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Hereditary Angioedema (HAE) is an autosomal dominant disorder characterized clinically by recurrent episodes of swelling involving subcutaneous tissues, gastrointestinal tract, and oro-pharyngeal area. Gene mutations are the most common genetic cause of HAE and observed in more than 90% of patients. More than 700 mutation variants have been described so far. Patients with angioedema who have no mutations in the gene for C1-INH and normal levels and activity of this inhibitor are labelled: normal C1 inhibitor HAE. These include genetic mutations in factor 12 gene, plasminogen gene, angiopoietin gene, kininogen 1, and myoferlin genes. The clinical manifestations of patients with these mutations are similar to with patients with C1-INH gene mutations. However, a later age of onset, oro-pharyngeal involvement, and higher female preponderance have been reported in these rare subtypes of hereditary angioedema. With the advent and increased accessibility of whole-exome sequencing, it is expected that new genetic defects and novel pathophysiological pathways will be identified in families with HAE of unknown cause or normal C1-INH angioedema. This review covers some of the recent advances in the field of HAE. The review focuses on pathophysiology of HAE beyond the well-known C1-INH deficiency phenotypes, including various biomarkers that can serve the diagnosis and management of these rare disorders.

Background Hereditary angioedema (HAE) comprises HAE with C1-inhibitor deficiency (C1-INH-HAE) and HAE with normal C1-INH activity (nl-C1-INH-HAE), due to mutations in factor XII (FXII-HAE), plasminogen (PLG-HAE), angiopoietin 1 (ANGPT1-HAE), kininogen 1 genes (KNG1-HAE), or angioedema of unknown origin (U-HAE). The Italian network for C1-INH-HAE (ITACA) created a registry including different forms of angioedema without wheals. Objective We analyzed clinical and laboratory features of a cohort of Italian subjects with nl-C1-INH-HAE followed by ITACA to identify specific biomarkers. Methods A total of 105 nl-C1-INH-HAE patients were studied. Plasma concentrations of cleaved high-molecular-weight kininogen (cHK), vascular endothelial growth factors (VEGFs), angiopoietins (Angs), and secreted phospholipase A2 enzymes (sPLA2 ) were evaluated. Results We identified 43 FXII-HAE patients, 58 U-HAE, and 4 ANGPT1-HAE. We assessed a prevalence of 1:1.4 × 106 for FXII-HAE and 1:1.0 × 106 for U-HAE. cHK levels in U-HAE patients were similar to controls in plasma collected using protease inhibitors cocktail (PIC), but they significantly increased in the absence of PIC. In FXII-HAE patients, cHK levels, in the absence of PIC, were significantly higher than in controls. We found a significant increase of VEGF-A, VEGF-C, and Ang1 levels in U-HAE patients compared to controls. In FXII-HAE, only VEGF-C levels were increased. Ang2 concentrations and sPLA2 activity were not modified. The levels of these mediators in ANGPT1-HAE patients were not altered. Conclusions Our results suggest that pathogenesis of FXII-, ANGPT1-, and U-HAE moves through an unbalanced control of kallikrein activity, with bradykinin as most likely mediator. VEGFs and Ang1 participate in the pathophysiology of U-HAE increasing the basal vascular permeability.

Globally, nearly 40 percent of all diabetic patients develop serious diabetic kidney disease (DKD). The identification of the potential early-stage biomarkers and elucidation of their underlying molecular mechanisms in DKD are required. In this study, we performed integrated bioinformatics analysis on the expression profiles GSE111154, GSE30528 and GSE30529 associated with early diabetic nephropathy (EDN), glomerular DKD (GDKD) and tubular DKD (TDKD), respectively. A total of 1,241, 318 and 280 differentially expressed genes (DEGs) were identified for GSE30258, GSE30529, and GSE111154 respectively. Subsequently, 280 upregulated and 27 downregulated DEGs shared between the three GSE datasets were identified. Further analysis of the gene expression levels conducted on the hub genes revealed SPARC (Secreted Protein Acidic And Cysteine Rich), POSTN (periostin), LUM (Lumican), KNG1 (Kininogen 1), FN1 (Fibronectin 1), VCAN (Versican) and PTPRO (Protein Tyrosine Phosphatase Receptor Type O) having potential roles in DKD progression. FN1, LUM and VCAN were identified as upregulated genes for GDKD whereas the downregulation of PTPRO was associated with all three diseases. Both POSTN and SPARC were identified as the overexpressed putative biomarkers whereas KNG1 was found as downregulated in TDKD. Additionally, we also identified two drugs, namely pidorubicine, a topoisomerase inhibitor (LINCS ID- BRD-K04548931) and Polo-like kinase inhibitor (LINCS ID- BRD-K41652870) having the validated role in reversing the differential gene expression patterns observed in the three GSE datasets used. Collectively, this study aids in the understanding of the molecular drivers, critical genes and pathways that underlie DKD initiation and progression. Keywords diabetic nephropathy, gene ontology, prognosis, biomarkers, differential gene expressions, diabetic kidney disease.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disease that exhibits sex differences on clinical presentation. The present study aimed to investigate the sex differences associated with ARVC by conducting an integrated bioinformatics analysis. The GSE29819 gene expression dataset was downloaded from the Gene Expression Omnibus database. The online analytical tool GEO2R was then used to screen for differentially expressed genes (DEGs), which were subsequently processed using enrichment analysis and protein‑protein interaction (PPI) network construction. Functional annotation of the DEGs was determined using ClueGO. The PPI network was constructed with Search Tool for the Retrieval of Interacting Genes, and was visualized with Cytoscape to identify the modules and hub genes. Compared with the female group, a total of 1,188 DEGs, of which 915 were upregulated and 273 were downregulated, were identified in the male group. The enrichment analysis revealed that in KEGG pathways, the upregulated DEGs were substantially enriched in the 'nicotine addiction' pathways, whereas the downregulated DEGS were mainly enriched in the 'ECM‑receptor interaction' and 'protein digestion and absorption' pathways. The PPI network contained 899 nodes and 1,627 edges, among which four significant modules were identified. In addition, kininogen 1, lysophosphatidic acid receptor 5, formyl peptide receptor (FPR) 2, adenylate cyclase 2, γ‑aminobutyric acid type B receptor subunit 2, FPR1, hydroxycarboxylic acid receptor 1, prostaglandin E receptor 3, cannabinoid receptor 1 and proenkephalin were identified as the top 10 hub genes. The key genes and related pathways identified in this study provide genetic insight into the diversity in phenotypes between female and male patients with ARVC, and may facilitate therapeutic individualization.

Hereditary angioedema is a rare inherited disorder characterized by recurrent episodes of the accumulation of fluids outside of the blood vessels, causing rapid swelling of tissues in the hands, feet, limbs, face, intestinal tract, or airway. Mutations in SERPING1, the gene that encodes C1-INH (C1 esterase inhibitor), are responsible for the majority of cases of hereditary angioedema. C1 esterase inhibitor (C1-INH) is a major regulator of critical enzymes that are implicated in the cascades of bradykinin generation, which increases the vascular permeability and allows the flow of fluids into the extracellular space and results in angioedema. Moreover, a dominantly inherited disease has been described that has a similar clinical picture to C1-INH-HAE (Hereditary angioedema due to C1 inhibitor deficiency), but with normal C1-INH level and activity. This new type of HAE has no mutation in the SERPING1 gene and it is classified as nC1-INH-HAE (HAE with normal C1-INH). Currently mutations in six different genes have been identified as causing nC1-INH-HAE: factor XII (F12), plasminogen (PLG), angiopoietin 1 (ANGPT1), Kininogen 1 (KNG1), Myoferlin (MYOF), and heparan sulfate (HS)-glucosamine 3-O-sulfotransferase 6 (HS3ST6). In this review we aim to summarize the recent advances in genetic characterization of angioedema and possible future prospects in the identification of new genetic defects in HAE. We also provide an overview of diagnostic applications of genetic biomarkers using NGS technologies (Next Generation Sequencing).

