Inhibition of TRPM8 function by prostacyclin receptor agonists requires coupling to Gq/11 proteins.

BACKGROUND AND PURPOSE: The TRPM8 ion channel is involved in innocuous cold sensing and has a potent anti-inflammatory action. Its activation by lower temperature or chemical agonists such as menthol and icilin induces analgesic effects, reversing hypersensitivity and reducing chronic pain. On the other hand, prostacyclin (PGI2) enhances pain and inflammation by activating the IP receptors. Due to the critical roles of TRPM8 and IP receptors in the regulation of inflammatory pain, and considering their overlapping expression pattern, we analysed the functional interaction between human TRPM8 and IP receptors. EXPERIMENTAL APPROACH: We transiently expressed human TRPM8 channels and IP receptors in HEK293T cells and carried out intracellular calcium and cAMP measurements. Additionally, we cultured neurons from the dorsal root ganglia (DRGs) of mice and determined the increase in intracellular calcium triggered by the TRPM8 agonist, icilin, in the presence of the IP receptor agonist cicaprost, the IP receptor antagonist Cay10441, and the Gq/11 inhibitor YM254890. KEY RESULTS: Activation of IP receptors by selective agonists (cicaprost, beraprost, and iloprost) inhibited TRPM8 channel function, independently of the Gs-cAMP pathway. The potent inhibition of TRPM8 channels by IP receptor agonists involved Gq/11 coupling. These effects were also observed in neurons isolated from murine DRGs. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate an unusual signalling pathway of IP receptors by coupling to Gq/11 proteins to inhibit TRPM8 channel function. This pathway may contribute to a better understanding of the role of TRPM8 channels and IP receptors in regulating pain and inflammation.

EDEM1 regulates the insulin mRNA level by inhibiting the endoplasmic reticulum stress-induced IRE1/JNK/c-Jun pathway.

Pancreatic beta cells produce and secrete insulin as a response to rises in blood glucose. Despite the advances in understanding glucose-regulated insulin transcription and translation the mechanisms triggering the synthesis of new insulin molecules are still incompletely described. In this report, we identify EDEM1 as a new modulator of insulin synthesis and secretion. In the presence of EDEM1, INS-1E cells secrete significantly more insulin upon glucose stimulation compared to control cells. We found that overexpression of EDEM1 inhibited the IRE1/JNK/c-Jun pathway, leading to an increase in the insulin mRNA level. Similarly, EDEM1 transduced human islets secreted significantly more insulin upon stimulation. Furthermore, EDEM1 improved insulin secretion restoring normoglycemia and glucose tolerance in diabetic rats. We propose EDEM1 as a regulator of the UPR via IRE1/XBP1s and IRE1/JNK/c-Jun signaling cascades and insulin transcription in pancreatic ß-cells, supporting EDEM1 as a potential target for the treatment of diabetes.

Methionine oxidation selectively enhances T cell reactivity against a melanoma antigen.

The impact of the peptide amino acids side-chain modifications on the immunological recognition has been scarcely explored. We investigate here the effect of methionine oxidation on the antigenicity of the melanoma immunodominant peptide 369-YMDGTMSQV-377 (YMD). Using CD8+ T cell activation assays, we found that the antigenicity of the sulfoxide form is higher when compared to the YMD peptide. This is consistent with free energy computations performed on HLA-A∗02:01/YMD/TCR complex showing that this is lowered upon oxidation, paired with a steep increase in order at atomic level. Oxidized YMD forms were identified at the melanoma cell surface by LC-MS/MS analysis. These results demonstrate that methionine oxidation in the antigenic peptides may generate altered peptide ligands with increased antigenicity, and that this oxidation may occur in vivo, opening up the possibility that high-affinity CD8+ T cells might be naturally primed in the course of melanoma progression, as a result of immunosurveillance.

New panel of biomarkers to discriminate between amelanotic and melanotic metastatic melanoma.

