Immunoinformatic and reverse vaccinology-based designing of potent multi-epitope vaccine against Marburgvirus targeting the glycoprotein.

Marburg virus (MARV) has been a major concern since its first outbreak in 1967. Although the deadly BSL-4 pathogen has been reported in few individuals with sporadic outbreaks following 1967, its rarity commensurate the degree of disease severity. The virus has been known to cause extreme hemorrhagic fever presenting flu-like symptoms (as implicated in COVID-19) with a 90% case fatality rate (CFR). After a number of plausible evidences, it has been observed that the virus usually originates from African fruit bat, Rousettus aegyptiacus, who themselves do not indicate any signs of illness. Thus, efforts have been made in the recent years for a universal treatment of the infection, but till date, no such vaccine or therapeutics could circumvent the viral pathogenicity. In an attempt to formulate a vaccine design computationally, we have explored the entire proteome of the virus and found a strong correlation of its glycoprotein (GP) in receptor binding and subsequent role in infection progression. The present study, explores the MARV glycoprotein GP1 and GP2 domains for quality epitopes to elicit an extended immune response design potential vaccine construct using appropriate linkers and adjuvants. Finally, the chimeric vaccine wass evaluated for its binding affinity towards the receptors via molecular docking and molecular dynamics simulation studies. The rare, yet deadly zoonotic infection with mild outbreaks in recent years has flustered an alarming future with various challenges in terms of viral diseases. Thus, our study has aimed to provide novel insights to design potential vaccines by using the predictive framework.

Mapping CircRNA-miRNA-mRNA regulatory axis identifies hsa_circ_0080942 and hsa_circ_0080135 as a potential theranostic agents for SARS-CoV-2 infection.

Non-coding RNAs (ncRNAs) can control the flux of genetic information; affect RNA stability and play crucial roles in mediating epigenetic modifications. A number of studies have highlighted the potential roles of both virus-encoded and host-encoded ncRNAs in viral infections, transmission and therapeutics. However, the role of an emerging type of non-coding transcript, circular RNA (circRNA) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has not been fully elucidated so far. Moreover, the potential pathogenic role of circRNA-miRNA-mRNA regulatory axis has not been fully explored as yet. The current study aimed to holistically map the regulatory networks driven by SARS-CoV-2 related circRNAs, miRNAs and mRNAs to uncover plausible interactions and interplay amongst them in order to explore possible therapeutic options in SARS-CoV-2 infection. Patient datasets were analyzed systematically in a unified approach to explore circRNA, miRNA, and mRNA expression profiles. CircRNA-miRNA-mRNA network was constructed based on cytokine storm related circRNAs forming a total of 165 circRNA-miRNA-mRNA pairs. This study implies the potential regulatory role of the obtained circRNA-miRNA-mRNA network and proposes that two differentially expressed circRNAs hsa_circ_0080942 and hsa_circ_0080135 might serve as a potential theranostic agents for SARS-CoV-2 infection. Collectively, the results shed light on the functional role of circRNAs as ceRNAs to sponge miRNA and regulate mRNA expression during SARS-CoV-2 infection.

Integrated analysis to study the interplay between post-translational modifications (PTM) in hepatitis C virus proteins and hepatocellular carcinoma (HCC) development.

Many PTMs dysregulation is known to be the major cause of many cancers including HCV induced HCC. PTMs of hepatitis C virus (HCV) regions NS3/4A, NS5A and NS5B are crucial for proper protein functions and replication that directly affect the generation of infectious virus particles and completion of its life cycle. In this study, we have performed comprehensive analysis of PTMs within HCV non-structural proteins (NS3/4A, NS5A and NS5B) through bioinformatics analysis to examine post-translational crosstalk between phosphorylation, palmitoylation, methylation, acetylation and ubiquitination sites in selected viral proteins. Our analysis has revealed many highly putative PTMs sites that are also conserved among major genotypes conferring the importance of these sites. We have also analysed viral 3D structures in their modified and unmodified forms to address extent and signatures of structural changes upon PTM. This study provides evidence that PTMs induce significant conformational changes and make viral proteins more stable. To find the potential role of PTMs in HCV induced HCC, docking analysis between selected viral proteins and p38-MAPK has been performed which also confirms their strong association with HCV induced HCC. The major findings proposed that PTMs at specific sites of HCV viral proteins could dysregulate specific pathways that cause the development of HCC.

Human Coronavirus Spike Protein Based Multi-Epitope Vaccine against COVID-19 and Potential Future Zoonotic Coronaviruses by Using Immunoinformatic Approaches.