The evolution in the knowledge of rare genetic diseases such as hereditary angioedema (HAE) has increased at a parallel pace with the development of new molecular tools. The deficiency of C1 inhibitor (C1-INH) has been recognized as the main cause of HAE (HAE-C1-INH) since the 1960s, but the discovery of the wide spectrum of mutations affecting the C1-INH gene (SERPING1) was possible only from the late 1980s, when Sanger sequencing became available and more accessible worldwide. Nevertheless, the involvement of other genes in HAE was discovered only in 2006 with the description of mutations in the F12 gene in patients with HAE and normal C1-INH. In the last 3 years, advanced next-generation sequencing techniques allowed the identification of mutations in 5 new genes being associated with HAE and normal C1-INH: ANGPT1 (angiopoietin-1), PLG (plasminogen), KNG1 (kininogen), MYOF (myoferlin), and HS3ST6 (heparan sulfate-glucosamine 3-O-sulfotransferase 6). The knowledge provided by the new era of genomic studies was pivotal in the discovery of mutations in new genes responsible for this complex pathogenesis. Genomics advances promise a better understanding of unknown mechanisms leading to HAE, the establishment of new molecular targets for novel therapeutic agents, and personalized treatment.

International audience; Background: Cognitive impairment is common in patients with PD. Core markers of Alzheimer's dementia have been related also to PD dementia, but no disease-specific signature to predict PD dementia exists to date.Objectives: The aim of this study was to investigate CSF markers associated with cognition in early PD.Methods: A high-throughput suspension bead array examined 216 proteins in CSF of 74 PD patients in the AETIONOMY project. Cognitive function was assessed with Repeatable Battery for the Assessment of the Neuropsychological Status, Montreal Cognitive Assessment, and Mini-Mental State Examination.Results: Of 69 patients with complete data, 34 had high (≥90) and 35 had low Repeatable Battery for the Assessment of the Neuropsychological Status total score (

BACKGROUND Immune-related genes (IRGs) are closely related to the incidence and progression of tumors, potentially indicating that IRGs play an important role in laryngeal squamous cell carcinoma (LSCC). MATERIAL AND METHODS An RNA sequencing dataset containing 123 samples was collected from The Cancer Genome Atlas. Based on immune-related differentially expressed genes (IRDEGs), a potential molecular mechanism of LSCC was explored through analysis of information in the Gene Ontology (GO) resource and the Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interactions (PPIs). A regulatory network of transcriptional regulators and IRDEGs was constructed to explore the underlying molecular mechanism of LSCC at the upstream level. Candidates from IRDEGs for signature were screened via univariate Cox analysis and using the least absolute shrinkage and selection operator (LASSO) technique. The IRDEG signature of LSCC was constructed by using a multivariate Cox proportional hazards model. RESULTS GO and KEGG analysis showed that IRDEGs may participate in the progression of LSCC through immune-related reactions. PPI analysis demonstrated that, among the IRDEGs in LSCC, the Kininogen 1; C-X-X motif chemokine ligand 10; elastase, neutrophil expressed; and LYZ genes are hub genes in the development of LSCC. At the upstream level, SPI1, SP140, signal transducer and activator of transcription 4, zinc finger E-box binding homeobox, and Ikaros family zinc finger 2 are the hub transcriptional regulators of IRDEGs. The risk score based on the IRDEG signature was able to distinguish prognosis in patients with LSCC and represents an independent prognostic risk factor for LSCC. CONCLUSIONS From the perspective of IRGs, we first constructed an IRDEG signature related to the prognosis of LSCC, which can be used as a novel marker to predict prognosis in patients with LSCC.

Multiple myeloma (MM) is a plasma cell-associated cancer and exists as the second most common hematological malignancy worldwide. Although researchers have been working on MM, a comprehensive quantitative Bone Marrow Interstitial Fluid (BMIF) and serum proteomic analysis from the same patients’ samples is not yet reported. The present study involves the investigation of alterations in the BMIF and serum proteome of MM patients compared to controls using multipronged quantitative proteomic approaches viz., 2D-DIGE, iTRAQ, and SWATH-MS. A total of 279 non-redundant statistically significant differentially abundant proteins were identified by the combination of three proteomic approaches in MM BMIF, while in the case of serum 116 such differentially abundant proteins were identified. The biological context of these dysregulated proteins was deciphered using various bioinformatic tools. Verification experiments were performed in a fresh independent cohort of samples using immunoblotting and mass spectrometry based SRM assays. Thorough data evaluation led to the identification of a panel of five proteins viz., haptoglobin, kininogen 1, transferrin, and apolipoprotein A1 along with albumin that was validated using ELISA in a larger cohort of serum samples. This panel of proteins could serve as a useful tool in the diagnosis and understanding of the pathophysiology of MM in the future.

Globally, the proportion of older adults is increasing. Older people face chronic conditions such as sarcopenia and functional decline, which are often associated with disability and frailty. Proteomics assay of potential serum biomarkers of frailty in older adults. Older adults were divided into non-frail and frail groups (n = 6 each; 3 males in each group) in accordance with the Chinese-Canadian Study of Health and Aging Clinical Frailty Scale. Adults were measured for grip power and the 6-min walk test for physical activity, and venous blood was sampled after adults fasted for 8 h. Ultra-high-performance liquid chromatography-tandem mass spectrometry was used for proteomics assay. The groups were compared for levels of biomarkers by t test and Pearson correlation analysis. Non-frail and frail subjects had mean age 77.5±0.4 and 77.7±1.6 years, mean height 160.5±1.3 and 156.6±2.9 cm and mean weight 62.5±1.2 and 62.8±2.9 kg, respectively. Physical activity level was lower for frail than non-frail subjects (grip power: 13.8±0.4 vs 26.1±1.2 kg; 6-min walk test: 215.2±17.2 vs 438.3±17.2 m). Among 226 proteins detected, for 31, serum levels were significantly higher for frail than non-frail subjects; serum levels of Ig kappa chain V-III region WOL, COX7A2, and albumin were lower. The serum levels of ANGT, KG and AT were 2.05-, 1.76- and 2.22-fold lower (all p < 0.05; Figure 1A, 2A and 3A) for non-frail than frail subjects and were highly correlated with grip power (Figure 1B, 2B and 3B). Our study found that ANGT, KG and AT levels are known to increase with aging, so degenerated vascular function might be associated with frailty. In total, 226 proteins were revealed proteomics assay; levels of angiotensinogen (ANGT), kininogen-1 (KG) and antithrombin III (AT) were higher in frail than non-frail subjects (11.26±2.21 vs 5.09±0.74; 18.42±1.36 vs 11.64±1.36; 22.23±1.64 vs 9.52±0.95, respectively, p < 0.05). These 3 factors were highly correlated with grip power (p < 0.05), with higher correlations between grip power and serum levels of ANGT (r = -0.89), KG (r = -0.90), and AT (r = -0.84). In conclusion, this is the first study to demonstrate a serum proteomic profile characteristic of frailty in older adults. Serum ANGT, KG and AT levels could be potential biomarkers for monitoring the development and progression of frailty in older adults.

Clear cell renal cell carcinoma (CCRCC) is a typical type of RCC with the worst prognosis among the common epithelial neoplasms of the kidney. However, its molecular pathogenesis remains unknown. Therefore, the aim of the present study was to screen for effective and potential pathogenic biomarkers of CCRCC. The gene expression profile of the GSE16441, GSE36895, GSE40435, GSE46699, GSE66270 and GSE71963 datasets were downloaded from the Gene Expression Omnibus database. First, the limma package in R language was used to identify differentially expressed genes (DEGs) in each dataset. The robust and strong DEGs were explored using the robust rank aggregation method. A total of 980 markedly robust DEGs were identified (429 upregulated and 551 downregulated). According to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, these DEGs exhibited an obvious enrichment in various cancer-related biological pathways and functions. The Search Tool for the Retrieval of Interacting Genes/Proteins database was used for the construction of a protein-protein interaction (PPI) network, the Cytoscape MCODE plug-in for module analysis and the cytoHubba plug-in to identify hub genes from the aforementioned DEGs. A total of four key modules were identified in the PPI network. A total of six hub genes, including C-X-C motif chemokine ligand 12, bradykinin receptor B2, adenylate cyclase 7, calcium sensing receptor (CASR), kininogen 1 and lysophosphatidic acid receptor 5, were identified. The DEG results of the hub genes were verified using The Cancer Genome Atlas database, and CASR was found to be significantly associated with the prognosis of patients with CCRCC. In conclusion, the present study provided new insight and potential biomarkers for the diagnosis and prognosis of CCRCC.

