Pentraxin Domain Research Articles

Improving the prediction of major adverse cardiovascular events in patients with hypertrophic cardiomyopathy by adding plasma SVEP1 to conventional clinical model including NT-proBNP

Abstract Background Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease with a prevalence between 1 in 200 to 500 [1]. HCM can lead to major adverse cardiovascular events (MACE) such as heart failure (HF) hospitalization and cardiac death [2,3]. A protein named "sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1" (SVEP1) is a large extracellular matrix protein whose activity can be measured in plasma [4,5]. SVEP1 promotes inflammation and atherosclerosis [4,5]. SVEP1 is associated with risk of HF hospitalization and cardiac death in patients with HF with reduced ejection fraction [6]. However, no prior study has assessed the prognostic value of SVEP1 in patients with HCM. Purpose To test the hypothesis that the addition of SVEP1 improves the predictive performance of the conventional model based on clinical risk factors plus NT-proBNP for future MACE in patients with HCM. Methods We performed a multicenter prospective cohort study of patients with HCM [7]. The outcome was MACE, defined as a composite of HF hospitalization or cardiac death [6]. After dividing the cohort into four groups using the median values of SVEP1 and NT-proBNP, we compared the risk of the outcome event using the Cox proportional hazards model adjusting for 15 clinical risk factors known to be associated with MACE in HCM (Figure 1) [8-11]. We also developed the Lasso-regularized Cox proportional hazards model to predict the time to first MACE by adding SVEP1 to the 15 clinical risk factors with or without NT-proBNP in the training cohort (randomly selected 75% of the cohort). We then compared the predictive performance in the test cohort (25% of the cohort) using the C-statistic of each model. Results During a median follow-up of 1.9 (25 percentile-75 percentile, 0.7-4.3) years, the outcome event occurred in 63 (10%) of 610 patients. The four groups stratified by the median of SVEP1 and NT-proBNP levels had different risks of the outcome event (log-rank p<0.001: Figure 1). Even in the groups with lower-than-median NT-proBNP levels, the high-SVEP1 group had higher risks of MACE compared with the low-SVEP1 group (adjusted hazard ratio 4.52, p=0.042: Figure 1). In the prediction of time to first MACE, the addition of SVEP1 improved the C-statistic of the clinical model (p<0.001) and that of the clinical plus NT-proBNP model (p=0.01: Figure 2). The clinical plus SVEP1 model also outperformed the clinical plus NT-proBNP model (p=0.005: Figure 2). Conclusions In the present multicenter prospective cohort study of 610 patients with HCM, SVEP1 improved MACE risk stratification when added to the conventional model consisting of clinical risk factors plus NT-proBNP. SVEP1 also exhibited a superior predictive ability than NT-proBNP to predict MACE in patients with HCM. These data collectively indicate that SVEP1 provides additional risk stratification and may be a better biomarker than NT-proBNP for the prognostication of patients with HCM.Figure 1.Kaplan-Meier curves for MACEFigure 2.Time-dependent AUROC curves

Aptamer Proteomics for Biomarker Discovery in Heart Failure With Preserved Ejection Fraction: The PARAGON-HF Proteomic Substudy.

Prognostic markers and biological pathways linked to detrimental clinical outcomes in heart failure with preserved ejection fraction (HFpEF) remain incompletely defined. We measured serum levels of 4123 unique proteins in 1117 patients with HFpEF enrolled in the PARAGON-HF (Efficacy and Safety of LCZ696 Compared to Valsartan, on Morbidity and Mortality in Heart Failure Patients With Preserved Ejection Fraction) trial using a modified aptamer proteomic assay. Baseline circulating protein concentrations significantly associated with the primary end point and the timing and occurrence of total heart failure hospitalization and cardiovascular death were identified by recurrent events regression, accounting for multiple testing, adjusted for age, sex, treatment, and anticoagulant use, and compared with published analyses in 2515 patients with heart failure with reduced ejection fraction from the PARADIGM-HF (Prospective Comparison of ARNI With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure) and ATMOSPHERE (Efficacy and Safety of Aliskiren and Aliskiren/Enalapril Combination on Morbidity-Mortality in Patients With Chronic Heart Failure) clinical trials. We identified 288 proteins that were robustly associated with the risk of heart failure hospitalization and cardiovascular death in patients with HFpEF. The baseline proteins most strongly related to outcomes included B2M (β-2 microglobulin), TIMP1 (tissue inhibitor of matrix metalloproteinase 1), SERPINA4 (serpin family A member 4), and SVEP1 (sushi, von Willebrand factor type A, EGF, and pentraxin domain containing 1). Overall, the protein-outcome associations in patients with HFpEF did not markedly differ as compared with patients with heart failure with reduced ejection fraction. A proteomic risk score derived in patients with HFpEF was not superior to a previous proteomic score derived in heart failure with reduced ejection fraction nor to clinical risk factors, NT-proBNP (N-terminal pro-B-type natriuretic peptide), or high-sensitivity cardiac troponin. Numerous serum proteins linked to metabolic, coagulation, and extracellular matrix regulatory pathways were associated with worse HFpEF prognosis in the PARAGON-HF proteomic substudy. Our results demonstrate substantial similarities among serum proteomic risk markers for heart failure hospitalization and cardiovascular death when comparing clinical trial participants with heart failure across the ejection fraction spectrum. URL: https://www.clinicaltrials.gov; Unique Identifiers: NCT01920711, NCT01035255, NCT00853658.

