Proteomic-Based Discovery of Predictive Biomarkers for Drug Therapy Response and Personalized Medicine in Chronic Immune Thrombocytopenia.

Purpose: ITP is the most prevalent autoimmune blood disorder. The lack of predictive biomarkers for therapeutic response is a major challenge for physicians caring of chronic ITP patients. This study is aimed at identifying predictive biomarkers for drug therapy responses. Methods: 2D gel electrophoresis (2-DE) was performed to find differentially expressed proteins. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) analysis was performed to identify protein spots. The Cytoscape software was employed to visualize and analyze the protein-protein interaction (PPI) network. Then, enzyme-linked immunosorbent assays (ELISA) were used to confirm the results of the proteins detected in the blood. The DAVID online software was used to explore the Gene Ontology and pathways involved in the disease. Results: Three proteins, including APOA1, GC, and TF, were identified as hub-bottlenecks and confirmed by ELISA. Enrichment analysis results showed the importance of several biological processes and pathway, such as the PPAR signaling pathway, complement and coagulation cascades, platelet activation, vitamin digestion and absorption, fat digestion and absorption, cell adhesion molecule binding, and receptor binding. Conclusion and Clinical Relevance. Our results indicate that plasma proteins (APOA1, GC, and TF) can be suitable biomarkers for the prognosis of the response to drug therapy in ITP patients.

Comparison of Native Hydatid Cyst Fluid (HCF), Lyophilized HCF, Antigen B (AgB) and Lyophilized AgB (LAgB) Originated from Echinococcus granulosus Sensu Stricto for Sero-Diagnosis of Active, Transitional and Inactive Human Liver Cystic Echinococcosis.

Background: Cystic echinococcosis (CE) is an important zoonotic parasitic disease caused by the larval stage or metacestode of the tapeworm Echinococcus granulosus sensu lato. Due to treatment protocols for different liver cysts, diagnosis of cyst stages is very important. Different antigens have been used for CE diagnosis. However, each one is more sensitive and effective for the diagnosis of specific CE stages is not known well. We aimed to compare Native Hydatid Cyst Fluid (HCF), Lyophilized Hydatid Cyst Fluid (LHCF), antigen B (AgB) and Lyophilized antigen B (LAgB) originated from E. granulosus sensu stricto (G1-G3) genotype, for sero- diagnosis of active, transitional and inactive human liver CE using ELISA technique. Methods: The HCF was collected aseptically from liver CE cysts of sheep slaughtered from 2018 to 2019 in Shiraz slaughterhouse, Southern, Iran. The cysts were characterized by PCR and sequencing for genotype specification. Four types of antigens were used: HCF, LHCF, AgB and LAgB originated from E. granulosus sensu stricto (G1-G3) genotype. Thirty-three serum samples from active, transitional, and inactive human cysts were collected. Overall, 48 samples from other parasitic diseases and 60 samples from healthy subjects as negative controls were checked using four antigens by ELISA method. Results: The best diagnostic sensitivity with 96.97% was observed by anti-LHCF IgG ELISA test. The best specificity with 95.37% was observed in ELISA test using LAgB. Conclusion: Simultaneous test of sera with anti-LHCF IgG ELISA and anti-LAgB IgG ELISA would be the best in the diagnosis of human liver cystic echinococcosis.

Proteomics investigation of human sera for determination of postoperative indicators of pulmonary cystic echinococcosis.

BACKGROUND: Cystic echinococcosis (CE)/hydatidosis is an important zoonotic parasitic disease caused by the larval stage of Echinococcus granulosus. The disease is a major health problem all over the world. Finding specific and sensitive biomarkers for follow-up of CE in patients after surgery is essential. Using proteomics methods, the present study aimed to evaluate post-surgical treatment by finding probable biomarker/s in the serum of human lungs CE. METHODS: A total of 24 human sera were tested. These sera included eight confirmed lung/s CE patients sera before surgery (BS), eight sera 12 months post-surgery (12MPS) as well as eight control sera from healthy people. Proteomics methods including 2DE and LC-MS/MS were performed on the specimens followed by bioinformatics analysis. Differentially expressed proteins (DEP) were detected and, separately integrated with protein-protein interaction (PPI) data to construct the PPI network. RESULTS: A total of 171 protein spots were detected in three groups including BS, 12MPS, and control groups; of which a total of 106 DEP have been expressed based on fold changes > = 2 and p-value < 0.05. More analysis was performed and a total of 10 protein spots were selected for identification by mass spectrometry showing the following proteins: APOA1, BGN, SPP2, EAF1, ACOXL, MRPL55, MCTP2, SEPTIN1, B4GALNT1, and ZNF843. Based on centrality parameters of the PPI network (degree and betweenness) five Hub-bottlenecks proteins with significant centrality values were found including APOA1, BGN, SPP2, EAF1, and ACOXL. CONCLUSION: This study showed five proteins as hub-bottleneck proteins; of which APOA1 was more prominent. It can be concluded that a change in expression of this protein in patients' sera could be used as an indicator tool for the achievement of lungs CE surgical therapy.

