into reported differences in the progression of necroptosis between mouse and human cells.Subject terms: Kinases, Necroptosis     

The Relationship Between Body Image Discrepancy and Body Mass Index Across Ethnic Groups

Objective: This study sought to examine at what body mass index (BMI) body image discrepancy (BD) was reported in a community sample of 389 white, Hispanic, and black women. In addition, we assessed the trajectory of the BMI–BD relationship as BMI increases by ethnic group. Research Methods and Procedures: All participants were assessed on height and weight and completed the Figure Rating Scale. Results: We found no difference in the proportion of women in each ethnic group reporting BD. However, white women experienced BD at a lower BMI level (BMI = 24.6), and below the criterion for overweight (BMI = 25). In contrast, black and Hispanic women did not report BD until they were overweight (BMIs of 29.2 and 28.5, respectively). Compared with black and white women, Hispanic women registered increases in BD at smaller increases in BMI. Discussion: These findings could have unhealthful implications for weight control behavior. The results encourage a closer look at ethnicity and BD, and their relationship to obesity and weight control.