Idiopathic multicentric Castleman disease - TAFRO results in high levels of mTOR activator SVEP1, tissue factor, and endotheliopathy

AbstractIdiopathic multicentric Castleman disease (iMCD) is an inflammatory disease associated with a cytokine storm, activation of the PI3K/AKT/mTOR pathway, coagulopathy, and increased risk of thrombosis. The mechanisms underlying these pathologic processes remain elusive. We studied novel markers of mTOR activation and thrombosis in 1 patient with typical features of iMCD with TAFRO (thrombocytopenia, anasarca, fevers, reticulin myelofibrosis, and organomegaly) syndrome (iMCD-TAFRO). Plasma levels of SVEP1 (Sushi, von Willebrand factor type A, epidermal growth factor, and pentraxin domain–containing 1 protein), a newly identified mTOR activator associated with cardiovascular diseases and dementia, in addition to cytokines, chemokines and components of the coagulation cascade and complement system were evaluated by enzyme-linked immunosorbent assay (ELISA) and arrays. Compared with healthy controls, a 15-fold increase in SVEP1 was observed. High levels of factor VIIa/antithrombin and microparticles expressing functional tissue factor (TF) were detected. The anticoagulants thrombomodulin and soluble endothelial protein C receptor were elevated, indicating shedding from endothelial cells. Plasminogen activator inhibitor 1 was increased, consistent with hypofibrinolysis, whereas high levels of C3b and C5a are in keeping with complement activation. Furthermore, markers of endothelial cell activation (e.g. von Willebrand factor, angiopoietin-2), cell adhesion molecules, and angiogenesis mediators were upregulated. SVEP1 emerges as a potential mechanism of mTOR activation in iMCD-TAFRO, while multiple pathways influence coagulopathy. Immunothrombosis emerges as a potential therapeutic target for iMCD.

Proteomic Associations of NT-proBNP (N-Terminal Pro-B-Type Natriuretic Peptide) in Heart Failure With Preserved Ejection Fraction.

NT-proBNP (N-terminal pro-B-type natriuretic peptide) levels are variably elevated in heart failure with preserved ejection fraction (HFpEF), even in the presence of increased left ventricular filling pressures. NT-proBNP levels are prognostic in HFpEF and have been used as an inclusion criterion for several recent randomized clinical trials. However, the underlying biologic differences between HFpEF participants with high and low NT-proBNP levels remain to be fully understood. We measured 4928 proteins using an aptamer-based proteomic assay (SOMAScan) in available plasma samples from 2 cohorts: (1) Participants with HFpEF enrolled in the PHFS (Penn Heart Failure Study; n=253); (2) TOPCAT (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist Trial) participants in the Americas (n=218). We assessed the relationship between SOMAScan-derived plasma NT-proBNP and levels of other proteins available in the SOMAScan assay version 4 using robust linear regression, with correction for multiple comparisons, followed by pathway analysis. NT-proBNP levels exhibited prominent proteome-wide associations in PHFS and TOPCAT cohorts. Proteins most strongly associated with NT-proBNP in both cohorts included SVEP1 (sushi, von Willebrand factor type-A, epidermal growth factor, and pentraxin domain containing 1; βTOPCAT=0.539; P<0.0001; βPHFS=0.516; P<0.0001) and ANGPT2 (angiopoietin 2; βTOPCAT=0.571; P<0.0001; βPHFS=0.459; P<0.0001). Canonical pathway analysis demonstrated consistent associations with multiple pathways related to fibrosis and inflammation. These included hepatic fibrosis and inhibition of matrix metalloproteases. Analyses using cut points corresponding to estimated quantitative concentrations of 360 pg/mL (and 480 pg/mL in atrial fibrillation) revealed similar proteomic associations. Circulating NT-proBNP levels exhibit prominent proteomic associations in HFpEF. Our findings suggest that higher NT-proBNP levels in HFpEF are a marker of fibrosis and inflammation. These findings will aid the interpretation of NT-proBNP levels in HFpEF and may guide the selection of participants in future HFpEF clinical trials.