Lung squamous cell carcinoma (LUSC) progression is accompanied by changes in protein levels that may be reflected in body fluids, such as plasma, bronchoalveolar lavage fluid (BALF) and urine. Certain proteins present in these biofluids can facilitate lung cancer diagnosis. Kininogen 1 (KNG1), osteopontin (OPN) and α-1-antitrypsin (AAT) are associated with tumorigenesis. The present study aimed to explore the combined monitoring of plasma, urine and BALF to gain insight into LUSC by monitoring the levels of the above three protein using ELISA. LUSC (n=31) and healthy controls with benign lung diseases (n=20) were enrolled in the study. KNG1 levels in plasma, BALF and urine were significantly higher in patients with LUSC patients than in controls (P

Background Depressive disorder is a debilitating psychiatric mental disease. However, no biological methods are used for the diagnosis of this disorder. Proteomic approaches for biomarker discovery may provide an important objective tool for diagnostics of depression. This study aimed to identify serum protein biomarkers for diagnosis of depressive disorder. Methods We screened for potential depression biomarkers in 175 serum samples from 86 patients and 89 healthy controls. Serum protein spectrums were detected by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Differentially expressed peptides among the two groups were analyzed and followed by sequence analysis to identify these peptides. Results Five peaks were found to have a significant different between the depression and healthy control groups. Among them, up-regulated m/z 1,466.21 and down-regulated m/z 1,944.99 are identified as the fractions of fibrinogen alpha chain and kininogen 1, respectively. Conclusions Fibrinogen and kininogen may be potential serum protein biomarkers in the diagnosis and prognosis of depressive disorders.

Women with inherited BRCA1 mutations are more likely to develop breast cancer (BC); however, not every carrier will progress to BC. The aim of this study was to identify and characterize circulating peptides that correlate with BC patients carrying BRCA1 mutations. Circulating peptides were enriched using our well-designed nanoporous silica thin films (NanoTraps) and profiled by mass spectrometry to identify among four clinical groups. To determine the corresponding proteolytic processes and their sites of activity, purified candidate peptidases and synthesized substrates were assayed to verify the processes predicted by the MERPOS database. Proteolytic processes were validated using patient serum samples. The peptides, KNG1K438-R457 and C 3fS1304-R1320, were identified as putative peptide candidates to differentiate BRCA1 mutant BC from sporadic BC and cancer-free BRCA1 mutant carriers. Kallikrein-2 (KLK2) is the major peptidase that cleaves KNG1K438-R457 from kininogen-1, and its expressions and activities were also found to be dependent on BRCA1 status. We further determined that KNG1K438-R457 is cleaved at its C-terminal arginine by carboxypeptidase N1 (CPN1). Increased KLK2 activity, with decreased CPN1 activity, results in the accumulation of KNG1K438-R457 in BRCA1-associated BC. Our work outlined a useful strategy for determining the peptide-petidase relationship and thus establishing a biological mechanism for changes in the peptidome in BRCA1-associated BC.

Yin-deficiency-heat (YDH) syndrome is a concept in Traditional Chinese Medicine (TCM) for describing subhealth status. However, there are few efficient diagnostic methods available for confirming YDH syndrome. To explore the novel method for diagnosing YDH syndrome, we applied iTRAQ to observe the serum protein profiles in YDH syndrome rats and confirmed protein levels by ELISA. A total of 92 differentially expressed proteins (63 upregulated proteins and 29 downregulated proteins), which were mainly involved in complement and coagulation cascades and glucose metabolism pathway, were identified by the proteomic experiments. Kininogen 1 (KNG1) was significantly increased (p<0.0001), while apolipoprotein C-III (APOC3,p<0.005) and paraoxonase 1 (PON1,p<0.001) were significantly decreased in the serum of YDH syndrome rats. The combination of KNG1, APOC3, and PON1 constituted a diagnostic model with 100.0% sensitivity and 85.0% specificity. The results indicated that KNG1, APOC3, and PON1 may act as potential biomarkers for diagnosing YDH syndrome. KNG1 may regulate cytokines and chemokines release in YDH syndrome, and the low levels of PON1 and APOC3 may increase oxidative stress and lipolysis in YDH syndrome, respectively. Our work provides a novel method for YDH syndrome diagnosis and also provides valuable experimental basis to understand the molecular mechanism of YDH syndrome.

Background: Colorectal cancer (CRC) at a current clinical level is still hardly diagnosed, especially with regard to nascent tumors, which are typically asymptotic. Searching for reliable biomarkers of early diagnosis is an extremely essential task. Identification of specific post-translational modifications (PTM) may also significantly improve net benefits and tailor the process of CRC recognition. We examined depleted plasma samples obtained from 41 healthy volunteers and 28 patients with CRC at different stages to conduct comparative proteome-scaled analysis. The main goal of the study was to establish a constellation of protein markers in combination with their PTMs and semi-quantitative ratios that may support and realize the distinction of CRC until the disease has a poor clinical manifestation. Results: Proteomic analysis revealed 119 and 166 proteins for patients in stages I&ndash, II and III&ndash, IV, correspondingly. Plenty of proteins (44 proteins) reflected conditions of the immune response, lipid metabolism, and response to stress, but only a small portion of them were significant (p &lt, 0.01) for distinguishing stages I&ndash, II of CRC. Among them, some cytokines (Clusterin (CLU), C4b-binding protein (C4BP), and CD59 glycoprotein (CD59), etc.) were the most prominent and the lectin pathway was specifically enhanced in patients with CRC. Significant alterations in Inter-alpha-trypsin inhibitor heavy chains (ITIH1, ITIH2, ITIH3, and ITIH4) levels were also observed due to their implication in tumor growth and the malignancy process. Other markers (Alpha-1-acid glycoprotein 2 (ORM2), Alpha-1B-glycoprotein (A1BG), Haptoglobin (HP), and Leucine-rich alpha-2-glycoprotein (LRG1), etc.) were found to create an ambiguous core involved in cancer development but also to exactly promote tumor progression in the early stages. Additionally, we identified post-translational modifications, which according to the literature are associated with the development of colorectal cancer, including kininogen 1 protein (T327-p), alpha-2-HS-glycoprotein (S138-p) and newly identified PTMs, i.e., vitamin D-binding protein (K75-ac and K370-ac) and plasma protease C1 inhibitor (Y294-p), which may also contribute and negatively impact on CRC progression. Conclusions: The contribution of cytokines and proteins of the extracellular matrix is the most significant factor in CRC development in the early stages. This can be concluded since tumor growth is tightly associated with chronic aseptic inflammation and concatenated malignancy related to loss of extracellular matrix stability. Due attention should be paid to Apolipoprotein E (APOE), Apolipoprotein C1 (APOC1), and Apolipoprotein B-100 (APOB) because of their impact on the malfunction of DNA repair and their capability to regulate mTOR and PI3K pathways. The contribution of the observed PTMs is still equivocal, but a significant decrease in the likelihood between modified and native proteins was not detected confidently.