Zoonotic coronaviruses (CoV) have emerged twice and have caused severe respiratory diseases in humans. Due to the frequent outbreaks of different human coronaviruses (HCoVs), the development of a pan-HCoV vaccine is of great importance. Various conserved epitopes shared by HCoVs are reported to induce cross-reactive T-cell responses. Therefore, this study aimed to design a multi-epitope vaccine, targeting the HCoV spike protein. Genetic analysis revealed that the spike region is highly conserved among SARS-CoV-2, bat SL-CoV, and SARS-CoV. By employing the immunoinformatic approach, we prioritized 20 MHC I and 10 MHCII conserved epitopes to design a multi-epitope vaccine. This vaccine candidate is anticipated to strongly elicit both humoral and cell-mediated immune responses. These results warrant further development of this vaccine into real-world application.

Brain-protective mechanisms of autophagy associated circRNAs: Kick starting self-cleaning mode in brain cells via circRNAs as a potential therapeutic approach for neurodegenerative diseases.

Altered autophagy is a hallmark of neurodegeneration but how autophagy is regulated in the brain and dysfunctional autophagy leads to neuronal death has remained cryptic. Being a key cellular waste-recycling and housekeeping system, autophagy is implicated in a range of brain disorders and altering autophagy flux could be an effective therapeutic strategy and has the potential for clinical applications down the road. Tight regulation of proteins and organelles in order to meet the needs of complex neuronal physiology suggests that there is distinct regulatory pattern of neuronal autophagy as compared to non-neuronal cells and nervous system might have its own separate regulator of autophagy. Evidence has shown that circRNAs participates in the biological processes of autophagosome assembly. The regulatory networks between circRNAs, autophagy, and neurodegeneration remains unknown and warrants further investigation. Understanding the interplay between autophagy, circRNAs and neurodegeneration requires a knowledge of the multiple steps and regulatory interactions involved in the autophagy pathway which might provide a valuable resource for the diagnosis and therapy of neurodegenerative diseases. In this review, we aimed to summarize the latest studies on the role of brain-protective mechanisms of autophagy associated circRNAs in neurodegenerative diseases (including Alzheimer's disease, Parkinson's disease, Huntington's disease, Spinal Muscular Atrophy, Amyotrophic Lateral Sclerosis, and Friedreich's ataxia) and how this knowledge can be leveraged for the development of novel therapeutics against them. Autophagy stimulation might be potential one-size-fits-all therapy for neurodegenerative disease as per considerable body of evidence, therefore future research on brain-protective mechanisms of autophagy associated circRNAs will illuminate an important feature of nervous system biology and will open the door to new approaches for treating neurodegenerative diseases.

The emerging role and significance of circular RNAs in viral infections and antiviral immune responses: possible implication as theranostic agents.

Circular RNAs (circRNAs) are ubiquitously expressed, covalently closed rings, produced by pre-mRNA splicing in a reversed order during post-transcriptional processing. Circularity endows 3'-5'-linked circRNAs with stability and resistance to exonucleolytic degradation which raises the question whether circRNAs may be relevant as potential therapeutic targets or agents. High stability in biological systems is the most remarkable property and a major criterion for why circRNAs could be exploited for a range of RNA-centred medical applications. Even though various biological roles and regulatory functions of circRNAs have been reported, their in-depth study is challenging because of their circular structure and sequence-overlap with linear mRNA counterparts. Moreover, little is known about their role in viral infections and in antiviral immune responses. We believe that an in-depth and detailed understanding of circRNA mediated viral protein regulations will increase our knowledge of the biology of these novel molecules. In this review, we aimed to provide a comprehensive basis and overview on the biogenesis, significance and regulatory roles of circRNAs in the context of antiviral immune responses and viral infections including hepatitis C virus infection, hepatitis B virus infection, hepatitis delta virus infection, influenza A virus infection, Epstein-Barr virus infection, kaposi's sarcoma herpesvirus infection, human cytomegalovirus infection, herpes simplex virus infection, human immunodeficiency virus infection, porcine epidemic diarrhoea virus infection, ORF virus infection, avian leukosis virus infection, simian vacuolating virus 40 infection, transmissible gastroenteritis coronavirus infection, and bovine viral diarrhoea virus infection. We have also discussed the critical regulatory role of circRNAs in provoking antiviral immunity, providing evidence for implications as therapeutic agents and as diagnostic markers.

Formal model of the interplay between TGFß1 and MMP-9 and their dynamics in hepatocellular carcinoma.

Transforming growth factor beta1 (TGFß1) and matrix metalloproteinase-9 (MMP-9) have been associated with migration and invasion in hepatocellular carcinoma (HCC). Recent studies have suggested a positive feedback loop between TGFß1 and MMP-9 mediated by the PI3K signaling pathway that confers acquired invasion and metastasis in HCC via induction of the epithelial-mesenchymal transition (EMT), which grows into invasive carcinoma. But the potential molecular mechanism of this loop on HCC has not been clarified yet. Therefore, this study is designed to explore the association between the two entities and their key determinants such as NFκB, TIMP-1, and hepatic stellate cells (HSCs). Hence, a qualitative modeling framework is implemented that predict the role of biological regulatory network (BRN) during recovery and HCC metastasis. Qualitative modeling predicts discrete trajectories, stable states, and cycles that highlight the paths leading to disease recovery and homeostasis, respectively. The deadlock stable state (1, 1, 1, 1, 1) predicts high expression of all the entities in the BRN, resulting in the progression of HCC. The qualitative model predicts 30 cycles representing significant paths leading to recovery and homeostasis and amongst these the most significant discrete cycle was selected based on the highest betweenness centralities of the discrete states. We further verified our model with network modeling and simulation analysis based on petri net modeling approach. The BRN dynamics were analyzed over time. The results implied that over the course of disease condition or homeostasis, the biological entities are activated in a variable manner. Taken together, our findings suggest that the TGFß1 and the MMP-9 feedback loop is critical in tumor progression, as it may aid in the development of treatment strategies that are designed to target both TGFß and MMP-9.