The emerging Janus face of SVEP1 in development and disease.

Sushi, von Willebrand factor type A, EGF, and pentraxin domain containing 1 (SVEP1) is a large extracellular matrix protein that is also detected in circulation. Recent plasma proteomic and genomic studies have revealed a large number of associations between SVEP1 and human traits, particularly chronic disease. These include associations with cardiac death and disease, diabetes, platelet traits, glaucoma, dementia, and aging; many of these are causal. Animal models demonstrate that SVEP1 is critical in vascular development and disease, but its molecular and cellular mechanisms remain poorly defined. Future studies should aim to characterize these mechanisms and determine the diagnostic, prognostic, and therapeutic value of measuring or intervening on this enigmatic protein.

The dimerized pentraxin-like domain of the adhesion G protein–coupled receptor 112 (ADGRG4) suggests function in sensing mechanical forces

Adhesion G protein-coupled receptors (aGPCRs) feature large extracellular regions with modular domains that often resemble protein classes of various function. The pentraxin (PTX) domain, which is predicted by sequence homology within the extracellular region of four different aGPCR members, is well known to form pentamers and other oligomers. Oligomerization of GPCRs is frequently reported and mainly driven by interactions of the seven-transmembrane region and N or C termini. While the functional importance of dimers is well-established for some class C GPCRs, relatively little is known about aGPCR multimerization. Here, we showcase the example of ADGRG4, an orphan aGPCR that possesses a PTX-like domain at its very N-terminal tip, followed by an extremely long stalk containing serine-threonine repeats. Using X-ray crystallography and biophysical methods, we determined the structure of this unusual PTX-like domain and provide experimental evidence for a homodimer equilibrium of this domain which is Ca2+-independent and driven by intermolecular contacts that differ vastly from the known soluble PTXs. The formation of this dimer seems to be conserved in mammalian ADGRG4 indicating functional relevance. Our data alongside of theoretical considerations lead to the hypothesis that ADGRG4 acts as an invivo sensor for shear forces in enterochromaffin and Paneth cells of the small intestine.

WR-PTXF, a novel short pentraxin, protects gut mucosal barrier and enhances the antibacterial activity in Carassius cuvieri × Carassius auratus red var.

The pentraxin family is an evolutionarily conserved group that plays an important role in innate immunity. C-reactive protein (CRP) and serum amyloid P component (SAP) are classical members of the short pentraxins and are known to be the major acute phase proteins. In this work, we have cloned a novel pentraxin fusion protein, WR-PTXF, from Carassius cuvieri × Carassius auratus red var. In fish, the biological function of PTXF is essentially unknown. For this purpose, we report the identification and analysis of WR-PTXF and elucidate its role in the antibacterial innate immunity. WR-PTXF contains 224 amino acids and shares 79.8% and 23.0% sequence identities with crucian carp CRP and SAP, respectively. Blast analysis shows that WR-PTXF and goldfish PTXF had the highest similarity (97.3%). WR-PTXF is expressed in multiple tissues and is upregulated by Aeromonas hydrophila infection. WR-PTXF contains a short pentraxin domain and recombinant WR-PTXF protein (rWR-PTXF) can bind the A. hydrophila in a concentration-dependent manner. Further, rWR-PTXF displays apparent bacteriostatic activity against A. hydrophila in vitro by enhancing the uptake of the bound bacteria by host phagocytes. When introduced in vivo, rWR-PTXF not only protects the gut mucosa but also limits the colonization of A. hydrophila in systemic immune organs. Consistently, knockdown of WR-PTXF significantly promotes bacterial dissemination in the tissues of host. These results indicate that WR-PTXF is a classic pattern recognition molecule that exerts a protective effect against bacterial infection.

SVEP1 is an endogenous ligand for the orphan receptor PEAR1

Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 (SVEP1) is an extracellular matrix protein that causally promotes vascular disease and associates with platelet reactivity in humans. Here, using a human genomic and proteomic approach, we identify a high affinity, disease-relevant, and potentially targetable interaction between SVEP1 and the orphan receptor Platelet and Endothelial Aggregation Receptor 1 (PEAR1). This interaction promotes PEAR1 phosphorylation and disease associated AKT/mTOR signaling in vascular cells and platelets. Mice lacking SVEP1 have reduced platelet activation, and exogenous SVEP1 induces PEAR1-dependent activation of platelets. SVEP1 and PEAR1 causally and concordantly relate to platelet phenotypes and cardiovascular disease in humans, as determined by Mendelian Randomization. Targeting this receptor-ligand interaction may be a viable therapeutic strategy to treat or prevent cardiovascular and thrombotic disease.