Hepatocellular carcinoma (HCC) is one of the most common malignant types of cancer, with a high mortality rate. Sorafenib is the sole approved oral clinical therapy against advanced HCC. However, individual patients exhibit varying responses to sorafenib and the development of sorafenib resistance has been a new challenge for its clinical efficacy. The current study identified gene biomarkers and key pathways in sorafenib-resistant HCC using bioinformatics analysis. Gene dataset GSE73571 was obtained from the Gene Expression Omnibus (GEO) database, including four sorafenib-acquired resistant and three sorafenib-sensitive HCC phenotypes. Differentially expressed genes (DEGs) were identified using the web tool GEO2R. Functional and pathway enrichment of DEGs were analyzed using the Database for Annotation, Visualization and Integrated Discovery and the protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins and Cytoscape. A total of 1,319 DEGs were selected, which included 593 upregulated and 726 downregulated genes. Functional and pathway enrichment analysis revealed DEGs enriched in negative regulation of endopeptidase activity, cholesterol homeostasis, DNA replication and repair, coagulation cascades, insulin resistance, RNA transport, cell cycle and others. Eight hub genes, including kininogen 1, vascular cell adhesion molecule 1, apolipoprotein C3, alpha 2-HS glycoprotein, erb-b2 receptor tyrosine kinase 2, secreted protein acidic and cysteine rich, vitronectin and vimentin were identified from the PPI network. In conclusion, the present study identified DEGs and key genes in sorafenib-resistant HCC, which further the knowledge of potential mechanisms in the development of sorafenib resistance and may provide potential targets for early diagnosis and new treatments for sorafenib-resistant HCC.

The serum protein markers of colorectal adenoma in patients with a family history of colorectal cancer have been rarely reported. Serum samples from colorectal adenoma patients with or without a family history of colorectal cancer and healthy controls were profiled using Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). The model to distinguish colorectal adenoma patients with a family history of colorectal cancer from atypical hereditary colorectal families (CRA-H) and sporadic colorectal adenoma patients without a family history of colorectal cancer (CRA-S) was established with 85.0% accuracy. The model distinguishing CRA-H from healthy individuals was established with 90.0% specificity and 86.7% sensitivity. Additionally, five peaks (2202, 5821, 3260, 2480, and 2218) showing differential expression in advanced colorectal adenoma patients with a family history of colorectal cancer were selected. The protein Kininogen 1 (KNG1) was identified in colorectal adenoma patients and validated using Western Blotting. KNG1 may be a biomarker for colorectal adenoma patients with a family history of colorectal cancer.

BACKGROUND This study investigated the protective effects of pharmaceutical CD26/dipeptidylpeptidase-4 (CD26/DPP-4) inhibitor in lung transplantation (LTx). Changes in protein expression associated with the treatment were screened and identified to evaluate the role of kininogen-1 in early-term ischemia/reperfusion (I/R) injury after LTx. MATERIAL AND METHODS Orthotopic single LTx was performed in syngeneic C57BL/6 mice, with a pharmaceutical CD26/DPP-4 inhibitor (vildagliptin, subcutaneous injection, 10 mg/kg, every 12 h) administered to the investigational group. All donors were perfused and preserved with low potassium dextran (LPD). Grafts were harvested at 60 h post-transplantation after 8 h of cold ischemia. Myeloperoxidase activity and wet/dry weight ratio were measured, followed by histopathological examination. Proteins were separated, analyzed, and identified using proteomics and database searches. The target proteins were validated by Western blot. Immunohistochemical studies were performed in the same lung specimen locus. RESULTS Investigational group (IN) versus control group (CON) comparison showed decreased myeloperoxidase enzymatic activity, as well as decreased edema and interstitial-alveolar inflammation. Proteomics results revealed 78 spots with significant differences in abundance between the 2 groups. Fifteen proteins were identified. Kininogen-1 was up-regulated in CON and down-regulated in IN, with contrasting results for the heat shock protein 70. Immunohistochemical results revealed significantly different staining with kininogen-1 in alveolar macrophages and inflammatory cells. CONCLUSIONS Combined vildagliptin and LPD significantly ameliorated I/R injury after LTx. This treatment may change local pulmonary protein levels. Moreover, proper application of proteins such as kininogen-1 may enhance the protective effects against I/R injury during transplantation.

Tengfu Jiangya Tablet (TJT) is a well accepted antihypertension drug in China and its major active components wereUncaria total alkaloidsandSemen Raphani soluble alkaloid. To further explore treatment effects mechanism of TJT on essential hypertension, a serum proteomic study was performed. Potential biomarkers were quantified in serum of hypertension individuals before and after taking TJT with isobaric tags for relative and absolute quantitation (iTRAQ) coupled two-dimensional liquid chromatography followed electrospray ionization-tandem mass spectrometry (2D LC-MS/MS) proteomics technique. Among 391 identified proteins with high confidence, 70 proteins were differentially expressed (fold variation criteria, >1.2 or

International audience; Vasoactive peptides are key early mediators of inflammation released through activation of different enzymatic systems. The mammalian kinin-kallikrein (K-KLK) system produces bradykinin (BK) through proteolytic cleavage of a kininogen precursor by enzymes named kallikreins. BK acts through specific ubiquitous G-protein coupled receptors (B1R and B2R) to participate in physiological processes and inflammatory responses, such as activation of mononuclear phagocytes. In chickens, the BK-like nonapeptide omithokinin (OK) has been shown to promote intracellular calcium increase in embryonic fibroblasts and to be vasodilatory in vivo. Also, one of its receptors (B2R) was already cloned. However, the participation of chicken K-KLK system components in the inflammatory response remains unknown and was therefore investigated. We first showed that B1R, B2R and kininogen 1 (KNG1) are expressed in unstimulated chicken tissues and macrophages. We next showed that chicken B1R and B2R are expressed at transcript and protein levels in chicken macrophages and are upregulated by E. coli LPS or avian pathogenic E. coli (APEC) infection. Interestingly, exogenous OK induced internalization and degradation of OK receptors protein, notably B2R. Also, OK induced intracellular calcium increase and potentiated zymosan-induced ROS production and Dextran-FITC endocytosis by chicken macrophages. Exogenous OK itself did not promote APEC killing and had no pro-inflammatory effect. However, when combined with LPS or APEC, OK upregulated cytokine/chemokine gene expression and NO production by chicken macrophages. This effect was not blocked by canonical non-peptide B1R or B2R receptor antagonists but was GPCR- and PI3K/Akt-dependent. In vivo, pulmonary colibacillosis led to upregulation of OK receptors expression in chicken lungs and liver. Also, colibacillosis led to significant upregulation of OK precursor KNG1 expression in liver and in cultured hepatocytes (LMH). We therefore provide hitherto unknown information on how OK and its receptors are involved in inflammation and infection in chickens.

Background:Diurnal variations and physiologic changes of urinary proteome have been suggested in the urinary proteomics field. However, no clear evidence has been demonstrated. The present study thus aimed to define changes in urinary proteome by physiological stimuli, i.e. caffeine intake and excessive water drinking, both of which cause physiologic diuresis.Methods:Urine samples were collected from 30 healthy individuals under three different conditions: (i) morning void as the control; (ii) after drinking a cup of coffee; and (iii) after drinking 1 L of water within 20 min. Thereafter, differentially excreted proteins were analyzed by 2-DE proteomics approach and validated by Western blotting and ELISA.Results:Spot matching, quantitative intensity analysis, and ANOVA followed by Tukey’s post-hoc multiple comparisons and the Bonferroni correction revealed significant differences in levels of five protein spots among three different conditions. These proteins were identified by quadrupole time-of-flight mass spectrometry (Q-TOF MS) and/or MS/MS analyses as kininogen 1 isoform 3, β-actin, prostaglandin D synthase (PGDS), fibrinogen α-chain and immunoglobulin light chain. Among these, the decreased level of immunoglobulin was successfully validated by Western blotting and ELISA.Conclusions:These data indicated that caffeine intake and excessive water drinking could affect urinary excretion of some proteins and may affect urinary proteome analysis.

