Diagnosing and staging epithelial ovarian cancer by serum glycoproteomic profiling.

BACKGROUND: There is a need for diagnostic tests for screening, triaging and staging of epithelial ovarian cancer (EOC). Glycoproteomics of blood samples has shown promise for biomarker discovery. METHODS: We applied glycoproteomics to serum of people with EOC or benign pelvic masses and healthy controls. A total of 653 analytes were quantified and assessed in multivariable models, which were tested in an independent cohort. Additionally, we analyzed glycosylation patterns in serum markers and in tissues. RESULTS: We identified a biomarker panel that distinguished benign lesions from EOC with sensitivity and specificity of 83.5% and 90.1% in the training set, and of 86.7 and 86.7% in the test set, respectively. ROC analysis demonstrated strong performance across a range of cutoffs. Fucosylated multi-antennary glycopeptide markers were higher in late-stage than in early-stage EOC. A comparable pattern was found in late-stage EOC tissues. CONCLUSIONS: Blood glycopeptide biomarkers have the potential to distinguish benign from malignant pelvic masses, and early- from late-stage EOC. Glycosylation of circulating and tumor tissue proteins may be related. This study supports the hypothesis that blood glycoproteomic profiling can be used for EOC diagnosis and staging and it warrants further clinical evaluation.

Mass Spectrometry Analysis of Glycopeptides Enriched by Anion Exchange-Mediated Methods Reveals PolyLacNAc-Extended N-Glycans in Integrins and Tetraspanins in Melanoma Cells.

Glycosylation is a key modulator of the functional state of proteins. Recent developments in large-scale analysis of intact glycopeptides have enabled the identification of numerous glycan structures that are relevant in pathophysiological processes. However, one motif found in N-glycans, poly-N-acetyllactosamine (polyLacNAc), still poses a substantial challenge to mass spectrometry-based glycoproteomic analysis due to its relatively low abundance and large size. In this work, we developed approaches for the systematic mapping of polyLacNAc-elongated N-glycans in melanoma cells. We first evaluated five anion exchange-based matrices for enriching intact glycopeptides and selected two materials that provided better overall enrichment efficiency. We then tested the robustness of the methodology by quantifying polyLacNAc-containing glycopeptides as well as changes in protein fucosylation and sialylation. Finally, we applied the optimal enrichment methods to discover glycopeptides containing polyLacNAc motifs in melanoma cells and found that integrins and tetraspanins are substantially modified with these structures. This study demonstrates the feasibility of glycoproteomic approaches for identification of glycoproteins with polyLacNAc motifs.

Inter- and transgenerational heritability of preconception chronic stress or alcohol exposure: Translational outcomes in brain and behavior.

Chronic stress and alcohol (ethanol) use are highly interrelated and can change an individual's behavior through molecular adaptations that do not change the DNA sequence, but instead change gene expression. A recent wealth of research has found that these nongenomic changes can be transmitted across generations, which could partially account for the "missing heritability" observed in genome-wide association studies of alcohol use disorder and other stress-related neuropsychiatric disorders. In this review, we summarize the molecular and behavioral outcomes of nongenomic inheritance of chronic stress and ethanol exposure and the germline mechanisms that could give rise to this heritability. In doing so, we outline the need for further research to: (1) Investigate individual germline mechanisms of paternal, maternal, and biparental nongenomic chronic stress- and ethanol-related inheritance; (2) Synthesize and dissect cross-generational chronic stress and ethanol exposure; (3) Determine cross-generational molecular outcomes of preconception ethanol exposure that contribute to alcohol-related disease risk, using cancer as an example. A detailed understanding of the cross-generational nongenomic effects of stress and/or ethanol will yield novel insight into the impact of ancestral perturbations on disease risk across generations and uncover actionable targets to improve human health.

Plasma glycoproteomic biomarkers identify metastatic melanoma patients with reduced clinical benefit from immune checkpoint inhibitor therapy.

The clinical success of immune-checkpoint inhibitors (ICI) in both resected and metastatic melanoma has confirmed the validity of therapeutic strategies that boost the immune system to counteract cancer. However, half of patients with metastatic disease treated with even the most aggressive regimen do not derive durable clinical benefit. Thus, there is a critical need for predictive biomarkers that can identify individuals who are unlikely to benefit with high accuracy so that these patients may be spared the toxicity of treatment without the likely benefit of response. Ideally, such an assay would have a fast turnaround time and minimal invasiveness. Here, we utilize a novel platform that combines mass spectrometry with an artificial intelligence-based data processing engine to interrogate the blood glycoproteome in melanoma patients before receiving ICI therapy. We identify 143 biomarkers that demonstrate a difference in expression between the patients who died within six months of starting ICI treatment and those who remained progression-free for three years. We then develop a glycoproteomic classifier that predicts benefit of immunotherapy (HR=2.7; p=0.026) and achieves a significant separation of patients in an independent cohort (HR=5.6; p=0.027). To understand how circulating glycoproteins may affect efficacy of treatment, we analyze the differences in glycosylation structure and discover a fucosylation signature in patients with shorter overall survival (OS). We then develop a fucosylation-based model that effectively stratifies patients (HR=3.5; p=0.0066). Together, our data demonstrate the utility of plasma glycoproteomics for biomarker discovery and prediction of ICI benefit in patients with metastatic melanoma and suggest that protein fucosylation may be a determinant of anti-tumor immunity.