Melanoma is a form of skin cancer that can rapidly invade distant organs. A distinctive feature of melanomas is their pigmentation status, as melanin is present in most skin melanomas, whilst many metastatic tumors could become amelanotic. Besides the obvious malfunction of the key genes of the melanin pathway, the amelanotic tumors could bear a characteristic molecular signature accounting for their aggressivity. Using mass spectrometry-based proteomics we report here a distinctive panel of biomarkers for amelanotic aggressive melanoma that differ from the less invasive pigmented cells. The developed method allows the label-free quantification of proteins identified by LC-MS/MS analysis. We found a set of proteins comprising AHNAK, MYOF, ANXA1, CAPN2, ASPH, EPHA2, THBS1, TGM2, ACTN4 along with proteins involved in cell adhesion/migration (integrins, PLEC, FSCN1, FN1) that are highly expressed in amelanotic melanoma. Accompanying the down regulation of pigmentation specific proteins such as tyrosinase and TYRP1, these biomarkers are highly specific for a type of highly invasive melanoma. Interestingly, the LC-MS/MS proteomics analysis in hypoxia revealed that the abundance of this specific set of proteins found in normoxia was rather unaltered in these conditions. These biomarkers could therefore predict a metastatic behaviour for the amelanotic cells in the early stages of the tumor development and thus serve in melanoma prognostic. Applying this algorithm to related databases including melanoma samples published by independent laboratories/public databases we confirm the specificity of the newly found signatures. Overall, we begin to unravel the molecular alterations in the amelanotic melanoma and how basic proteomics offers insights into how to assess the clinical, pathological and misdiagnosis differences between the main subtypes of melanoma.

Affinity Proteomics and Deglycoproteomics Uncover Novel EDEM2 Endogenous Substrates and an Integrative ERAD Network.

Various pathologies result from disruptions to or stress of endoplasmic reticulum (ER) homeostasis, such as Parkinson's disease and most neurodegenerative illnesses, diabetes, pulmonary fibrosis, viral infections, and cancers. A critical process in maintaining ER homeostasis is the selection of misfolded proteins by the ER quality-control system for destruction via ER-associated degradation (ERAD). One key protein proposed to act during the first steps of misfolded glycoprotein degradation is the ER degradation-enhancing α-mannosidase-like protein 2 (EDEM2). Therefore, characterization of the EDEM2-associated proteome is of great interest. We took advantage of using melanoma cells overexpressing EDEM2 as a cancer model system, to start documenting at the deglycoproteome level (N-glycosites identification) the emerging link between ER homeostasis and cancer progression. The dataset created for identifying the EDEM2 glyco clients carrying high mannose/hybrid N-glycans provides a comprehensive N-glycosite analysis mapping over 1000 N-glycosites on more than 600 melanoma glycoproteins. To identify EDEM2-associated proteins, we used affinity proteomics and proteome-wide analysis of sucrose density fractionation in an integrative workflow. Using intensity and spectral count-based quantification, we identify seven new EDEM2 partners, all of which are involved in ER quality-control system and ERAD. Moreover, we defined novel endogenous candidates for EDEM2-dependent ERAD by combining deglycoproteomics, stable isotope labeling with amino acids in cell culture-based proteomics, and biochemical methods. These included tumor antigens and several ER-transiting endogenous melanoma proteins, including integrin alpha-1 and protocadherin 2, the expression of which was negatively correlated with that of EDEM2. Tumor antigens are key in the antigen presentation process, whereas integrin alpha-1 and protocadherin 2 are involved in melanoma metastasis and invasion. EDEM2 could therefore have a regulatory role in melanoma through the modulation of degradation and trafficking in these glycoproteins. The data presented herein suggest that EDEM2 is involved in ER homeostasis to a greater extent than previously suggested.

EDEM3 Domains Cooperate to Perform Its Overall Cell Functioning.