Objective: In this study, we aimed to search for noninvasive predictive biomarkers for prenatal diagnosis of Down’s syndrome (DS). Methods: Maternal serum samples from five DS-affected pregnant women and five DS-unaffected women were analyzed by 2D gel electrophoresis and MALDI-TOF mass spectrometry to screen for potential predictive biomarkers of DS. Then, differential levels of dGTPase, β2-glycoprotein I (β2-GPI), complement factor H-related protein 1 precursor (CFHR1) and kininogen 1 isoform 2 were further verified by western blotting tests in another independent group. Results: Statistical analysis results revealed 29 protein spots whose levels differed significantly in the DS-affected pregnancies group. Of these, the eight most differentially expressed in DP were identified successfully. Among these, levels of dGTPase, CFHR1 and kininogen 1 were elevated significantly, whereas β2-GPI was reduced in DP. Discussion: These preliminarily verified proteins might serve as potential predictive biomarkers fo...

BackgroundSchizophrenia (SCZ) is a complex and severe mental illness. There is a lack of effective biomarkers for SCZ diagnosis. The aim of this study was to explore the possibility of using serum peptides for the diagnosis of SCZ as well as analyze the association of variants in genes coding for these peptides and SCZ.MethodsAfter bead-based fractionation, the matrix-assisted laser desorption ionization/time-of-flight mass spectrometry technique was used to identify peptides that showed different expressions between 166 SCZ patients and 201 healthy controls. Differentially expressed peptides were verified in a second set of samples (81 SCZ patients and 103 healthy controls). The association of SCZ and three tagSNPs selected in genes coding for differentially expressed peptides was performed in 1,126 SCZ patients and 1,168 controls.ResultsThe expression level of peptides with m/z 1,945.07 was significant lower in SCZ patients than in healthy controls (P< 0.000001). The peptide with m/z 1,945.07 was confirmed to be a fragment of Kininogen-1. In the verification tests, Kininogen-1 had a sensitivity of 95.1% and a specificity of 97.1% in SCZ prediction. Among the three tagSNPs (rs13037490, rs2983639, rs2983640) selected in the Cystatin 9 gene (CST9) which encodes peptides including Kininogen-1, tagSNP rs2983640 had its genotype distributions significantly different between SCZ patients and controls under different genetic models (P< 0.05). Haplotypes CG (rs2983639–rs2983640) and TCG (rs13037490–rs2983639–rs2983640) were significantly associated with SCZ (CG: OR = 1.21, 95% CI [1.02–1.44],P= 0.032; TCG: OR = 24.85, 95% CI [5.98–103.17],P< 0.0001).ConclusionsThe present study demonstrated that SCZ patients had decreased expression of Kininogen-1 and genetic variants in Kininogen-1 coding geneCST9were significantly associated with SCZ. The findings from both protein and genetic association studies suggest that Kininogen-1 could be a biomarker of SCZ.

Locoism threatens the sustainable development of animal husbandry in areas around the world with intensified desertification, especially in the western United States, western China, Canada, and Mexico, among other countries. This study was conducted to discover potential serum biomarkers in locoweed-poisoned rabbits and lay a foundation for early diagnosis of locoism. We performed iTRAQ labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS), comparing locoweed-poisoned rabbits and healthy controls. A total of 78 differentially-expressed proteins (fold change > 1.5 or < 0.67) were identified in the locoweed-poisoned rabbits compared to healthy controls. We found that 57.70% of differentially-expressed proteins were functionally related, and through bioinformatics analysis, we were able to construct a network mainly in complement and coagulation cascades. Significant differences in thrombospondin 4 (THBS4), kininogen 1 (KNG1), hemoglobin (HBB), and complement factor I (CFI) between locoweed poisoned animals and controls were found (P < 0.05) and validated by western blotting. These results suggested that locoweed could damage neurocytes, lower immunity, and form thrombi in rabbits. Our study proposes potential biomarkers for locoism diagnosis and also provides a new experimental basis to understand the pathogenesis of locoism.

Background Type 2 diabetes mellitus (T2DM) is a complex, pandemic disease contributing towards the global burden of health issues. To date, there are no simple clinical tests for the early detection of T2DM. Method To identify potential peptide biomarkers for such applications, 406 sera of T2DM patients (n = 206) and healthy controls (n = 200) were analyzed using MALDI-TOF MS with a cross-sectional case-control design. Result Six peptides (peaks m/z 1452.9, 1692.8, 1946.0, 2115.1, 2211.0 and 4053.6) were identified as candidate biomarkers for T2DM. A diagnostic model constructed with the 6 peptides was able to discriminate T2DM patients from healthy controls, with an accuracy of 82.20%, sensitivity of 82.50%, and specificity of 77.80% in the validation set. Peptide peaks m/z 1452.9 and 1692.8 were identified as fragments of the complement C3f, while peptide peaks m/z 1946.0, 2115.0, and 2211.0 were identified as the fragments of kininogen 1 isoform 1 precursor. Conclusion This study reinforces proteomic analyses as a potential technique for defining significant clinical peptide biomarkers, providing a simple and convenient diagnostic model for T2DM in clinical examination. This article is protected by copyright. All rights reserved

Implementation of therapy for acute kidney injury (AKI) depends on successful prediction of individual patient prognosis. Clinical markers as serum creatinine (sCr) have limitations in sensitivity and early response. The aim of the study was to identify novel molecules in urine which show altered levels in response to AKI and investigate their value as predictors of recovery. Changes in the urinary proteome were here investigated in a cohort of 88 subjects (55 AKI patients and 33 healthy donors) grouped in discovery and validation independent cohorts. Patients'urine was collected at three time points: within the first 48 h after diagnosis(T1), at 7 days of follow-up(T2) and at discharge of Nephrology(T3). Differential gel electrophoresis was performed and data were confirmed by Western blot (WB), liquid chromatography/mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA). Retinol binding protein 4 (RBP4) and kininogen-1 (KNG1) were found significantly altered following AKI. RBP4 increased at T1, and progressively decreased towards normalization. Maintained decrease was observed for KNG1 from T1. Individual patient response along time revealed RBP4 responds to recovery earlier than sCr. In conclusion, KNG1 and RBP4 respond to AKI. By monitoring RBP4, patient's recovery can be anticipated pointing to a role of RBP4 in prognosis evaluation., Funding: from Instituto de Salud Carlos III: FIS PI11/01401, PI13/01873, FIS IF08/3667-1, CP09/00229, PI13/00047, PI10/00624, ISCIII-RETIC REDinREN RD012/0021. FEDER funds, Comunidad de Madrid/CIFRA S2010/BMD-2378, Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM) to AO, IDCSalud (3371/002) and Fundación Conchita Rábago de Jiménez Díaz, Proteomic Facility from Universidad Complutense de Madrid-Fundación Parque Científico de Madrid (UCM-FPCM), Spain, a member of ProteoRed-ISCIII Network member of ProteoRed- ISCIII Network

Despite tremendous efforts in disclosing the pathophysiological and epidemiological factors associated with liver fibrogenesis, non-invasive diagnostic measures to estimate the clinical outcome and progression of liver fibrogenesis are presently limited. Therefore, there is a mandatory need for methodologies allowing the reasonable and reliable assessment of the severity and/or progression of hepatic fibrogenesis. We here performed proteomic serum profiling by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in 179 samples of patients chronically infected with hepatitis C virus and 195 control sera. Multidimensional analysis of spectra allowed the definition of algorithms capable to distinguish class-specific protein expression profiles in serum samples. Overall about 100 peaks could be detected per single spectrum. Different algorithms including protein peaks in the range of 2000 and 10,000 Da were generated after pre-fractionation on a weak cation exchange surface. A specificity of 93% with a sensitivity of 86% as mean of the test set results was found, respectively. The nature of three of these protein peaks that belonged to kininogen-1 and thymosin-β(4) was further analysed by tandem mass spectrometry (MS)/MS. We further found that kininogen-1 mRNA was significantly down-regulated in cirrhotic livers. We have identified kininogen-1 and thymosin-β(4) as potential new biomarkers for human chronic hepatitis C and conclude that serum profiling is a reliable technique to identify hepatitis-associated expression patterns. Based on the high throughput capability, the identified differential protein panel may serve as a diagnostic marker and warrants further validation in larger cohorts.