A proximity labeling method for protein-protein interactions on cell membrane.

Antibodies targeting specific antigens are widely utilized in biological research to investigate protein interactions or to quantify target antigens. Here, we introduce antigen-antibody proximity labeling (AAPL), a novel method to map the antigen interaction sites as well as interactors of antibody-targeted proteins. As a proof of concept, AAPL was demonstrated using sodium/potassium transporting ATPase (ATP1A1) and epidermal growth factor receptor 2 (ERBB2)-specific antibodies that were modified with an Fe(iii) catalytic probe. Once bound to their target proteins, Fe(iii)-induced catalytic oxidation occurred in proximity of the antigen's epitope. Oxidative proteomic analysis was then used to determine the degree of oxidation, the site of oxidation within the targeted antigen, and the interacting proteins that were in close proximity to the targeted antigen. An AAPL score was generated for each protein yielding the specificity of the oxidation and proximity of the interacting protein to the target antigen. As a final demonstration of its utility, the AAPL approach was applied to map the interactors of liver-intestine-cadherin (CDH17) in colon cancer cells.

Serum Glycoprotein Markers in Nonalcoholic Steatohepatitis and Hepatocellular Carcinoma.

Fatty liver disease progresses through stages of fat accumulation and inflammation to nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis, and eventually hepatocellular carcinoma (HCC). Currently available diagnostic tools for HCC lack sensitivity and specificity. In this study, we investigated the use of circulating serum glycoproteins to identify a panel of potential prognostic markers that may be indicative of progression from the healthy state to NASH and further to HCC. Serum samples were processed and analyzed using a novel high-throughput glycoproteomics platform. Our initial dataset contained healthy, NASH, and HCC serum samples. We analyzed 413 glycopeptides, representing 57 abundant serum proteins, and compared among the three phenotypes. We studied the normalized abundance of common glycoforms and found 40 glycopeptides with statistically significant differences in abundances in NASH and HCC compared to controls. Summary level relative abundances of core-fucosylated, sialylated, and branched glycans containing glycopeptides were higher in NASH and HCC as compared to controls. We replicated some of our findings in an independent set of samples of individuals with benign liver conditions and HCC. Our results may be of value in the management of liver diseases. Data generated in this work can be downloaded from MassIVE (https://massive.ucsd.edu) with identifier MSV000088809.

University students' awareness of causes and risk factors of miscarriage: a cross-sectional study.

BACKGROUND: Spontaneous miscarriage is the most common complication of pregnancy, occurring in up to 20% of pregnancies. Despite the prevalence of miscarriage, little is known regarding peoples' awareness and understanding of causes of pregnancy loss. The aim of this study was to explore university students' understanding of rates, causes and risk factors of miscarriage. METHODS: A cross-sectional study including university students. An online questionnaire was circulated to all students at the University College Cork using their university email accounts in April and May 2016. Main outcomes included identification of prevalence, weeks of gestation at which miscarriage occurs and causative risk factors for miscarriage. RESULTS: A sample of 746 students were included in the analysis. Only 20% (n = 149) of students correctly identified the prevalence of miscarriage, and almost 30% (n = 207) incorrectly believed that miscarriage occurs in less than 10% of pregnancies. Female were more likely to correctly identify the rate of miscarriage than men (21.8% versus 14.5%). However, men tended to underestimate the rate and females overestimate it. Students who did not know someone who had a miscarriage underestimated the rate of miscarriage, and those who were aware of some celebrities who had a miscarriage overestimated the rate. Almost 43% (n = 316) of students correctly identified fetal chromosomal abnormalities as the main cause of miscarriage. Females, older students, those from Medical and Health disciplines and those who were aware of a celebrity who had a miscarriage were more likely to identify chromosomal abnormalities as a main cause. However, more than 90% of the students believed that having a fall, consuming drugs or the medical condition of the mother was a causative risk factor for miscarriage. Finally, stress was identified as a risk factor more frequently than advanced maternal age or smoking. CONCLUSION: Although almost half of the participants identified chromosomal abnormalities as the main cause of miscarriage, there is still a lack of understanding about the prevalence and most important risk factors among university students. University represents an ideal opportunity for health promotion strategies to increase awareness of potential adverse outcomes in pregnancy.

Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain.