EDEM3 recognizes and directs misfolded proteins to the ER-associated protein degradation (ERAD) process. EDEM3 was predicted to act as lectin or as a mannosidase because of its homology with the GH47 catalytic domain of the Man1B1, but the contribution of the other regions remained unresolved. Here, we dissect the molecular determinants governing EDEM3 function and its cellular interactions. LC/MS analysis indicates very few stable ER interactors, suggesting EDEM3 availability for transient substrate interactions. Sequence analysis reveals that EDEM3 consists of four consecutive modules defined as GH47, intermediate (IMD), protease-associated (PA), and intrinsically disordered (IDD) domain. Using an EDEM3 knock-out cell line, we expressed EDEM3 and domain deletion mutants to address EDEM3 function. We find that the mannosidase domain provides substrate binding even in the absence of mannose trimming and requires the IMD domain for folding. The PA and IDD domains deletions do not impair the trimming, but specifically modulate the turnover of two misfolded proteins, NHK and the soluble tyrosinase mutant. Hence, we demonstrate that EDEM3 provides a unique ERAD timing to misfolded glycoproteins, not only by its mannose trimming activity, but also by the positive and negative feedback modulated by the protease-associated and intrinsically disordered domain, respectively.

EDEM1 Drives Misfolded Protein Degradation via ERAD and Exploits ER-Phagy as Back-Up Mechanism When ERAD Is Impaired.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the main mechanism of targeting ER proteins for degradation to maintain homeostasis, and perturbations of ERAD lead to pathological conditions. ER-degradation enhancing α-mannosidase-like (EDEM1) was proposed to extract terminally misfolded proteins from the calnexin folding cycle and target them for degradation by ERAD. Here, using mass-spectrometry and biochemical methods, we show that EDEM1 is found in auto-regulatory complexes with ERAD components. Moreover, the N-terminal disordered region of EDEM1 mediates protein-protein interaction with misfolded proteins, whilst the absence of this domain significantly impairs their degradation. We also determined that overexpression of EDEM1 can induce degradation, even when proteasomal activity is severely impaired, by promoting the formation of aggregates, which can be further degraded by autophagy. Therefore, we propose that EDEM1 maintains ER homeostasis and mediates ERAD client degradation via autophagy when either dislocation or proteasomal degradation are impaired.

Epitope located N-glycans impair the MHC-I epitope generation and presentation.

The degradation process of the antigens specific to MHC-I presentation depends mainly on the proteasomal proteases in the cytosol. However, since many antigens are glycoproteins, including tumor antigens or viruses envelope proteins, their glycosylation status could also affect their processing and presentation. Here, we investigate the processing of tyrosinase, a multiple glycosylated tumor antigen overexpressed in human malignant melanoma. By LC-MS/MS analysis of human tyrosinase expressed in a melanoma cell, we show that all seven sites of tyrosinase are at least partially N-glycosylated. Using human CD8+ T-cell clones specific for the tyrosinase epitope YMDGTMSQV (369-377), including an N-glycosylation site, we found that transfectants of single and triple N-glycosylation mutants are recognized by specific T cells. Importantly, single, triple, and the aglycosylated tyrosinase mutants lacking the epitope located N-glycosylation site (N371D) were able to trigger higher CD8+ T-cell activation. The LC/MS analysis showed significant increase of the amount of YMDGTMSQV peptide resulted from accelerated oligomerization and degradation of aglycosylated mutants. The generation of the antigenic peptide by the antigen processing machinery is therefore largely independent of tyrosinase N-glycosylation. However, while distal N-glycans had no effect on the epitope generation, the mutants lacking the N371 glycan generated the antigenic peptide more efficiently. We conclude that epitope located N-glycans limit the ability of human tyrosinase to provide HLA-A2-restricted antigen for recognition by specific CD8+ T cells.

Combinatorial MAPLE gradient thin film assemblies signalling to human osteoblasts.

There is increased interest in smart bioactive materials to control tissue regeneration for the engineering of cell instructive scaffolds. We introduced combinatorial matrix-assisted pulsed laser evaporation (C-MAPLE) as a new method for the fabrication of organic thin films with a compositional gradient. Synchronized C-MAPLE of levan and oxidized levan was employed to assemble a two-compound biopolymer film structure. The gradient of the film composition was validated by fluorescence microscopy. In this study, we investigated the cell response induced by the compositional gradient using imaging of early osteoblast attachment and analysis of signalling phosphoprotein expression. Cells attached along the gradient in direct proportion to oxidized levan concentration. During this process distinct areas of the binary gradient have been shown to modulate the osteoblasts' extracellular signal-regulated kinase signalling with different propensity. The proposed fabrication method results in the preparation of a new bioactive material, which could control the cell signalling response. This approach can be extended to screen new bioactive interfaces for tissue regeneration.