The most common and toxic form of aflatoxin, aflatoxin B(1) (AFB(1)), is produced by molds growing on crops. Use of moldy corn can result in high concentrations of AFB(1) in swine diets, which could potentially lead to an increased incidence of aflatoxicosis, a disease associated with decreased health and performance through reduced feed intake, reduced BW gain, and impaired liver function. The objective of this study was to determine the effects of AFB(1) on the hepatic gene expression of growing barrows. Ninety Duroc × Yorkshire crossbred barrows (age = 35 ± 5 d; initial BW = 14.2 ± 3.0 kg) were allocated to 9 pens with 10 pigs per pen, and randomly assigned in a 3 × 3 factorial arrangements of treatments to receive diets containing 0 µg/kg of AFB(1), 250 µg/kg of AFB(1), or 500 µg/kg of AFB(1) for 7, 28, or 70 d. Because performance was most affected in animals administered AFB(1) for an extended period, liver samples from d 70 animals were used for RNA-sequencing analysis. Of 82,744 sequences probed, 179 had transcripts that were highly correlated (r ≥ |0.8|; P < 0.0001) with treatment. Of the 179 significant transcripts, 46 sequences were negatively and 133 sequences positively related to treatment. Forty-three unique functional groups were identified. Genes within the apoptosis regulation functional group were selected for 1) confirmation of d 70 gene expression differences using real-time reverse-transcription (RT)-PCR (n = 4 genes), and 2) investigation of d 7 expression to identify early responses to AFB(1) (n = 15 genes) using real-time RT-PCR. Expression of the 4 apoptosis genes selected for confirmation, cyclin-dependent kinase inhibitor 1A, zinc finger matrin type 3, kininogen 1, and pim-1 oncogene, was confirmed with real-time RT-PCR. Of the 15 genes tested in d 7 liver samples, 4 were differentially expressed: cyclin-dependent kinase inhibitor 1A; zinc finger matrin type 3; tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide; and apoptosis enhancing nuclease. Results from this study demonstrate that administration of an AFB(1)-contaminated diet to growing barrows alters hepatic gene expression, and in particular apoptosis genes.

Objectives: The objectives were to determine if the skin secretion of the European yellow-bellied toad ( Bombina variegata ), in common with other related species, contains a bradykinin inhibitor peptide and to isolate and structurally characterize this peptide. Materials and Methods: Lyophilized skin secretion obtained from this toad was subjected to reverse phase HPLC fractionation with subsequent bioassay of fractions for antagonism of the bradykinin activity using an isolated rat tail artery smooth muscle preparation. Subsequently, the primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy, and molecular cloning, following which a synthetic replicate was chemically synthesised for bioassay. Results: A single peptide of molecular mass 2300.92 Da was resolved in HPLC fractions of skin secretion and its primary structure determined as IYNAIWP-KH-NK-KPGLL-. Database interrogation with this sequence indicated that this peptide was encoded by skin kininogen-1 previously cloned from B. variegata . The blank cycles were occupied by cysteinyl (C) residues and the peptide was located toward the C-terminus of the skin kininogen, and flanked N-terminally by a classical -KR- propeptide convertase processing site. The peptide was named IC-20 in accordance (I = N-terminal isoleucine, C = C-terminal cysteine, 20 = number of residues). Like the natural peptide, its synthetic replicate displayed an antagonism of bradykinin-induced arterial smooth muscle relaxation. Conclusion: IC-20 represents a novel bradykinin antagonizing peptide from amphibian skin secretions and is the third such peptide found to be co-encoded with bradykinins within skin kininogens.

Background CRISP-3 was previously shown to be bound to α1B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such that involve cell cultures, binding proteins present in sera might interfere in the experiments. Methods We examined sera from five different animal species for CRISP-3 binding proteins using gel filtration and ligand blotting. We developed a rapid method for isolation of proteins that bind to human CRISP-3 and identified the isolated proteins by mass spectrometry and N-terminal sequencing. Results We identified A1BG as a CRISP-3 binding protein in sera from cow, horse and rabbit. CRISP-3 bound kininogen 1 in mouse serum, whereas rat serum showed no CRISP-3 binding activity. In equine serum, we furthermore detected a possible CRISP, already bound to A1BG. General significance It seems to be a common mechanism that A1BGs bind CRISPs, also across species. Apart from the possible physiological implications hereof, complex binding of CRISPs by A1BG (and other proteins) may interfere with the detection and function of CRISPs, when these are studied in the presence of animal sera.

Prekallikrein in human citrated plasma was activated by incubation with acetone, 17 or 25% (v/v). EDTA-2Na was added before or after the evaporation of acetone. Determinations of the relative activities of the enzyme preparations were based on the release of kinin from the 3 functional states of kininogen in human citrated EDTA-plasma as described by BRISEID et al. (1973). Incubations were carried out at 37° or at 0°. The kininogen 1/kininogen 2 activityratios were the same at 37° and 0°, indicating that the kininogenase activities tested were the same. The relative activities of the different kallikrein preparations varied with the type of kininogen used, possibly reflecting the presence of more than one kininogenase. Purification of the kallikrein combined with determinations of kininogen 1/kininogen 2 activity-ratios must be carried out to solve the problem.

IgA vasculitis (IgAV), previously named as Henoch-Schonlein purpura, is the most common systematic vasculitis with unknown etiology. Lack of appropriate study system and/or animal model limits the understanding of its molecular pathogenesis and hinders the identification of targets for rational therapy, especially for its long-term complication, IgAV nephritis (IgAVN). In this study, we applied comparative analysis of serum proteomes to obtain an insight about disease pathogenesis. This study has utilized high sensitivity nanoscale ultra performance liquid chromatography-mass spectrometry (nanoLC-MS/MS) to investigate the alterations in serum proteomic profiles in patients with IgAV (n=6), IgAVN (n=6) and healthy subjects (n=7). The differentially expressed proteins were subjected to functional pathway analysis by PANTHER and DAVID software. We identified 107 differentially expressed proteins among three different groups, and functional analysis suggested that, in addition to earlier reported pathways, such as acute phase response, immune response, complement and blood coagulation pathways, hemostasis and Wnt signaling pathway were probably involved in pathogenesis of IgAV. A few differentially abundant proteins identified, such as C4a, serum amyloid A, angiotensinogen, and kininogen 1, were further validated by ELISA. More importantly, we found that angiotensinogen concentration is correlated with IgAVN and could be used as a potential marker for the progression of IgAV. This is the first report of analyzing the proteomic alterations in IgAV patients and the differentially proteins identified in this study may enhance understanding of the pathology of IgAV and a few of them may be used to monitor disease progression.

Autism is a complex developmental disability for which no specific diagnostic markers have been identified so far. The present study aimed to evaluate whether there is any abnormal protein(s) excreted in the urine of autistic children by proteome analysis which may act as diagnostic marker.Urine proteome analysis was carried out in first void urine samples of autistic and normal children (n=30) in the age group of 4-12 years by 2D-PAGE followed by MALDI-TOF-MS analysis.Comparison of 2D-PAGE gels revealed that many urinary proteins are expressed differentially in autistic children. Total numbers of spots observed were 250 and 159 in autism and normal samples respectively, out of which 95 matches were observed. In addition, 3 spots of abnormally expressed peptides were selected, excised and analyzed. Peptide sequence with significant match score was for kininogen-1 (KNG-1)-50 (spot-1), IgG1 heavy chain variable region-35(spot-2) and mannan-binding lectin serine protease-2 isoform-2 precursor-45(spot-3). All the autistic children showed significant increase (p0.001) in urinary kininogen level measured quantitatively by ELISA, when compared to normal children.Increased urinary kininogen-1 level in all the autistic children and the possibility of this protein as a diagnostic marker need further investigation.