Aptamer proteomics for biomarker discovery in heart failure with reduced ejection fraction

Abstract Background Though current heart failure (HF) biomarkers are highly prognostic, systematically characterizing associations between circulating proteins and risk of subsequent events may improve clinical risk prediction and illuminate new biological pathways. Large-scale assays measuring thousands of proteins now enable unbiased proteomic investigation in clinical trials. Purpose To identify and replicate serum proteins associated with HF events in patients with chronic HF with reduced ejection fraction (HFrEF), and to develop and validate a proteomic risk score. Methods Serum levels of 4076 proteins were measured at baseline in the ATMOSPHERE (n=1261, 487 events over 6 years) and PARADIGM-HF (n=1257, 287 events over 4 years) trials of chronic HFrEF using a modified aptamer-based proteomics assay. Proteins associated with the primary endpoint, HF hospitalization or cardiovascular death, were identified in the ATMOSPHERE discovery cohort (false discovery rate&amp;lt;0.05) by Cox regression adjusted for age, sex, treatment arm, and anticoagulant use, and replicated in PARADIGM-HF (Bonferroni-corrected p&amp;lt;0.05). A proteomic risk score was derived in ATMOSPHERE using Cox LASSO penalized regression and evaluated in PARADIGM-HF compared to the MAGGIC clinical risk score and N-terminal pro-B-type natriuretic peptide (NT-proBNP). For proteins associated with the primary endpoint, pathway analysis was conducted using Ingenuity Pathway analysis and an exploratory two-sample Mendelian randomization was performed using genetic and outcome data from both trials and protein quantitative trait loci from deCODE to infer which identified proteins contribute to HF prognosis. Results We identified 377 serum proteins associated with the primary endpoint in ATMOSPHERE and replicated 167 in PARADIGM-HF. Prognostic proteins included known HF biomarkers Growth Differentiation Factor 15, NT-proBNP, and Angiopoietin-2, and also a previously unrecognized HF biomarker: Sushi, Von Willebrand Factor Type A, EGF And Pentraxin Domain Containing 1 (SVEP1) (HR 1.60 [95% CI 1.44–1.79] per standard deviation [SD], p=2x10–17) (Table 1). Proteins related to hepatic fibrosis, granulocyte adhesion, and inhibition of matrix metalloproteinases were over-represented. A 64-protein risk score derived in ATMOSPHERE predicted clinical events in PARADIGM-HF with greater discrimination (c-statistic 0.70) than the MAGGIC clinical score (c-statistic 0.61), NT-proBNP (c-statistic 0.65), or both (c-statistic 0.66) (Figure 1). Genetically predicted levels of NT-proBNP, WISP2, FSTL1, and CTSS were associated with the primary endpoint by Mendelian randomization. Conclusions We identify SVEP1, an extracellular matrix protein known to cause inflammation in vascular smooth muscle cells, as a previously unrecognized HF biomarker. A 64-protein score improved risk discrimination compared with NT-proBNP and may assist in identifying high-risk patients for clinical trials or disease management programs. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): The ATMOSPHERE and PARADIGM-HF trials were sponsored by Novartis

CRP Involved in Nile Tilapia (Oreochromis niloticus) against Bacterial Infection.

Simple SummaryC-reactive protein (CRP) is an acute-phase protein that can be used as an early diagnostic marker for inflammation. Few CRPs have been isolated from teleost, and the specific immunological functions and mechanism of fish CRP have not been well-studied. Therefore, in this research, a CRP gene from Nile tilapia was identified, and its roles during bacterial infection were investigated. The current results revealed that CRP participated in anti-bacterial immune response through agglutinating bacterial, regulating phagocytosis and inflammation. Hopefully, our data might be beneficial in further study to understand the protective mechanism of fish CRP against bacterial infection.C-reactive protein (CRP) is an acute-phase protein that can be used as an early diagnostic marker for inflammation, which is also an evolutionarily conserved protein and has been identified from arthropods to mammals. However, the roles of CRP during the immune response of Nile tilapia (Oreochromis niloticus) remain unclear. In this study, a CRP gene from Nile tilapia (On-CRP) was identified, and its roles in response to bacterial infection were investigated in vivo or in vitro. On-CRP was found to contain an open reading frame of 675 bp, encoding a polypeptide of 224 amino acids with the conservative pentraxin domain. On-CRP shares more than 50% of its identity with other fish species, and 30% of its identity with mammals. The transcriptional level of On-CRP was most abundant in the liver and its transcripts can be remarkably induced following Streptococcus agalactiae and Aeromonas hydrophila infection. Furthermore, in vitro analysis indicated that the recombinant protein of On-CRP improved phagocytic activity of monocytes/macrophages, and possessed a bacterial agglutination activity in a calcium-dependent manner. Both in vivo and in vitro experiments indicated that On-CRP could promote inflammation and activate the complement pathway. However, a direct relationship between CRP and several immune pathways could not be confirmed. The present data lays a theoretical foundation to further explore the mechanism of how CRP protects fish against bacterial infection.

