Global O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow.

A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.

Alterations in the kallikrein-kinin system predict death after heart transplant.

Heart transplantation remains the definitive treatment for end stage heart failure. Because availability is limited, risk stratification of candidates is crucial for optimizing both organ allocations and transplant outcomes. Here we utilize proteomics prior to transplant to identify new biomarkers that predict post-transplant survival in a multi-institutional cohort. Microvesicles were isolated from serum samples and underwent proteomic analysis using mass spectrometry. Monte Carlo cross-validation (MCCV) was used to predict survival after transplant incorporating select recipient pre-transplant clinical characteristics and serum microvesicle proteomic data. We identified six protein markers with prediction performance above AUROC of 0.6, including Prothrombin (F2), anti-plasmin (SERPINF2), Factor IX, carboxypeptidase 2 (CPB2), HGF activator (HGFAC) and low molecular weight kininogen (LK). No clinical characteristics demonstrated an AUROC > 0.6. Putative biological functions and pathways were assessed using gene set enrichment analysis (GSEA). Differential expression analysis identified enriched pathways prior to transplant that were associated with post-transplant survival including activation of platelets and the coagulation pathway prior to transplant. Specifically, upregulation of coagulation cascade components of the kallikrein-kinin system (KKS) and downregulation of kininogen prior to transplant were associated with survival after transplant. Further prospective studies are warranted to determine if alterations in the KKS contributes to overall post-transplant survival.

The family of kallikrein-related peptidases and kinin peptides as modulators of epidermal homeostasis.

The epidermis is the outermost skin layer and is part of one of the largest organs in the body; it is supported by the dermis, a network of fibrils, blood vessels, pilosebaceous units, sweat glands, nerves, and cells. The skin as a whole is a protective shield against numerous noxious agents, including microorganisms and chemical and physical factors. These functions rely on the activity of multiple growth factors, peptide hormones, proteases, and specific signaling pathways that are triggered by the activation of distinct types of receptors sited in the cell membranes of the various cell types present in the skin. The human kallikrein family comprises a large group of 15 serine proteases synthesized and secreted by different types of epithelial cells throughout the body, including the skin. At this site, they initiate a proteolytic cascade that generates the active forms of the proteases, some of which regulate skin desquamation, activation of cytokines, and antimicrobial peptides. Kinin peptides are formed by the action of plasma and tissue kallikreins on kininogens, two plasma proteins produced in the liver and other organs. Although kinins are well known for their proinflammatory abilities, in the skin they are also considered important modulators of keratinocyte differentiation. In this review, we summarize the contributions of the kallikreins and kallikrein-related peptidases family and those of kinins and their receptors in skin homeostasis, with special emphasis on their pathophysiological role.

The effect of bradykinin on the pro-inflammatory response of human adipocytes.

The proper functioning of adipose tissue is one of the factors in maintaining energy homeostasis. Adipocytes not only store lipids but also produce active molecules such as adipokines and adipocytokines, which are involved in many functions of adipose tissue, including the secretion of hormones that regulate energy and lipid metabolism. Inflammation has been shown to underlie the deregulation of adipose tissue function. Bradykinin belongs to a family of pro-inflammatory kinin peptides that are abundant in most tissues and biological fluids. This study aimed to determine the ability to produce kinin peptides and characterize the effect of bradykinin on pro-inflammatory responses in adipocytes. The Chub-S7 human preadipocyte line was differentiated to show specific properties for adipose tissue cells. The differentiated cells expressed genes that encode proteins such as kininogen, kallikrein, and prolylcarboxypeptidase that are involved in the production of kinins and also showed the expression of kinin receptors. The response of adipocytes to bradykinin was examined in relation to kinin concentration and the presence of kininase inhibitors. The high concentration of bradykinin induced a moderate increase in lipid accumulation, increased release of pro-inflammatory cytokines, and altered gene expression of molecules involved in adipocyte function, such as adiponectin, lipoprotein lipase, and other transcription factors. This study suggests an important role for kinin peptides in inducing inflammatory responses in adipocytes, which can modify the function of adipose tissue and ultimately lead to diseases related to disturbance of energy homeostasis. The results obtained may enrich our understanding of the mechanisms underlying obesity-related disorders.