Expression and subcellular localization of RAGE in melanoma cells.

The receptor for advanced glycation end products (RAGE) is involved in multiple stages of tumor development and malignization. To gain further knowledge on the RAGE role in tumor progression, we investigated the receptor expression profile and its subcellular localization in melanoma cells at different stages of malignancy. We found that RAGE clustered at membrane ruffles and leading edges, and at sites of cell-to-cell contact in primary melanoma cells (e.g., MelJuSo), in contrast with a more dispersed localization in metastatic cells (e.g., SK-Mel28). RAGE silencing by RNAi selectively inhibited migration of MelJuSo cells, whilst having no influence on SK-Mel28 cell migration, in a "wound healing" assay. Western blot detection of RAGE showed a more complex RAGE oligomerization in MelJuSo cells compared to melanocytes and SK-Mel28 cells. By competing the binding of antibodies with recombinant soluble RAGE, an oligomeric form running at approximately 200 kDa was detected, as it was the monomeric RAGE of 55-60 kDa. SDS-PAGE electrophoresis under reducing versus nonreducing conditions indicated that the oligomer of about 200 kDa is formed by disulfide bonds, but other interactions are likely to be important for RAGE multimerization in melanoma cells. Immunofluorescence microscopy revealed that treatment with two cholesterol-chelating drugs, nystatin and filipin, significantly affected RAGE localization in MelJuSo cells. SK-Mel28 cells showed a reduced RAGE glycosylation and association with cholesterol-rich membranes and also a considerable downregulation of the soluble forms. Our results indicate that RAGE isoform expression and subcellular localization could be important determinants for the regulation of its function in tumor progression.

Value of dopachrome tautomerase detection in the assessment of melanocytic tumors.

Dopachrome tautomerase (DCT) and tyrosinase (Tyr) are melanogenic enzymes and structurally related melanosomal proteins. The present study investigates DCT expression comparatively with Tyr, the most tested melanoma biomarker, aiming to evaluate DCT potential in the assessment of melanocytic tumors and gain insights into the molecular and pathological characterization of DCT-phenotype in tumor progression. DCT and Tyr are simultaneously analyzed in melanoma cell lines by semiquantitative RT-PCR, western blot, and N-glycan analysis, and in cell populations of melanocytic tumors by immunohistofluorescence using a novel anti-hDCT antibody against an extended sequence within DCT luminal domain. DCT, unlike Tyr, is fully processed along the secretory pathway in both pigmented and amelanotic melanoma cells. In 53 nevi and 116 primary malignant melanomas, 81% and 52%, respectively, are DCT+/Tyr+, showing that DCT is a stable antigen, retained by most tumors and partially expressed in Tyr-negative cell populations. The DCT/Tyr disjunction is a process correlated with melanocyte neoplastic transformation and malignant progression. A tumor architecture--DCT-phenotype-containing DCT+/Tyr- cell populations selected into the innermost dermis from double-positive cells is detected in 35% of DCT+/Tyr+ specimens. The DCT-phenotype is associated with enhanced neurotization in benign nevi and with ulceration in thin malignant melanomas. The intradermal DCT+/Tyr- clones in superficial melanomas acquire the expression and specific subcellular distribution of unfavorable prognostic markers. DCT assessment shows specific antigen patterns with potential significance in the outcome of melanocytic lesions, connecting DCT, a mediator of a melanoma stress-resistant pathway, and an antiapoptotic molecule to DCT- phenotypes that are possibly more stable and stress resistant.

AP-3 and Rabip4' coordinately regulate spatial distribution of lysosomes.