Following up of Surgical Treated Human Liver Cystic Echinococcosis: A Proteomics Approach.

BACKGROUND: Cystic echinococcosis (CE) is one of the most important parasitic zoonosis in the world. Post-surgery follow up in CE patients is an important non-solved problem up to now. Therefore, the investigations on this problematic issue would be very applicable in the view of CE clinical treatment. METHODS: A total of 24 confirmed liver CE patients sera including eight sera before surgery (BS), eight sera three months post-surgery (3MPS), and eight sera six months post-surgery (6MPS) were used in the present study. Proteomics methods including 2DE and LC-MS/MS were performed on the specimens followed by bioinformatics analysis such as Gene Ontology (GO) and Protein-Protein Interaction (PPI) network analysis. RESULTS: A total of 235 proteins were detected of which 12 differentially expressed proteins (DEP) were identified by LC-MS/MS in all sera. The proteins were presented in BS and suppressed after surgery as follows: HPX, SERPINA1, SERPINC1, CP, HBD, and HBA2. Comparisons of the protein expression in sera of patients BS, 3MPS, and 6MPS revealed that GC, IGJ, AHSG, CD5L, FGG, and APOC3 have been overexpressed in 3MPS and 6MPS. PPI network analysis demonstrated that SERPINC1 and AHSG with more connection in the network could be considered as hub proteins and potential prognostic biomarkers in response to surgical treatment of liver CE. CONCLUSION: Application of proteomics methods on patient's sera could be used as a novel biomarker tool for following-up liver CE patients. In this regards, proteomics and, application of bioinformatics analysis including GO and PPI showed that SERPINC1, AHSG and HPX are of more value as a potential follow up biomarkers in response to surgical treatment.

Proteomic characterization of hydatid cyst fluid: two-dimensional electrophoresis (2-DE) setup through optimizing protein extraction.

OBJECTIVE: Proper characterization of hydatid cyst fluid (HCF) is useful for diagnostic and follow up purposes of cystic echinococcosis/hydatidosis, which is an important zoonotic disease. In this regard, proteomics methods are very helpful. The present study was conducted to compare three protein extraction methods for HCF collected from sheep liver hydatid cysts including, trichloracetic acid (TCA)/Acetone precipitation, TCA/Acetone along with dialysis, and combination of 2-D Clean-up Kit and dialysis followed by two-dimensional electrophoresis (2-DE), to achieve better resolution in the proteomic characterization of HCF proteins. RESULTS: The 2-DE of TCA/Acetone products showed a lot of smears in the background of gels; TCA/Acetone with dialysis showed greatly reduced smears while the 2-D Clean-up Kit together with dialysis showed sharp spots and least smears. Three-dimensional images of separated spots created by Progenesis SameSpots software showed the best result was achieved by 2-D Clean-up Kit and dialysis.

Comparison between Echinococcus granulosus sensu stricto (G1) and E. canadensis (G6) mitochondrial genes (cox1 and nad1) and their related protein models using experimental and bioinformatics analysis.