The colony-stimulating factor 1 receptor (CSF1R) is a key regulator of myeloid lineage cells. Genetic loss of the CSF1R blocks the normal population of resident microglia in the brain that originates from the yolk sac during early development. However, the role of CSF1R signaling in microglial homeostasis in the adult brain is largely unknown. To this end, we tested the effects of selective CSF1R inhibitors on microglia in adult mice. Surprisingly, extensive treatment results in elimination of ∼99% of all microglia brain-wide, showing that microglia in the adult brain are physiologically dependent upon CSF1R signaling. Mice depleted of microglia show no behavioral or cognitive abnormalities, revealing that microglia are not necessary for these tasks. Finally, we discovered that the microglia-depleted brain completely repopulates with new microglia within 1 week of inhibitor cessation. Microglial repopulation throughout the CNS occurs through proliferation of nestin-positive cells that then differentiate into microglia.

Ethanol-induced conditioned partner preference in female mice.

Drinking behavior and social context are intimately intertwined. Peer relations can promote drinking. Conversely, alcohol promotes social interaction. The present study tested female mice for ethanol-induced conditioned partner preference. Ovariectomized (OVX) C57Bl/6 females with chronic estradiol replacement (OVX+E) received saline or ethanol (1, 2 or 4 g/kg) ip and were paired 4 × for 30 min each with 1 of 2 stimulus females. The test female was paired with the CS- stimulus female following saline, and was paired with the CS+ female following ethanol. After pairing, we measured proximity of the test female to the CS+ and CS- females in a 10' test. In a second study, OVX and OVX+E females were tested for conditioned partner preference (CS+ vs. CS-) in response to 2.5 g/kg ethanol. In separate groups of mice, both test and stimulus females (IS+) received ethanol during pairing to determine if test mice develop conditioned partner preference for another intoxicated mouse. OVX+E test females showed conditioned partner preference for the CS+ female in response to ethanol at 2g/kg (change in preference score for CS+: +86.6 ± 30.0 s/10 min), but not at 0, 1 or 4 g/kg. At 2.5 g/kg ethanol, OVX+E females developed conditioned partner preference for either IS+ (+63.6 ± 24.0 s) or CS+ females (+93.8 ± 27.1 s). OVX test females demonstrated ethanol-induced conditioned partner preference only for the IS+ female (+153.8 ± 32.0 s). These data demonstrate that ethanol promotes social preference in female mice, and that estradiol enhances this effect.

Assessment of replication rates of human keratinocytes in engineered skin substitutes grafted to athymic mice.

Stable closure of skin wounds with engineered skin substitutes (ESS) requires indefinite mitotic capacity to generate the epidermis. To evaluate whether keratinocytes in ESS exhibit the stem cell phenotype of label retention, ESS (n = 6-9/group) were pulsed with 5-bromo-2'-deoxyuridine (BrdU) in vitro, and after grafting to athymic mice (n = 3-6/group). Pulse and immediate chase in vitro labeled virtually all basal keratinocytes at day 8, with label uptake decreasing until day 22. Label retention in serial chase decreased more rapidly from day 8 to day 22, with a reorganization of BrdU-positive cells into clusters. Similarly, serial chase of labeled basal keratinocytes in vivo decreased sharply from day 20 to day 48 after grafting. Label uptake was assessed by immediate chases of basal keratinocytes, and decreased gradually to day 126, while total labeled cells remained relatively unchanged. These results demonstrate differential rates of label uptake and retention in basal keratinocytes of ESS in vitro and in vivo, and a proliferative phenotype with potential for long-term replication in the absence of hair follicles. Regulation of a proliferative phenotype in keratinocytes of ESS may improve the biological homology of tissue-engineered skin to natural skin, and contribute to more rapid and stable wound healing.

Protein kinase CK2 modulates IL-6 expression in inflammatory breast cancer.

Inflammatory breast cancer is driven by pro-angiogenic and pro-inflammatory cytokines. One of them Interleukin-6 (IL-6) is implicated in cancer cell proliferation and survival, and promotes angiogenesis, inflammation and metastasis. While IL-6 has been shown to be upregulated by several oncogenes, the mechanism behind this phenomenon is not well characterized. Here we demonstrate that the pleotropic Serine/Threonine kinase CK2 is implicated in the regulation of IL-6 expression in a model of inflammatory breast cancer. We used siRNAs targeted toward CK2 and a selective small molecule inhibitor of CK2, CX-4945, to inhibit the expression and thus suppress the secretion of IL-6 in in vitro as well as in vivo models. Moreover, we report that in a clinical trial, CX-4945 was able to dramatically reduce IL-6 levels in plasma of an inflammatory breast cancer patient. Our data shed a new light on the regulation of IL-6 expression and position CX-4945 and potentially other inhibitors of CK2, for the treatment of IL-6-driven cancers and possibly other diseases where IL-6 is instrumental, including rheumatoid arthritis.