The RUN and FYVE domain proteins rabip4 and rabip4' are encoded by RUFY1 and differ in a 108 amino acid N-terminal extension in rabip4'. Their identical C terminus binds rab5 and rab4, but the function of rabip4s is incompletely understood. We here found that silencing RUFY1 gene products promoted outgrowth of plasma membrane protrusions, and polarized distribution and clustering of lysosomes at their tips. An interactor screen for proteins that function together with rabip4' yielded the adaptor protein complex AP-3, of which the hinge region in the ß3 subunit bound directly to the FYVE domain of rabip4'. Rabip4' colocalized with AP-3 on a tubular subdomain of early endosomes and the extent of colocalization was increased by a dominant negative rab4 mutant. Knock-down of AP-3 had an ever more dramatic effect and caused accumulation of lysosomes in protrusions at the plasma membrane. The most peripheral lysosomes were localized beyond microtubules, within the cortical actin network. Our results uncover a novel function for AP-3 and rabip4' in regulating lysosome positioning through an interorganellar pathway.

C-terminus glycans with critical functional role in the maturation of secretory glycoproteins.

The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs--one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I.

Levan nanostructured thin films by MAPLE assembling.

Synthesis of nanostructured thin films of pure and oxidized levan exopolysaccharide by matrix-assisted pulsed laser evaporation is reported. Solutions of pure exopolysaccharides in dimethyl sulfoxide were frozen in liquid nitrogen to obtain solid cryogenic pellets that have been used as targets in pulsed laser evaporation experiments with a KrF* excimer source. The expulsed material was collected and assembled onto glass slides and Si wafers. The contact angle studies evidenced a higher hydrophilic behavior in the case of oxidized levan structures because of the presence of acidic aldehyde-hydrogen bonds of the coating formed after oxidation. The obtained films preserved the base material composition as confirmed by Fourier transform infrared spectroscopy. They were compact with high specific surface areas, as demonstrated by scanning electron and atomic force microscopy investigations. In vitro colorimetric assays revealed a high potential for cell proliferation for all coatings with certain predominance for oxidized levan.

Biocompatibility and bioactivity enhancement of Ce stabilized ZrO(2) doped HA coatings by controlled porosity change of Al(2) O(3) substrates.

Al(2) O(3) substrates with controlled porosity were manufactured from nanosized powders obtained by plasma processing. It was observed that when increasing the sintering temperature the overall porosity was decreasing, but the pores got larger. In a second step, Ce stabilized ZrO(2) doped hydroxyapatite coatings were pulsed laser deposited onto the Al(2) O(3) substrates. It was shown that the surface morphology, consisting of aggregates and particulates in micrometric range, was altered by the substrate porosity and interface properties, respectively. TEM studies evidenced that Ce stabilized ZrO(2) doped HA particulates ranged from 10 to 50 nm, strongly depending on the Al(2) O(3) porosity. The coatings consisted of HA nanocrystals embedded in an amorphous matrix quite similar to the bone structure. These findings were congruent with the increased biocompatibility and bioactivity of these layers confirmed by enhanced growing and proliferation of human mesenchymal stem cells.

Tailoring immobilization of immunoglobulin by excimer laser for biosensor applications.

The sheltered transfer and immobilization of rabbit anti-human antiserum immunoglobulin G (IgG) by matrix-assisted pulsed laser evaporation (MAPLE) are reported. The iced targets submitted to laser irradiation consisted of 0.2-2 mg/mL IgG blended or not with lipid (L-α-phosphatidylcholine dipalmitoyl) dissolved in distilled water-based saline buffer. Thin IgG coatings were obtained at room temperature onto glass, fused silica, or silicon substrates. Ten thousand subsequent laser pulses of 0.33, 0.5, or 0.67 J/cm(2) fluence were applied for the synthesis of each sample. Morphology and composition of the thin films were studied by optical, scanning, and atomic force microscopy and Fourier transformed infrared spectrometry. Optical labeling methods such as spectrofluorimetry and fluorescence microscopy were selected to verify the biosensor transduction principle because of their high sensitivity for detecting low amounts of antigen (IgG). Protein immobilization to the substrate surface was demonstrated for all obtained structures after immersion in the donkey anti-rabbit secondary antibody solution. The IgG transfer and immobilization onto substrates were improved by addition of lipid to MAPLE solutions.