A mechanism for hereditary angioedema caused by a lysine 311-to-glutamic acid substitution in plasminogen.

Patients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311 to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311 vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311 induces BK generation independently of PKa and FXIIa. Plm-Glu311 cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311 substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311 in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311 cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311 is pathogenic in the context of human Plm when human kininogens are the substrates.

High Molecular Weight Kininogen: A Review of the Structural Literature.

Kininogens are multidomain glycoproteins found in the blood of most vertebrates. High molecular weight kininogen demonstrate both carrier and co-factor activity as part of the intrinsic pathway of coagulation, leading to thrombin generation. Kininogens are the source of the vasoactive nonapeptide bradykinin. To date, attempts to crystallize kininogen have failed, and very little is known about the shape of kininogen at an atomic level. New advancements in the field of cryo-electron microscopy (cryoEM) have enabled researchers to crack the structure of proteins that has been refractory to traditional crystallography techniques. High molecular weight kininogen is a good candidate for structural investigation by cryoEM. The goal of this review is to summarize the findings of kininogen structural studies.

Investigation of proteins important for microcirculation using in vivo microdialysis after glucose provocation: a proteomic study.

Insulin has metabolic and vascular effects in the human body. What mechanisms that orchestrate the effects in the microcirculation, and how the responds differ in different tissues, is however not fully understood. It is therefore of interest to search for markers in microdialysate that may be related to the microcirculation. This study aims to identify proteins related to microvascular changes in different tissue compartments after glucose provocation using in vivo microdialysis. Microdialysis was conducted in three different tissue compartments (intracutaneous, subcutaneous and intravenous) from healthy subjects. Microdialysate was collected during three time periods; recovery after catheter insertion, baseline and glucose provocation, and analyzed using proteomics. Altogether, 126 proteins were detected. Multivariate data analysis showed that the differences in protein expression levels during the three time periods, including comparison before and after glucose provocation, were most pronounced in the intracutaneous and subcutaneous compartments. Four proteins with vascular effects were identified (angiotensinogen, kininogen-1, alpha-2-HS-glycoprotein and hemoglobin subunit beta), all upregulated after glucose provocation compared to baseline in all three compartments. Glucose provocation is known to cause insulin-induced vasodilation through the nitric oxide pathway, and this study indicates that this is facilitated through the interactions of the RAS (angiotensinogen) and kallikrein-kinin (kininogen-1) systems.

Type 2 Diabetes Coagulopathy Proteins May Conflict With Biomarkers Reflective of COVID-19 Severity.

Objective: Detailed proteomic analysis in a cohort of patients with differing severity of COVID-19 disease identified biomarkers within the complement and coagulation cascades as biomarkers for disease severity has been reported; however, it is unclear if these proteins differ sufficiently from other conditions to be considered as biomarkers. Methods: A prospective, parallel study in T2D (n = 23) and controls (n = 23). A hyperinsulinemic clamp was performed and normoglycemia induced in T2D [4.5 ± 0.07 mmol/L (81 ± 1.2 mg/dl)] for 1-h, following which blood glucose was decreased to ≤2.0 mmol/L (36 mg/dl). Proteomic analysis for the complement and coagulation cascades were measured using Slow Off-rate Modified Aptamer (SOMA)-scan. Results: Thirty-four proteins were measured. At baseline, 4 of 18 were found to differ in T2D versus controls for platelet degranulation [Neutrophil-activating peptide-2 (p = 0.014), Thrombospondin-1 (p = 0.012), Platelet factor-4 (p = 0.007), and Kininogen-1 (p = 0.05)], whilst 3 of 16 proteins differed for complement and coagulation cascades [Coagulation factor IX (p < 0.05), Kininogen-1 (p = 0.05), and Heparin cofactor-2 (p = 0.007)]; STRING analysis demonstrated the close relationship of these proteins to one another. Induced euglycemia in T2D showed no protein changes versus baseline. At hypoglycemia, however, four proteins changed in controls from baseline [Thrombospondin-1 (p < 0.014), platelet factor-4 (p < 0.01), Platelet basic protein (p < 0.008), and Vitamin K-dependent protein-C (p < 0.00003)], and one protein changed in T2D [Vitamin K-dependent protein-C, (p < 0.0002)]. Conclusion: Seven of 34 proteins suggested to be biomarkers of COVID-19 severity within the platelet degranulation and complement and coagulation cascades differed in T2D versus controls, with further changes occurring at hypoglycemia, suggesting that validation of these biomarkers is critical. It is unclear if these protein changes in T2D may predict worse COVID-19 disease for these patients. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT03102801.