Abstract 496: The Cardiometabolic Disease Risk Protein SVEP1 Activates AKT/mTOR By Signaling Through The Orphan Receptor PEAR1

Background: Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 (SVEP1) is an extracellular matrix protein that circulates in plasma and is causally related to cardiovascular disease, hypertension, and type 2 diabetes. A recent genome wide association study (GWAS) also implicates SVEP1 in platelet reactivity. The gene most strongly associated with platelet reactivity in the GWAS is Platelet and Endothelial Cell Receptor 1 ( PEAR1 ), a gene that encodes an orphan receptor tyrosine kinase-like protein that also associates with cardiovascular disease. Little is known about the molecular interactions and disease mechanisms of these proteins, despite their associations with cardiovascular disease and platelet reactivity. Methods: We used Mendelian Randomization (MR) and phenome-wide association studies to identify candidate SVEP1 protein interactions. We tested candidate interactions using recombinant proteins with molecular, cellular, and ex vivo assays. Results: A coding variant within the ectodomain of PEAR1 alters plasma levels of SVEP1 in humans (p=1.7x10^-17), suggesting these proteins may interact. MR demonstrates that genetically determined, increased plasma PEAR1 levels causally associate with decreased plasma SVEP1 (p=1.3x10^-11), further supporting an interaction. PEAR1 co-immunoprecipitates with SVEP1. Exposure to SVEP1 induces PEAR1 phosphorylation and activation of AKT/mTOR signaling in endothelial cells, vascular smooth muscle cells, and platelets. siRNA knockdown of PEAR1 abrogates this signaling. Endothelial cells adhere and proliferate in response to immobilized SVEP1. Platelets isolated from mice lacking SVEP1 have lower activation relative to controls and phenocopy platelets from mice lacking PEAR1. Conclusions: Despite robust evidence for the role of SVEP1 and PEAR1 in cardiovascular disease, critical gaps remain in our understanding of their disease mechanisms. Here, we identify SVEP1 as the first biological ligand of PEAR1 and a potent activator of AKT/mTOR signaling. The interaction between SVEP1 and PEAR1 is particularly intriguing, given the prominent role of AKT/mTOR in cardiovascular disease, and disrupting the interaction may be therapeutically beneficial.

Dysregulation of Amphiregulin stimulates the pathogenesis of cystic lymphangioma

Along with blood vessels, lymphatic vessels play an important role in the circulation of body fluid and recruitment of immune cells. Postnatal lymphangiogenesis commonly occurs from preexisting lymphatic vessels by sprouting, which is induced by lymphangiogenic factors such as vascular endothelial growth factor C (VEGF-C). However, the key signals and cell types that stimulate pathological lymphangiogenesis, such as human cystic lymphangioma, are less well known. Here, we found that mouse dermal fibroblasts that infiltrate to sponges subcutaneously implanted express VEGF-D and sushi, Von Willebrand factor type A, EGF, and pentraxin domain containing 1 (SVEP1) in response to PDGFRβ signal. In vitro, Pdgfrb knockout (β-KO) fibroblasts had reduced expression of VEGF-D and SVEP1 and overproduced Amphiregulin. Dysregulation of these three factors was involved in the cyst-like and uneven distribution of lymphatic vessels observed in the β-KO mice. Similarly, in human cystic lymphangioma, which is one of the intractable diseases and mostly occurs in childhood, fibroblasts surrounding cystic lymphatics highly expressed Amphiregulin. Moreover, fibroblast-derived Amphiregulin could induce the expression of Amphiregulin in lymphatic endothelial cells. The dual source of Amphiregulin activated EGFR expressed on the lymphatic endothelial cells. This exacerbation cascade induced proliferation of lymphatic endothelial cells to form cystic lymphangioma. Ultimately, excessive Amphiregulin produced by fibroblasts surrounding lymphatics and by lymphatic endothelial cells per se results in pathogenesis of cystic lymphangioma and will be a fascinating therapeutic target of cystic lymphangioma.