Differentiation of mesenchymal stem cells onto highly adherent radio frequency-sputtered carbonated hydroxylapatite thin films.

In this work, an improved version of the radio frequency magnetron sputtering (RF-MS) technique was used to prepare highly adherent B-type carbonated hydroxylapatite (B-CHA) thin films. Fourier transform infrared spectroscopy (FTIR) and grazing incidence X-ray diffraction studies proved that the coatings maintained the composition and revealed the polycrystalline structure of HA. Scanning electron microscopy analysis showed that the CHA films are rough and exhibit a homogeneous microstructure. Energy-dispersive X-ray spectroscopy (EDX) mapping demonstrated a uniform distribution of the Ca and P cations while a Ca/P ratio of 1.8 was found. In addition, the FTIR experiments showed a remarkable reproducibility of the nanostructures. Human mesenchymal stem cells (hMSCs), in vitro differentiated osteoblasts, and explanted bone cells were grown over the surface of CHA coatings for periods between a few hours and 21 days. Osteoprogenitor cells maintained viability and characteristic morphology after adhesion on CHA coatings. Ki67-positive osteoblasts were the evidence of cell proliferation events. Cells showed positive staining for markers of osteoblast phenotype such as collagen type I, bone sialoprotein and osteonectin. Our data showed the formation of mineralized foci by differentiation of hMSCs to human primary osteoblasts after cultivation in osteogenic media on RF-sputtered films. The results demonstrate the capacity of B-type CHA coating to support MSCs adhesion and osteogenic differentiation ability.

Encapsulated cargo internalized by fusogenic liposomes partially overlaps the endoplasmic reticulum.

Few endocytosed ligands, including bacterial toxins and simian virus 40 (SV40) have been shown to reach the endoplasmic reticulum (ER) in mammalian cells. Using calcein and fluorescently labelled lactoferrin encapsulated in fusogenic liposomes we found that the cargo uses a microtubule-based pathway with ER delivery. Endocytic uptake of the lipid vesicles was cholesterol dependent in all cell lines tested, including the caveolin-1-deficient human hepatoma 7 cell line. The ligand was transported in non-caveosome organelles requiring acidic pH for maturation, but able to escape the lysosomal route. These organelles were not recycling endosomes either, as shown by the lack of co-localization with recycling transferrin. Co-localization with the ER-tracker, orange fluorescent protein with KDEL signal retention and cholera toxin in live microscopy revealed an ER distribution of the fluorescent ligand. Brefeldin A, which prevents Golgi-dependent retrograde trafficking, does not disrupt the cargo delivery to the ER. This new endocytic pathway making use of acidic endosome-like organelles is an alternative to the reported SV40 caveolae pathways. Exploiting a cellular route linking the cell surface to the ER, fusogenic liposomes may become efficient drug delivery vehicles for ER stress and diseases.

Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG.

Ovarian cancer is the fourth most common cancer in women in the Western world. In a pilot scale study, we highlight changes in the total serum glycome of patients with advanced ovarian cancer that might shed insight into disease pathogenesis. These changes include increases in levels of core fucosylated, agalactosyl biantennary glycans (FA2) and sialyl Lewis x (SLe(x)). To investigate further which proteins contribute to these alterations, we developed technology to analyze simultaneously the glycosylation of protein glycoforms contained in single spots excised from a 2D gel (<1 ng protein). The acute-phase proteins, haptoglobin, alpha1-acid glycoprotein, and alpha1-antichymotrypsin from patients contained elevated levels of subsets of glycoforms containing SLe(x). We also established that IgG heavy chains from patients contained twice the level of FA2 compared with healthy controls. Serum CA125 is the only biomarker that is used routinely, and there is a need for complementary markers that will improve both sensitivity and specificity. There was some preliminary indication that combinations of changes in the serum glycome might improve the separation of ovarian cancer and benign tumors; however, a larger study using data receiver operating characteristic curves will be required to draw any firm conclusions.