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Monu,1,2 Prachi Agnihotri,1 Mohd Saquib,1,2 Ashish Sarkar,1,2 Debolina Chakraborty,1,2 Uma Kumar,3 Sagarika Biswas1 1Council of Scientific and Industrial Research -Institute of Genomics & Integrative Biology (CSIR–IGIB), Delhi, 110007, India; 2Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India; 3All India Institute of Medical Sciences, New Delhi, 110029, IndiaCorrespondence: Sagarika BiswasIntegrative and Functional Biology Department CSIR- Institute of Genomics & Integrative Biology, Mall Road, Delhi, 110 007, IndiaTel +91 11 27667602Fax +91-11-27667471; +9818004740Email sagarika.biswas@igib.res.inObjective: Rheumatoid arthritis (RA) is a chronic autoimmune, inflammatory joint disease. The identification of multifaceted etiological changes at the protein level in RA remains an important need. We aimed to identify differential proteins (DPs) and gene profiles to uncover inflammatory indicators and their association to RA pathogenesis.Methods: 2-DE and SWATH-MS were used to identify DPs in RA and healthy control plasma. Fluorescence phenylboronate gel electrophoresis (Flu-PAGE) with mass spectrometry was used for protein glycation in RA plasma. Disease specificity of identified DPs was confirmed by ELISA and Western blot analysis. The gene expressions of selected DPs were evaluated by qRT-PCR in PBMCs of RA, systemic lupus erythematosus (SLE), spondyloarthritis (SpA), and osteoarthritis (OA). The functional implication of glycated protein was determined by in- silico and validated by in vitro analysis in fibroblast-like synoviocytes.Results: A total of 150 DPs (127 increased and 23 decreased) were identified by 2-DE and SWATH-MS analysis in RA plasma compared to healthy control (HC). Nine proteins were identified as glycated by Flu-PAGE LC-MS/MS. Transthyretin (TTR), serotransferrin, and apolipoprotein-A1 (Apo-A1) were found to be differential and glycated. ELISA and Western blot results revealed the disease-specific increased expression of TTR and RAGE in RA. The qRT-PCR results signify the aberrant gene expression of TTR and RAGE, found to be associated with RA when compared with SLE, SpA, and OA PBMCs. TTR-RAGE interactions were predicted by in-silico and validated by in-vitro analysis using RA-FLS. The increased levels of pro-inflammatory cytokines IL-6, IL-1β, TNF-α, and differently expressed TTR and RAGE were confirmed in fibroblast-like synoviocytes under inflammatory conditions.Conclusion: Our findings showed that the level of TTR was increased in RA plasma, along with an altered glycation rate. TTR and RAGE aberrant gene expression in PBMCs are the key events associated with RA, and TNF-α activates the NF-KB pathways and promote TTR and RAGE differential expressions that may have pathogenic/inflammatory significance.Keywords: rheumatoid arthritis, differential proteins, post-translational modifications, transthyretin, advanced glycation end products, receptor for advanced glycation end products

Ashish Sarkar,1,2 Monu,1,2 Vijay Kumar,3 Rajesh Malhotra,3 Hemant Pandit,4 Elena Jones,4 Frederique Ponchel,4 Sagarika Biswas1 1Department of Integrative and Functional Biology, CSIR-Institute of Genomics and Integrative Biology, New Delhi, 110007, India; 2Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, 201002, India; 3All India Institute of Medical Sciences, New Delhi, 110029, India; 4Leeds Institute of Rheumatic and Musculoskeletal Medicine, School of Medicine, University of Leeds, Leeds, UKCorrespondence: Sagarika BiswasDepartment of Integrative and Functional Biology, CSIR-Institute of Genomics and Integrative Biology, Near Jubilee Hall, Mall Road, New Delhi, 110007, IndiaTel +91 11-2766-2581Email sagarika.biswas@igib.res.inIntroduction: Circulating plasma proteins play an important role in various diseases, and analysis of the plasma proteome has led to the discovery of various disease biomarkers. Osteoarthritis (OA) is the most common chronic joint disease, mostly affecting people of older age. OA typically starts as a focal disease (in a single compartment, typically treated with unicompartmental knee replacement), and then progresses to the other compartments (if not treated in time, typically treated with total knee replacement). For this, identification of differential proteins was carried out in plasma samples of OA cases and compared with healthy controls. The aim of this study was to identify circulatory differentially expressed proteins (DEPs) in knee-OA patients undergoing total knee replacement or unicompartmental knee replacement compared to healthy controls and assess their role, in order to have better understanding of the etiology behind OA pathophysiology.Methods: DEPs were identified with two-dimensional gel electrophoresis (2DE) and isobaric tags for relative and absolute quantification (iTRAQ), followed by liquid chromatography with tandem mass spectrometry. Validation of DEPs was carried out using Western blot and ELISA. Posttranslational modifications were checked after running native gel using purified protein from patients, followed by detection of autoantibodies.Results: In total, 52 DEPs were identified, among which 45 were distinct DEPs. Haptoglobin (Hp) was identified as one of the most significantly upregulated proteins in OA (P=0.005) identified by both 2DE and iTRAQ. Decreased levels of Hp tetramers and increased levels of autoantibodies against Hpβ were observed in OA plasma.Conclusion: Our data suggest that poor clearance of free hemoglobin and low levels of Hp tetramers may be associated with OA pathogenesis and inflammation.Keywords: osteoarthritis, blood, haptoglobin, hemoglobin, biomarker, inflammation

Exposure to radiation causes severe alterations of protein expression level inside the cell, thus it may influence the biological events and stress response. In the present investigation, we have demonstrated the effect of radiation on mice lung tissues.Two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF was used to check the expression changes in lung proteome profile of strain 'A' female mice after exposure to lethal doses of gamma irradiation at different time periods (24 and 48 h). Identified proteins were analysed for their altered expression and were further validated by Western blotting and enzyme-linked immunosorbent assay (ELISA).Nine significant differentially expressed proteins were identified from irradiated lungs tissues. The expression level of zinc finger protein was found to be up regulated at 24 h irradiation in comparison to 48 h irradiation.Zinc finger protein may be considered as a radiation responsive protein. Alteration in its expression pattern may primarily affect binding specificity of the protein that can further result in the interference in transcriptional control of multiple stress responsive genes.