Analysis of cold activation of the contact system in hereditary angioedema with normal C1 inhibitor.

Hereditary angioedema (HAE) attacks are caused by excessive activation of the contact system. Understanding how the contact system is activated in HAE, especially in patients with normal C1 inhibitor (HAEnCI), is essential to effectively treat this disease. Contact system activation involves the cleavage of several proteins including Factor XII (FXII), high molecular weight kininogen (HK), prekallikrein, sgp120 (ITIH4) and C1 inhibitor (C1-INH) before the subsequent generation of bradykinin that mediates HAE. In this study, we evaluated the fragmentation and enzymatic activity of contact system proteins in HAEnCI plasma samples before and after contact system activation induced by incubation in the cold. Our results show that in contrast to normal plasma, cold activation induced contact system activation in the majority of the HAEnCI patient samples we tested, in which each contact system protein exhibited fragmentation, FXII and kallikrein enzymatic activity increased, and C1-INH functional activity decreased. HAEnCI samples with low FXII concentrations or functional activity were not affected by cold activation. One HAEnCI sample with a plasminogen gene mutation activated the fibrinolytic system, as shown by an increase in concentration of plasma D dimers. Our results suggest that cold activation seems to be initiated by the cleavage of prekallikrein, and that it needs FXII in order to occur. Reported to be susceptible to excessive contact system activation after incubation in the cold, we further applied this system of study to the evaluation of plasma from women undergoing estrogen treatment. Similar to plasma from HAEnCI patients, excessive contact system activation was demonstrated.

Plasmin-mediated cleavage of high-molecular-weight kininogen contributes to acetaminophen-induced acute liver failure.

Acetaminophen (APAP)-induced liver injury is associated with activation of coagulation and fibrinolysis. In mice, both tissue factor-dependent thrombin generation and plasmin activity have been shown to promote liver injury after APAP overdose. However, the contribution of the contact and intrinsic coagulation pathways has not been investigated in this model. Mice deficient in individual factors of the contact (factor XII [FXII] and prekallikrein) or intrinsic coagulation (FXI) pathway were administered a hepatotoxic dose of 400 mg/kg of APAP. Neither FXII, FXI, nor prekallikrein deficiency mitigated coagulation activation or hepatocellular injury. Interestingly, despite the lack of significant changes to APAP-induced coagulation activation, markers of liver injury and inflammation were significantly reduced in APAP-challenged high-molecular-weight kininogen-deficient (HK-/-) mice. Protective effects of HK deficiency were not reproduced by inhibition of bradykinin-mediated signaling, whereas reconstitution of circulating levels of HK in HK-/- mice restored hepatotoxicity. Fibrinolysis activation was observed in mice after APAP administration. Western blotting, enzyme-linked immunosorbent assay, and mass spectrometry analysis showed that plasmin efficiently cleaves HK into multiple fragments in buffer or plasma. Importantly, plasminogen deficiency attenuated APAP-induced liver injury and prevented HK cleavage in the injured liver. Finally, enhanced plasmin generation and HK cleavage, in the absence of contact pathway activation, were observed in plasma of patients with acute liver failure due to APAP overdose. In summary, extrinsic but not intrinsic pathway activation drives the thromboinflammatory pathology associated with APAP-induced liver injury in mice. Furthermore, plasmin-mediated cleavage of HK contributes to hepatotoxicity in APAP-challenged mice independently of thrombin generation or bradykinin signaling.

The Role of Copper (II) on Kininogen Binding to Tropomyosin in the Presence of a Histidine-Proline-Rich Peptide.