Functional investigation of the coronary artery disease gene SVEP1

A missense variant of the sushi, von Willebrand factor type A, EGF and pentraxin domain containing protein 1 (SVEP1) is genome-wide significantly associated with coronary artery disease. The mechanisms how SVEP1 impacts atherosclerosis are not known. We found endothelial cells (EC) and vascular smooth muscle cells to represent the major cellular source of SVEP1 in plaques. Plaques were larger in atherosclerosis-prone Svep1 haploinsufficient (ApoE−/−Svep1+/−) compared to Svep1 wild-type mice (ApoE−/−Svep1+/+) and ApoE−/−Svep1+/− mice displayed elevated plaque neutrophil, Ly6Chigh monocyte, and macrophage numbers. We assessed how leukocytes accumulated more inside plaques in ApoE−/−Svep1+/− mice and found enhanced leukocyte recruitment from blood into plaques. In vitro, we examined how SVEP1 deficiency promotes leukocyte recruitment and found elevated expression of the leukocyte attractant chemokine (C-X-C motif) ligand 1 (CXCL1) in EC after incubation with missense compared to wild-type SVEP1. Increasing wild-type SVEP1 levels silenced endothelial CXCL1 release. In line, plasma Cxcl1 levels were elevated in ApoE−/−Svep1+/− mice. Our studies reveal an atheroprotective role of SVEP1. Deficiency of wild-type Svep1 increased endothelial CXCL1 expression leading to enhanced recruitment of proinflammatory leukocytes from blood to plaque. Consequently, elevated vascular inflammation resulted in enhanced plaque progression in Svep1 deficiency.

The Basic Characteristics of the Pentraxin Family and Their Functions in Tumor Progression.

The pentraxin is a superfamily of proteins with the same domain known as the pentraxin domain at C-terminal. This family has two subgroups, namely; short pentraxins (C-reactive protein and serum amyloid P component) and long pentraxins (neuronal pentraxin 1, neuronal pentraxin 2, neuronal pentraxin receptor, pentraxin 3 and pentraxin 4). Each group shares a similar structure with the pentameric complexes arranged in a discoid shape. Previous studies revealed the functions of different pentraxin family members. Most of them are associated with human innate immunity. Inflammation has commonly been associated with tumor progression, implying that the pentraxin family might also participate in tumor progression. Therefore, we reviewed the basic characteristics and functions of the pentraxin family and their role in tumor progression.

Proteomic biomarkers in mid-trimester amniotic fluid associated with adverse pregnancy outcomes in patients with systemic lupus erythematosus.

We aimed to explore the proteomic profiles of mid-trimester amniotic fluid in pregnant women with systemic lupus erythematosus (SLE) according to the occurrence of adverse pregnancy outcome (APO). The study population included 35 pregnant women with SLE who underwent clinically indicated amniocentesis at 15–24 weeks of gestation. Patients were divided into two groups according to pregnancy outcomes: SLE patients without APO (Group 1) and SLE patients with APO (Group 2). Stored samples of amniotic fluid were analyzed using mass spectrometry (MS)-based proteomics with two-step approach, consisting of discovery and verification phase. In the discovery phase, 44 proteins were differentially expressed between Group 1 and Group 2. In the verification phase, differentially expressed proteins (DEPs) were verified in independent samples using DIA method. Four proteins including filamin A (FLNA), sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 (SVEP1), lecithin-cholesterol acyltransferase (LCAT), and transglutaminase 2 (TGM2) were differentially expressed both in discovery and verification phase. To select the best combination of proteins for discriminating two groups, three-fold cross validation (CV) with repetition of one hundred times was performed. The multi-marker model with three biomarkers (SVEP1, LCAT, TGM2) had a high discriminatory power to distinguish between the two groups (the area under the receiver operating characteristic, AUROC = 0.946, p <0.001). Our results indicate that the expression of FLNA, SVEP1, LCAT, and TGM2 in mid-trimester amniotic fluid was increased in SLE patients with APO (Group 2). A large-scale prospective study is warranted to verify this finding.

Identification of genes associated with gastric cancer survival and construction of a nomogram to improve risk stratification for patients with gastric cancer.