BACKGROUND: Cystic echinococcosis (CE) as a zoonotic parasitic disease, remains a health challenge in many parts of the world. There are different species of Echinococcus granulosus sensu lato with different pathogenicity and host preferences.Different procedures have been applied for characterization of Echinococcus taxa in which two mitochondrial genes, cox1 and nad1 have been used more common. They have been able to differentiate E. granulosus sensu stricto and E. canadensis species in different hosts. The affinity of E. granulosus sensu stricto and E. canadensis species for localizing different organs seems to be different. To what such affinity and related pathogenicity could be related, is not known, so far. Bioinformatics analysis may be helpful to interpret such difference by investigating the genes and their related protein models between different species infecting human and animals. The current work was designed to study the differences between E. granulosus s.s. and E. canadensis species mitochondrial genes (cox1 and nad1) and related protein models of CE cysts by experimental and bioinformatics analysis. MATERIALS AND METHODS: Different human and animal CE cysts were collected and their DNA was extracted and sequenced based on their cox1 and nad1 genes. In order to determine the E. granulosus s.s. and E. canadensis species of the samples, BLAST analysis was performed on sequenced genes. Three sequences were selected for analysis and were deposited in GenBank. Moreover, the sequence number of KT988116.1 which belonged to E. canadensis from our already deposited in GenBank was also selected. Alignment and phylogenetic analysis were performed on the sequences using BioEdit and MEGA7 software. The raw sequences of translated proteins belonged to the mentioned genes were obtained from Protein database in NCBI. The secondary structure was determined by PSIPRED Protein Sequence Analysis Workbench. The tertiary models of COX1 and NAD1 proteins in both genotypes were constructed using Modeler 9.12 software and their physicochemical features were computed using ProtParam tool in ExPASY server. RESULTS: BLAST analysis on sequenced genes showed that the samples belonged to E. granulosus s.s. and E. canadensis species. These sequences were deposited in GenBank with accession numbers: JN579173.1, KF437811.1, and KY924632.1. The results showed that proteins of COX1 of E. granulosus s.s., COX1of E. canadensis, NAD1of E. granulosus s.s. and NAD1of E. canadensis species, consisted of 135, 122, 120 and 124 amino acids, respectively. The aligned sequences of translated proteins belonged to COX1 and NAD1 enzymes in E. granulosus s.s. and E. canadensis species were different; such that alignment COX1 sequence between E. granulosus s.s. and E. canadensis species showed that amino acids were different in 6 positions. This difference for NAD1 sequences were different in 19 positions. The secondary structure determined by PSIPRED showed differences in coil, strand and helix chains in COX1 and NAD1 proteins in E. granulosus s.s. and E. canadensis species. Comparison between three-dimensional structures (3D) of COX1 protein model in E. granulosus s.s. and E. canadensis species demonstrated an additional helix with two conserved iron binding sites in the COX1 protein of E. granulosus s.s. species. CONCLUSION: E. granulosus s.s. and E. canadensis species differences are reflected in two important proteins: COX1 and NAD1. These differences are demonstrable in the 3D structure of proteins of both strains. So, the present study is adding to our understanding of the difference in molecular sequences between the E. granulosus s.s. (G1) and E. canadensis (G6) which may be used for interpreting the difference between the pathogenicity and localization affinity in these two important helminthic zoonosis.

Comparative Two-dimensional Gel Electrophoresis Maps for Amastigote-like Proteomes of Iranian Leishmania Tropica and Leishmania Major Isolates.

BACKGROUND: Leishmania major and Leishmania tropica are the main causative agents of cutaneous leishmaniasis. Proteomics as a novel approaches could be used to evaluate protein expression levels in different stages of Leishmania species. We compare the protein contents of amastigote-like forms in L. tropica and L. major using two-dimensional gel electrophoresis (2-DE) and bioinformatics methods. MATERIALS AND METHODS: Leishmania parasites were isolated from the lesions of Iranian patients and identified using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). Five isolates of each two species were cultured in specific media to obtain amastigote-like forms to be prepared for proteomics study. Total protein contents were separated using 2-DE. The gels were stained by silver nitrate and scan was imaged. The protein spots with different expression changes in each gel were analyzed using Progenesis SameSpots software. RESULTS: A total of 354 protein spots were detected in both amastigote-like forms. Comparative analysis of protein spots with different expressions in the two amastigote-like form species showed 173 highly expressed spots of which 74 L. tropica and 99 L. major proteins were spotted with fold≥2. Also, 16 and 20 new protein spots were uniquely found in L. tropica and L. major, respectively. Clustering of different detected proteins using correlation analysis divided the proteins into two clusters based on their expression level. Furthermore, clustering results were confirmed by principal component analysis. CONCLUSION: Using proteomics methods specially 2-DE and statistical analysis demonstrated significant changes in protein expression levels in amastigote-like forms of L. tropica and L. major isolates.

Proteome evaluation of human cystic echinococcosis sera using two dimensional gel electrophoresis.

AIM: Detection of protein expression changes in human cystic echinococcosis sera by 2D gel electrophoresis. BACKGROUND: Diagnosis and successful treatment of cystic echinococcosis (CE) is a major challenge, up to now. Identification of related expressed proteins using proteomics tools and bioinformatics analysis of patients' sera have not been investigated, so far. METHODS: Sera from eight confirmed CE patients and three healthy controls were collected, tested by 2-DE for total protein separation of serum and analyzed using proteomics and bioinformatics methods. The gels were stained by Coomassie blue followed by scan imaging of the gels. The protein spots in each gel were analyzed using progenesis same spots software. Proteins names were obtained from TagIdent server. RESULTS: A total of 263 protein spots with different expression were detected in both normal and diseased samples. Comparison between diseased and normal gels showed the expression of 45 up-regulated protein spots with fold≥2 in diseased gel of which 10 were new proteins with statistical difference by normal gel (p-value<0.05). On the other hand, the expression of 50 down-regulated protein spots were observed of which 11 proteins have been suppressed. Clustering of all detected sera proteins (263) using correlation analysis, divided the proteins into 2 clusters based on up-regulated and down-regulated expression of proteins. Clustering results were approved by principal component analysis (PCA). CONCLUSION: Significant protein expression changes in human CE sera which is demonstrable by application of proteomics and bioinformatics analysis makes it an impressing tool for diagnosis of CE patients.