The antiangiogenic activity of the H/P domain of histidine-proline-rich glycoprotein is mediated by its binding with tropomyosin, a protein exposed on endothelial cell-surface during the angiogenic switch, in presence of zinc ions. Although it is known that copper ion serum concentration is significantly increased in cancer patients, its role in the interaction of H/P domain with tropomyosin, has not yet been studied. In this paper, by using ELISA assay, we determined the modulating effect of TetraHPRG peptide, a sequence of 20 aa belonging to H/P domain, on the binding of Kininogen (HKa) with tropomyosin, both in absence and presence of copper and zinc ions. A potentiometric study was carried out to characterize the binding mode adopted by metal ions with TetraHPRG, showing the formation of complex species involving imidazole amide nitrogen atoms in metal binding. Moreover, circular dichroism showed a conformational modification of ternary systems formed by TetraHPRG, HKa and copper or zinc. Interestingly, slight pH variation influenced the HKa-TetraHPRG-tropomyosin binding. All these results indicate that both metal ions are crucial in the interaction between TetraHPRG, tropomyosin and HKa.

Methylene blue may have a role in the treatment of COVID-19.

In this paper, we raise the hypothesis that Methylene Blue may be a treatment option for Corona Virus Disease of 2019 specially when combined with Non Steroid Anti-Inflammatory Drugs. In previous publications including ours, the role of kininogen system has been postulated. A correlation between clinical findings of the disease and this mechanism has been drawn to denote a pivotal role of kininogen-kallikrein system in pathophysiology of the disease. Therein the possible role of Icatibant, Ecallantide and Aprotinin in the treatment of this disease has been raised. Here we want to emphasize on an important post-receptor mechanism of bradykinin that is Nitric Oxide. We came to this aim because we found out how access to these novel treatment nominees may be expensive and unaffordable. For this reason we are focusing on possible role of an old albeit "mysterious" drug namely Methylene Blue. This medication may abort effects of Bradykinin by inhibition of Nitric Oxide synthase inhibitor and promote oxygen saturation while it is inexpensive and ubiquitously accessible. Clinical studies cannot be over emphasized.

Hereditary angioedema: Investigational therapies and future research.

The future therapies for hereditary angioedema will likely involve the development of oral agents as alternatives to parenteral administration of drugs, specific targeting of proteins and/or enzymes that are not yet possible (e.g., factor XIIa), new agents that target the ß2 receptor with sustained action properties, testing of products to determine whether the ß1 receptor contributes significantly to attacks of angioedema, disrupting protein synthesis by using RNA technology as an alternative to enzyme inhibition, and, finally, gene therapy to attempt to cure the disease. Complete inhibition of attacks may well require sustained blood levels of C1 inhibitor that exceed 85% of normal, and it may be possible to delete the prekallikrein gene (analogous to familial prekallikrein deficiency), which is the one factor that might alleviate bradykinin formation, even by factor XII-independent initiating mechanisms, with the possible exception of Mannose Associated Serine Protease 1 (MASP-1) cleavage of high molecular weight kininogen (HK). Deletion of the light chain of high-molecular-weight kininogen would eliminate all possibilities for bradykinin formation, except tissue kallikrein cleavage of low-molecular-weight kininogen to support normal physiologic function to at least 50%.

Sufentanil attenuates inflammation and oxidative stress in sepsis-induced acute lung injury by downregulating KNG1 expression.

The present study aimed to investigate the effects of sufentanil on sepsis-induced acute lung injury (ALI), and identify the potential molecular mechanisms underlying its effect. In order to achieve this, a rat sepsis model was established. Following treatment with sufentanil, the lung wet/dry (W/D) weight ratio was calculated. Histopathological analysis was performed via hematoxylin and eosin staining. Levels of inflammatory factors in bronchoalveolar lavage fluid were determined via ELISA. Furthermore, malondialdehyde (MDA) content and the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in tissue homogenates were assessed using commercial kits. Western blot analysis was performed to determine kininogen-1 (KNG1) protein expression. In addition, alveolar epithelial type II cells (AEC II) were stimulated with lipopolysaccharide (LPS) to mimic ALI. The levels of inflammation and oxidative stress were evaluated following overexpression of KNG1. Protein expression levels of nuclear factor-κB (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling were determined via western blot analysis. The results of the present study demonstrated that sufentanil alleviated histopathological injury and the W/D ratio in lung tissue. Following treatment with sufentanil, levels of inflammatory factors also decreased, accompanied by decreased concentrations of MDA, and increased activities of SOD, CAT and GSH-Px. Notably, KNG1 was decreased in lung tissues following treatment with sufentanil. Furthermore, overexpression of KNG1 attenuated the inhibitory effects of sufentanil on LPS-induced inflammation and oxidative stress in AEC II. Sufentanil markedly downregulated NF-κB expression, while upregulating Nrf2 and HO-1 expression levels, which was reversed following overexpression of KNG1. Taken together, the results of the present study suggested that sufentanil may alleviate inflammation and oxidative stress in sepsis-induced ALI by downregulating KNG1 expression.