Background— The renin–angiotensin–aldosterone system (RAAS) is critical for regulation of blood pressure and fluid balance and influences cardiovascular remodeling. Dysregulation of the RAAS contributes to cardiovascular and renal morbidity. The genetic architecture of circulating RAAS components is incompletely understood. Methods and Results— We meta-analyzed genome-wide association data for plasma renin activity (n=5275), plasma renin concentrations (n=8014), and circulating aldosterone (n=13289) from ≤4 population-based cohorts of European and European-American ancestry, and assessed replication of the top results in an independent sample (n=6487). Single-nucleotide polymorphisms (SNPs) in 2 independent loci displayed associations with plasma renin activity at genome-wide significance ( P −8 ). A third locus was close to this threshold (rs4253311 in kallikrein B [KLKB1], P =5.5×10 −8 ). Two of these loci replicated in an independent sample for both plasma renin and aldosterone concentrations (SNP rs5030062 in kininogen 1 [KNG1]: P =0.001 for plasma renin, P =0.024 for plasma aldosterone concentration; and rs4253311 with P NEBL gene reached genome-wide significance for plasma renin concentration in the discovery sample (top SNP rs3915911; P =8.81×10 −9 ), but did not replicate ( P =0.81). No locus reached genome-wide significance for aldosterone. SNPs rs5030062 and rs4253311 were not related to blood pressure or renal traits; in a companion study, variants in the kallikrein B locus were associated with B-type natriuretic peptide concentrations in blacks. Conclusions— We identified 2 genetic loci ( kininogen 1 and kallikrein B ) influencing key components of the RAAS, consistent with the close interrelation between the kallikrein–kinin system and the RAAS.

Chronic kidney disease (CKD) is a significant and costly public health problem with increasing prevalence in most societies and the need for improved diagnosis. The aim of this study was to demonstrate how it is possible to decipher a combination of markers from a compendium of differentially-expressed proteins using available statistical tools when more than two study groups are involved. Screening for potential urinary biomarker profile stratifying early and later stages of CKD were performed. Sixteen patients with defined CKD staging were selected and compared with ten healthy individuals. Urinary proteins were quantified using theiTRAQ method and analysed with ProteinPilot, GenEx and Meta Core software. Four proteins (out of 194) (apolipoprotein D; protein AMBP; zinc-alpha-2-glycoprotein; and kininogen 1) were identified as critical for CKD stage separation. In conclusion, this preliminary work provides evidence that several unique urinary proteins are involved in early and later stages of CKD and suggests that a selected combination of biomarkers could be used to profile patients into different CKD stages. Further validation studies are now needed.

Adiponectin is a fat cell-secreted hormone with antidiabetic and anti-inflammatory activities. The reduced adiponectin levels are associated with obesity-related metabolic syndrome. Replenishment of this hormone into animal models can improve insulin sensitivity, decrease blood glucose and lipid levels, and prevent the development of atherosclerosis and fatty liver injury. Despite these findings, the underlying molecular mechanisms remain largely unknown. Here, we have used affinity chromatography to purify the protein complexes that are associated with adiponectin in human serum. The nature of these adiponectin-binding proteins was analyzed by MS/MS. Eight proteins from the adiponectin-containing protein mixtures have been identified. Many of them, including thrombospondin-1 (TSP-1), histidine-rich glycoprotein, kininogen 1, and alpha 2 macroglobulin (alpha2M), are well-known glycoproteins involved in the regulation of inflammation, angiogenesis, and tissue remodeling. Coimmunoprecipitation and radioligand competitive-binding assays confirmed the direct interactions between adiponectin and alpha2M, or TSP-1. Moreover, these specific bindings were also detected in the serum samples derived from both healthy human subjects and patients with type 2 diabetes. In summary, our study demonstrated that, in the circulation, adiponectin forms protein complexes with other serum proteins. These proteins might serve as the physiological-binding partners of adiponectin and regulate its bioavailability and biological activities.

Background Serum markers represent potential tools for the detection of colorectal cancer (CRC). The aim of this study was to obtain proteomic expression profiles and identify serum markers for the early detection of CRC. Methods Proteomic profiles of serum samples collected from 35 healthy volunteers, 35 patients with advanced colorectal adenoma (ACA), and 40 patients with CRC were compared using Clinprot technology. Using enzyme-linked immunosorbent assays (ELISAs), 366 sera samples were additionally analyzed, and immunohistochemistry studies of 400 tissues were used to verify the expression of kininogen-1 and its value in the early detection of CRC. Results Predicting models were established among the three groups, and kininogen-1 was identified as a potential marker for CRC using Clinprot technology. ELISAs also detected significantly higher serum kininogen-1 levels in ACA and CRC patients compared to controls (P

Background The aim was to validate whether kininogen 1 (KNG1) or insulin-like growth factor binding protein 6 (IGFBP-6) are serum biomarkers of proliferative vitreoretinopathy (PVR). Methods Samples from vitreous and corresponding serum samples were collected from patients with PVR. The donor vitreous samples and serum samples from healthy volunteers and volunteers who had undergone vitrectomies for other conditions were used as controls. The samples were subsequently analysed using Western blotting (WB) and enzyme-linked immunosorbent assay. Results The Western blotting outcomes indicated both IGFBP-6 and KNG1 could be specifically detected in the vitreous and serum samples of patients with PVR. The concentrations of KNG1 and IGFBP-6 were significantly higher in both vitreous and serum samples from patients with severe PVR than in the samples from patients with moderate PVR. The serum concentrations of KNG1 or IGFBP-6 had decreased by the post-vitrectomy examinations. The receiver operating characteristic (ROC) analyses when the concentrations of IGFBP-6 or KNG1 were greater than 181.4 pg/ml or 441.75 ng/ml, respectively, predicted severe PVR with both a sensitivity and specificity of over 70 per cent. When the concentrations of IGFBP-6 or KNG1 were greater than 98.5 pg/ml or 88.5 ng/ml, respectively, they predicted the PVR prognosis with both a sensitivity and specificity of 80 per cent. Conclusions KNG1 and IGFBP-6 may be candidate serum biomarkers of PVR.

Background Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures. Results We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting. Conclusions Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.

Background Diagnosis of ovarian carcinoma is in urgent need for new complementary biomarkers for early stage detection. Proteins that are aberrantly excreted in the urine of cancer patients are excellent biomarker candidates for development of new noninvasive protocol for early diagnosis and screening purposes. In the present study, urine samples from patients with ovarian carcinoma were analysed by two-dimensional gel electrophoresis and the profiles generated were compared to those similarly obtained from age-matched cancer negative women. Results Significant reduced levels of CD59, kininogen-1 and a 39 kDa fragment of inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), and enhanced excretion of a 19 kDa fragment of albumin, were detected in the urine of patients with ovarian carcinoma compared to the control subjects. The different altered levels of the proteins were confirmed by Western blotting using antisera and a lectin that bind to the respective proteins. Conclusion CD59, kininogen-1 and fragments of ITIH4 and albumin may be used as complementary biomarkers in the development of new noninvasive protocols for diagnosis and screening of ovarian carcinoma.

Proliferative vitreoretinopathy (PVR) is the most common cause of anatomic failure in retinal detachment surgery. To understand the molecular mechanisms, vitreous proteomes of patients with PVR were investigated by two-dimensional-nano-liquid chromatography coupled with tandem mass spectrometry. Vitreous samples of moderate PVR (grade B), and severe PVR (grade C or D) were aspirated during pars plana vitrectomy before infusion. In the current study, 129, 97 and 137 proteins were identified in vitreous of normal control, moderate and severe PVR, respectively. In PVR vitreous samples, complement components, serine proteinase inhibitors, and extracellular proteins were up-regulated or appeared, while normal cytoskeleton and metabolism proteins were down-regulated or disappeared. It was noteworthy that the proteins involved in transcription and translation regulation increased in vitreous with PVR. Among 102 PVR-specific proteins, kininogen 1 was specifically detected in both vitreous and the corresponding serum. Therefore, it can be concluded that PVR is a complicated pathology process with great amount of proteins involved in metabolism dysfunction, immune reactions, and cytoskeleton remolding. Kininogen 1 may be a candidate biomarker of PVR. Further investigations of these special proteins will provide additional targets for treatment or prevention of ocular proliferative diseases.