The present study aimed to identify genes associated with gastric cancer survival and improve risk stratification for patients with gastric cancer. Transcriptomic and clinicopathological data from 443 gastric cancer samples were retrieved from The Cancer Genome Atlas database. The DESeq R package was applied to screen for differentially expressed genes between Tumor-Node-Metastasis (TNM) stage (I vs. IV) and histological grade (G3 vs. G1 and G2). A total of seven genes were common to both comparisons; spondin 1 (SPON1); thrombospondin 4 (THBS4); Sushi, Von Willebrand factor type A, EGF and pentraxin domain containing 1 (SVEP1); prickle planar cell polarity protein 1 (PRICKLE1); ATP binding cassette subfamily A member 8 (ABCA8); Slit guidance ligand 2 (SLIT2); and EGF containing fibulin extracellular matrix protein 1 (EFEMP1), were selected as candidate survival-associated genes for further analysis. The prognostic value of these genes was assessed according to a literature review and Kaplan-Meier survival analysis. In addition, a multivariate Cox regression analysis revealed PRICKLE1 expression to be an independent prognostic factor for patients with gastric cancer. Furthermore, a predictive nomogram was generated using PRICKLE1 expression, patient age and TNM stage to assess overall survival (OS) rate at 1, 3 and 5 years, with an internal concordance index of 0.65. External validation was conducted in an independent cohort of 59 patients with gastric cancer, and high consistency between the predicted and observed results for OS was exhibited. Overall, the current findings suggest that PRICKLE1 expression may serve as an independent prognostic factor that can be integrated with age and TNM stage in a nomogram able to predict OS rate in patients with gastric cancer.

Retrieval-Driven Hippocampal NPTX2 Plasticity Facilitates the Extinction of Cocaine-Associated Context Memory

BackgroundPostretrieval extinction attenuates the pathological memory associated with psychiatric states such as drug addiction in both humans and rodents. The extinction of a learned response requires gene transcription and protein synthesis after memory retrieval in a time-dependent manner, yet the precise physiological basis after retrieval to allow extinction to neutralize a learned behavior is not fully understood. MethodsIn a cocaine conditioned place preference paradigm, we used a ribosomal tagging strategy to measure the translational state of hippocampal pyramidal neurons after the retrieval of cocaine-associated context memory. Using approaches of electrophysiology, neuronal tracing, and a doxycycline-dependent robust activity marking system, we investigated the cellular and molecular basis of retrieval-induced plasticity that facilitated the extinction. ResultsBioinformatics analysis discovered the specific translational regulation of signaling pathways by retrieval and revealed Nptx2 as the hub gene. Manipulating Nptx2 in dorsal hippocampus bidirectionally regulated the extinction of cocaine-associated context memory as well as the retrieval-driven synaptic remodeling. The pentraxin (PTX) domain of NPTX2 recruited GluA1-AMPA receptors and enhanced the extinction and excitatory synaptic transmission that was prevented by overexpressing carboxyl cytoplasmic tail of GluA1. Furthermore, Nptx2 in retrieval-activated neurons was required for the extinction. ConclusionsThe retrieval-driven upregulation of Nptx2 contributes to the synaptic remodeling in dorsal hippocampus and facilitates the extinction of cocaine-associated context memory, indicating a potential target for the treatment of cue-induced cocaine seeking.

857: Proteomic biomarkers in mid-trimester amniotic fluid for adverse outcomes in pregnant women with SLE

Pregnant women with systemic lupus erythematosus (SLE) are at increased risk for adverse pregnancy outcomes (APOs) due to abnormal placentation. Identifying patients destined for APO early in pregnancy would significantly improve their obstetric outcomes. We sought to identify potential proteomic biomarkers in mid-trimester amniotic fluid of pregnant women with SLE for the prediction of APOs. The study population included 35 pregnant women with SLE who underwent clinically indicated amniocentesis at 15-24 weeks of gestation. Patients were divided into two groups: 19 women with normal pregnancy outcome (Group 1=control group) and 16 women who developed APOs (Group 2=APO group). APO was defined as the development of at least one of the followings: preeclampsia, fetal death in utero, neonatal death before hospital discharge, indicated preterm birth <36 weeks, and small for gestational age (<5th percentile) at birth. Stored samples of amniotic fluids were analyzed using mass spectrometry-based proteomics. In a discovery phase, we analyzed 9 patients (4 in Group 1 and 5 in Group 2) with data-dependent acquisition (DDA) approach. In a verification phase, we analyzed the remained 26 patients (15 in Group 1 and 11 in Group 2) using data- independent acquisition (DIA) method. Hierarchical clustering was performed based on the Euclidean distance and the average linkage. In the discovery phase, 44 proteins were differentially expressed between Group 1 and Group 2. In a verification phase, differentially expressed proteins (DEPs) were verified in independent samples using DIA method. Four proteins including FLNA (filamin A), SVEP1 (sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1), LCAT (lecithin-cholesterol acyltransferase), and TGM2 (transglutaminase 2) were differentially expressed both in discovery and verification phase. In order to select the best combination of proteins for predicting adverse outcomes, three-fold cross validation (CV) with repetition of 100 times was performed. The multi-marker model with 3 biomarkers (SVEP1, LCAT, TGM2) had a high discriminatory power to distinguish between the two groups (the area under the receiver operating characteristic, AUROC = 0.855, p<0.001). Our result indicates that increased expression of SVEP1, LCAT, and TGM2 in mid-trimester amniotic fluid may predict APOs in women with SLE. A large scale prospective study is warranted to verify this finding.