Factor XII and kininogen asymmetric assembly with gC1qR/C1QBP/P32 is governed by allostery.

The contact system is composed of factor XII (FXII), prekallikrein (PK), and cofactor high-molecular-weight kininogen (HK). The globular C1q receptor (gC1qR) has been shown to interact with FXII and HK. We reveal the FXII fibronectin type II domain (FnII) binds gC1qR in a Zn2+-dependent fashion and determined the complex crystal structure. FXIIFnII binds the gC1qR trimer in an asymmetric fashion, with residues Arg36 and Arg65 forming contacts with 2 distinct negatively charged pockets. gC1qR residues Asp185 and His187 coordinate a Zn2+ adjacent to the FXII-binding site, and a comparison with the ligand-free gC1qR crystal structure reveals the anionic G1-loop becomes ordered upon FXIIFnII binding. Additional conformational changes in the region of the Zn2+-binding site reveal an allosteric basis for Zn2+ modulation of FXII binding. Mutagenesis coupled with surface plasmon resonance demonstrate the gC1qR Zn2+ site contributes to FXII binding, and plasma-based assays reveal gC1qR stimulates coagulation in a FXII-dependent manner. Analysis of the binding of HK domain 5 (HKD5) to gC1qR shows only 1 high-affinity binding site per trimer. Mutagenesis studies identify a critical G3-loop located at the center of the gC1qR trimer, suggesting steric occlusion as the mechanism for HKD5 asymmetric binding. Gel filtration experiments reveal that gC1qR clusters FXII and HK into a higher-order 500-kDa ternary complex. These results support the conclusion that extracellular gC1qR can act as a chaperone to cluster contact factors, which may be a prelude for initiating the cascades that drive bradykinin generation and the intrinsic pathway of coagulation.

Pharmacokinetics, Pharmacodynamics, and Exposure-Response of Lanadelumab for Hereditary Angioedema.

Hereditary angioedema (HAE) with C1 inhibitor deficiency is a rare disorder characterized by unpredictable, potentially life-threatening recurrent angioedema attacks. Lanadelumab is a fully human monoclonal antibody with selective binding to active plasma kallikrein, and prevents the formation of cleaved high molecular weight kininogen (cHMWK) and bradykinin, thereby preventing HAE attacks. The clinical pharmacology of lanadelumab was characterized following subcutaneous administration in 257 subjects (24 healthy subjects and 233 patients with HAE). The pharmacokinetics of lanadelumab were described using a one-compartment model with first-order rate of absorption and linear clearance, showing slow absorption and a long half-life (14.8 days). A covariate analysis retained body weight and health status on apparent clearance (CL/F) and body weight on volume of distribution (V/F). Population estimates of CL/F and V/F were 0.0249 L/hour (0.586 L/day) and 12.8 L, respectively. An indirect-response Imax model showed 53.7% maximum suppression in cHMWK formation with a low potential for interactions with concomitant medications (analgesic, anti-inflammatory, and antirheumatic medications). A 300 mg dose administered Q2W was associated with a mean steady-state minimum concentration (Cmin,ss ; 25.4 µg/mL) that was ~ 4.5-fold higher than the half-maximal inhibitory concentration for cHMWK reduction (5.71 µg/mL). Exposure-response analyses suggest that 300 mg Q2W dosing was associated with a significantly reduced HAE attack rate, prolonged time to first attack after treatment initiation, and lower need for concomitant medications. The response was comparable across patient body weight groups. Findings from this analysis support the dosing rationale for lanadelumab to prevent attacks in patients with HAE.