Osteonecrosis of the femoral head (ONFH) is a skeletal disorder characterized by ischemic deterioration, bone marrow edema and eventually femoral head collapse. The systemic regulation of ONFH in adult patients has not been examined. Serum proteomic is an innovative tool that potentially detects simultaneous expressions of serum proteins in pathological contexts. We compared the serum proteome profiles of 11 adult patients with ONFH (3 females and 8 males) and 11 healthy volunteers (3 females and 8 males). The proteins in the aliquots of sera were subjected to isoelectric focusing, two-dimensional gel electrophoresis and silver staining. The protein spots were matched and quantified using an imaging analysis system. The differentially expressed protein spots were subjected to in-gel trypsin digestion. The peptide mass fingerprints were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF) and a bioinformation search. We found that ONFH patients showed significantly higher abundances of kininogen 1 variant, complement factor C3 precursor, and complement factor H and lower levels of antithrombin III chain B, apolipoprotein A--IV precursor, and gelsolin isoform alpha precursor. These proteins of interest were reported to modulate thrombotic/fibrinolytic reactions, oxidative stress, vessel injury, tissue necrosis or cell apoptosis in several tissue types under pathological contexts. Taken together, the occurrence of ONFH was associated with various serum protein expressions. Our high--throughput serum proteomic findings indicated that multiple pathological reactions presumably occurred in ONFH.

Previous peptidomic analyses of the defensive skin secretion from the North American pickerel frog, Rana palustris, have established the presence of canonical bradykinin and multiple bradykinin-related peptides (BRPs). As a consequence of the multiplicity of peptides identified and their diverse primary structures, it was speculated that they must represent the products of expression of multiple genes. Here, we present unequivocal evidence that the majority of BRPs (11/13) identified in skin secretion by the peptidomic approach can be generated by differential site-specific protease cleavage from a single common precursor of 321 amino acid residues, named skin kininogen 1, whose primary structure was deduced from cloned skin secretion-derived cDNA. The organization of skin kininogen 1 consists of a hydrophobic signal peptide followed by eight non-identical domains each encoding a single copy of either canonical bradykinin or a BRP. Two additional splice variants, encoding precursors of 233 (skin kininogen 2) or 189 amino acid residues (skin kininogen 3), were also cloned and were found to lack BRP-encoding domains 5 and 6 or 4, 5 and 6, respectively. Thus, generation of peptidome diversity in amphibian defensive skin secretions can be achieved in part by differential protease cleavage of relatively large and multiple-encoding domain precursors reflecting a high degree of transcriptional economy.

As the pathogenesis of the HELLP-syndrome is unknown, a complex consideration regarding the changes in the plasma as the main transport medium in the body is of great benefit because it is well available and can rapidly be investigated in the clinics. Besides that, the liver which is early affected in HELLP-syndrome produces the main part of plasma proteins. For the purpose of our study plasma protein abundances from patients with HELLP-syndrome and from control individuals were determined before and after delivery. In the differential analysis using two-dimensional gel electrophoresis, six areas with variable protein spot intensities were detected. The reference gel that we developed for HELLP plasma samples integrates the changes of plasma proteins when comparing HELLP patients to healthy women prior to and after delivery. A specific plasma protein profile for the HELLP-syndrome was generated involving protein areas that contain inter-alpha-trypsin inhibitor heavy chain H4, kininogen 1, fibrinogen gamma chain, transthyretin, haptoglobins, and serum amyloid A with statistically significant expression differences when compared to controls. The most striking difference between the majority of the gels from HELLP patients and the gels from non-HELLP samples were clearly overexpressed protein spots at about 11 kDa which were identified as serum amyloid A (SAA). This differential expression was validated and quantitatively assayed by ELISA measurements against human SAA in plasma. Our results show that significant differences in SAA expressions between healthy controls and HELLP patients were obtained, that could function as markers for the HELLP-syndrome. According to our data it is possible to draw a line of separation with no overlap between the HELLP group for which SAA plasma levels were found to be above 3.51 mg/L and the non-HELLP groups in which SAA plasma levels were below 3.51 mg/L. It now is possible to clinically elucidate if the differentially expressed proteins are suited for longitudinal studies concerning both, to function as markers or perhaps even as disease predictors that might become relevant for diagnostic tests.

Introduction: Pregnant women with sickle cell disease (SCD) are at higher risk of obstetrical complications, such as preeclampsia and preterm delivery. The placental circulation is susceptible to vaso-occlusion, which may contribute to fibrosis, necrosis and infarction of the placental villi. Restricted uteroplacental circulation is associated with low birth weight and fetal growth restriction. The mechanisms by which SCD affects placental physiology are largely unknown, and the chronic inflammatory state may be involved in this pathological process. The aim of this study was to evaluate the expression of mediators of inflammatory response in the placenta of pregnant women with SCD. Methods: This is an ongoing case control study that has recruited 4 pregnant patients with hemoglobin SC disease and 4 healthy control pregnant women paired for gestational age and route of delivery. Placental tissue was collected for total RNA extraction and synthesis of cDNA. Samples were run on a qPCR Array (Inflammatory Response and Autoimmunity RT2 Profiler, Qiagen), which includes 84 genes. Gene expression levels were normalized to housekeeping genes (ACTB, B2M, and RPLP0), and will be further validated by RT-qPCR. Results: Two patients underwent cesarean delivery and one patient delivered preterm. The analysis of placental gene expression showed that 16 genes were upregulated. The top 5 most upregulated genes were CCL24 (18.1 fold), TLR9 (8.6 fold), inducible nitric oxide synthase NOS2 (6.8 fold), IL17A (5.4 fold), and CCL16 (4.8 fold). Upregulated genes also included other chemokines and their receptors (CCL21, CCL22, CXCL3, and CCR7), interleukins (IL1RAP, IL5, and IL9), another toll-like receptor (TLR3), transcription factor BCL6, CD40 ligand (CD40LG), and kininogen 1 (KNG1). Interestingly, the two genes found to be downregulated were CRP (C reactive protein) and IFNG (interferon gamma). Conclusion: This is the first study of differential gene expression in placenta of patients with SCD. Despite patients with hemoglobin SC disease being clinically less severe than homozygous sickle cell anemia, we have found that placental tissue in this subset of patients displays increased expression of several genes involved in inflammatory response. Our data support local inflammatory changes in the placenta of these patients that might explain the development of complications in SCD pregnancies. Although the association between obstetric complications and SCD has been long described, our data should contribute to better define the mechanisms affecting the placental physiology that lead to complications, such as preeclampsia. Disclosures No relevant conflicts of interest to declare.

Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears. Published version

1. A method is described for determination of kininogen 1 (substrate mainly for plasma kallikrein) and kininogen 2 (substrate for glandular kallikrein) independently in cat plasma.2. In anaesthetized cats the arterial inflow to, and venous outflow from, the submandibular salivary gland were isolated: a roller pump giving constant volume inflow was interposed in the arterial circuit.3. Venous blood was collected at rest, during and after stimulation of the chorda tympani, and its content of kininogens 1 and 2 were estimated. Kininogen 2 was reduced up to 60% by chorda stimulation, whereas the level of kininogen 1 was unchanged.4. On close arterial infusion of bradykinin or histamine in amounts which produce large vascular effects, including increased capillary permeability, the venous blood levels of both kininogens 1 and 2 were unchanged.5. It is concluded that the selective loss of kininogen 2 on chorda stimulation results from the release of kallikrein into the tissue spaces and reflects the extent of kinin formation within the gland.