Identification and characterization of a novel zebrafish (Danio rerio) pentraxin-carbonic anhydrase.

BackgroundCarbonic anhydrases (CAs) are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H+ ions. Vertebrate genomes generally contain gene loci for 15–21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only secretory protein of the enzymatically active isoforms. We discovered that non-mammalian CA VI contains a C-terminal pentraxin (PTX) domain, a novel combination for both CAs and PTXs.MethodsWe isolated and sequenced zebrafish (Danio rerio) CA VI cDNA, complete with the sequence coding for the PTX domain, and produced the recombinant CA VI–PTX protein. Enzymatic activity and kinetic parameters were measured with a stopped-flow instrument. Mass spectrometry, analytical gel filtration and dynamic light scattering were used for biophysical characterization. Sequence analyses and Bayesian phylogenetics were used in generating hypotheses of protein structure and CA VI gene evolution. A CA VI–PTX antiserum was produced, and the expression of CA VI protein was studied by immunohistochemistry. A knock-down zebrafish model was constructed, and larvae were observed up to five days post-fertilization (dpf). The expression of ca6 mRNA was quantitated by qRT-PCR in different developmental times in morphant and wild-type larvae and in different adult fish tissues. Finally, the swimming behavior of the morphant fish was compared to that of wild-type fish.ResultsThe recombinant enzyme has a very high carbonate dehydratase activity. Sequencing confirms a 530-residue protein identical to one of the predicted proteins in the Ensembl database (ensembl.org). The protein is pentameric in solution, as studied by gel filtration and light scattering, presumably joined by the PTX domains. Mass spectrometry confirms the predicted signal peptide cleavage and disulfides, and N-glycosylation in two of the four observed glycosylation motifs. Molecular modeling of the pentamer is consistent with the modifications observed in mass spectrometry. Phylogenetics and sequence analyses provide a consistent hypothesis of the evolutionary history of domains associated with CA VI in mammals and non-mammals. Briefly, the evidence suggests that ancestral CA VI was a transmembrane protein, the exon coding for the cytoplasmic domain was replaced by one coding for PTX domain, and finally, in the therian lineage, the PTX-coding exon was lost. We knocked down CA VI expression in zebrafish embryos with antisense morpholino oligonucleotides, resulting in phenotype features of decreased buoyancy and swim bladder deflation in 4 dpf larvae.DiscussionThese findings provide novel insights into the evolution, structure, and function of this unique CA form.

Multiple gene and transcript variants encoding trout C-polysaccharide binding proteins are differentially but strongly induced after infection with Aeromonas salmonicida

Two ‘trout C-polysaccharide-binding proteins,’ TCBP1 and -2, with relevance to early inflammatory events have been discovered in the last century. The present study characterises the respective cDNA sequences from rainbow trout (Oncorhynchus mykiss), including multiple TCBP1 transcript variants. These variants are generated either by the use of alternative splice sites or the exclusion of exons. The longest mRNA isoform, TCBP1-1, encodes a 245-aa protein with a large signal peptide and a complement component C1q domain. The shortest mRNA isoform, TCBP1-5, contains a premature termination codon and hence fails to encode a functional factor. The 224-aa-long TCBP2 protein consists of a comparably shorter signal peptide and a pentraxin domain. Evolutionary analyses clearly separated TCBP1 and -2 because of distinctive protein motifs. Expression profiling in the liver, spleen, and head kidney tissues of healthy trout revealed that TCBP2 mRNA concentrations were higher than the concentrations of all five TCBP1 mRNA isoforms together. The hepatic levels of these TCBP1 variants increased significantly upon infection with Aeromonas salmonicida, whereas TCBP2 transcript levels rose moderately. As the biological function of TCBP1 is barely understood, we tagged this factor with the green fluorescent protein and visualised its expression in HEK-293 cells. Overexpression of TCBP1 increased the level of active NF-κB factors and induced cell death, indicating its involvement in proapoptotic NF-κB-dependent signalling routes.