The kallikrein-kinin pathway as a mechanism for auto-control of brown adipose tissue activity.

Brown adipose tissue (BAT) is known to secrete regulatory factors in response to thermogenic stimuli. Components of the BAT secretome may exert local effects that contribute to BAT recruitment and activation. Here, we found that a thermogenic stimulus leads to enhanced secretion of kininogen (Kng) by BAT, owing to induction of kininogen 2 (Kng2) gene expression. Noradrenergic, cAMP-mediated signals induce KNG2 expression and release in brown adipocytes. Conversely, the expression of kinin receptors, that are activated by the Kng products bradykinin and [Des-Arg9]-bradykinin, are repressed by thermogenic activation of BAT in vivo and of brown adipocytes in vitro. Loss-of-function models for Kng (the circulating-Kng-deficient BN/Ka rat) and bradykinin (pharmacological inhibition of kinin receptors, kinin receptor-null mice) signaling were coincident in showing abnormal overactivation of BAT. Studies in vitro indicated that Kng and bradykinin exert repressive effects on brown adipocyte thermogenic activity by interfering the PKA/p38 MAPK pathway of control of Ucp1 gene transcription, whereas impaired kinin receptor expression enhances it. Our findings identify the kallikrein-kinin system as a relevant component of BAT thermogenic regulation that provides auto-regulatory inhibitory signaling to BAT.

Identifying the potential protein biomarkers of preterm birth in amniotic fluid.

OBJECTIVE: Preterm birth severely threatens neonatal health and life. Although the detailed mechanism of preterm birth is not well understood, accurately predicting preterm birth can help people make preparations in advance, greatly reducing the subsequent health risk of neonates. Therefore, in this study, we aimed to identify potential protein biomarkers of preterm birth in amniotic fluid (AF). MATERIALS AND METHODS: We first enrolled pregnant subjects and collected their AF samples when they underwent amniocentesis at the second trimester of gestation. After delivery, the collected AF samples were classified into a full-term birth (sample size n = 21) set or preterm birth (n = 36) set, followed by 2-D DIGE and MS/MS assays. RESULTS: By doing so, we identified seven potential protein biomarkers of preterm birth, three of which were further validated in all samples with ELISA, including Apolipoprotein A-IV (Apoa4), Lumican (Lum) and Kininogen-1 (Kng1). As a result, all three potential biomarkers were significantly differently expressed between preterm and full-term birth AF samples. Furthermore, without prior classification, we found that these three biomarkers were positively correlated with gestation age (correlation coefficient ranging from 0.25 to 0.38) and were able to predict the occurrence of preterm birth. CONCLUSION: In this study, by examining amniotic fluid, we identified three biomarker proteins that may facilitate the identification of preterm birth. There three proteins were never reported to be related to preterm birth. Their pathogenesis roles in preterm birth deserve further investigations by using in vitro cell model or in vivo animal model assays.

Peptidylarginine Deiminase of Porphyromonas gingivalis Modulates the Interactions between Candida albicans Biofilm and Human Plasminogen and High-Molecular-Mass Kininogen.

Microorganisms that create mixed-species biofilms in the human oral cavity include, among others, the opportunistic fungus Candida albicans and the key bacterial pathogen in periodontitis, Porphyromonas gingivalis. Both species use arsenals of virulence factors to invade the host organism and evade its immune system including peptidylarginine deiminase that citrullinates microbial and host proteins, altering their function. We assessed the effects of this modification on the interactions between the C. albicans cell surface and human plasminogen and kininogen, key components of plasma proteolytic cascades related to the maintenance of hemostasis and innate immunity. Mass spectrometry was used to identify protein citrullination, and microplate tests to quantify the binding of modified plasminogen and kininogen to C. albicans cells. Competitive radioreceptor assays tested the affinity of citrullinated kinins to their specific cellular receptors. The citrullination of surface-exposed fungal proteins reduced the level of unmodified plasminogen binding but did not affect unmodified kininogen binding. However, the modification of human proteins did not disrupt their adsorption to the unmodified fungal cells. In contrast, the citrullination of kinins exerted a significant impact on their interactions with cellular receptors reducing their affinity and thus affecting the role of kinin peptides in the development of inflammation.