Model-based in silico analysis of the PI3K/Akt pathway: the elucidation of cross-talk between diabetes and breast cancer.

BACKGROUND: A positive association between diabetes and breast cancer has been identified by various epidemiological and clinical studies. However, the possible molecular interactions between the two heterogeneous diseases have not been fully determined yet. There are several underlying mechanisms which may increase the risk of breast cancer in diabetic patients. INTRODUCTION: In this study, we focused on the role of O-GlcNAc transferase (OGT) enzyme in the regulation of phosphatidylinositol-3 kinase (PI3K) pathway through activation/deactivation of Akt protein. The efficiency of insulin signaling in adipocytes is reduced as a result of OGT overexpression which further attenuates Akt signaling; as a result, the efficiency of insulin signaling is reduced by downregulation of insulin-responsive genes. On the other hand, increased expression of OGT results in Akt activation in breast cancer cells, leading to enhanced cell proliferation and inhibition of the apoptosis. However, the interplay amongst these signaling pathways is still under investigation. METHODS: In this study, we used Petri nets (PNs) to model and investigate the role of PI3K and OGT pathways, acting as key players in crosstalk between diabetes and breast cancer, resulting in progression of these chronic diseases. Moreover, in silico perturbation experiments were applied on the model to analyze the effects of anti-cancer agents (shRNA and BZX) and anti-diabetic drug (Metformin) on the system. RESULTS: Our PN model reflects the alterations in protein expression and behavior and the correlation between breast cancer and diabetes. The analysis proposed two combination therapies to combat breast cancer progression in diabetic patients including combination of OGTmRNA silencing and OGT inhibitor (BZX) as first combination and BZX and Metformin as the second. CONCLUSION: The PN model verified that alterations in O-GlcNAc signaling affect both insulin resistance and breast cancer. Moreover, the combination therapy for breast cancer patients consisting of anti-diabetic drugs such as Metformin along with OGT inhibitors, for example BZX, can produce better treatment regimens.

Exploring NS3/4A, NS5A and NS5B proteins to design conserved subunit multi-epitope vaccine against HCV utilizing immunoinformatics approaches.

Hepatitis C virus (HCV) vaccines, designed to augment specific T-cell responses, have been designated as an important aspect of effective antiviral treatment. However, despite the current satisfactory progress of these vaccines, extensive past efforts largely remained unsuccessful in mediating clinically relevant anti-HCV activity in humans. In this study, we used a series of immunoinformatics approaches to propose a multiepitope vaccine against HCV by prioritizing 16 conserved epitopes from three viral proteins (i.e., NS34A, NS5A, and NS5B). The prioritised epitopes were tested for their possible antigenic combinations with each other along with linker AAY using structural modelling and epitope-epitope interactions analysis. An adjuvant (ß-defensin) at the N-terminal of the construct was added to enhance the immunogenicity of the vaccine construct. Molecular dynamics (MD) simulation revealed the most stable structure of the proposed vaccine. The designed vaccine is potentially antigenic in nature and can form stable and significant interactions with Toll-like receptor 3 and Toll-like receptor 8. The proposed vaccine was also subjected to an in silico cloning approach, which confirmed its expression efficiency. These analyses suggest that the proposed vaccine can elicit specific immune responses against HCV; however, experimental validation is required to confirm the safety and immunogenicity profile of the proposed vaccine construct.

Model of the adaptive immune response system against HCV infection reveals potential immunomodulatory agents for combination therapy.

A regulated immune system employs multiple cell types, diverse variety of cytokines and interacting signalling networks against infections. Systems biology offers a promising solution to model and simulate such large populations of interacting components of immune systems holistically. This study focuses on the distinct components of the adaptive immune system and analysis, both individually and in association with HCV infection. The effective and failed adaptive immune response models have been developed followed by interventions/perturbations of various treatment strategies to get better assessment of the treatment responses under varying stimuli. Based on the model predictions, the NK cells, T regulatory cells, IL-10, IL-21, IL-12, IL-2 entities are found to be the most critical determinants of treatment response. The proposed potential immunomodulatory therapeutic interventions include IL-21 treatment, blocking of inhibitory receptors on T-cells and exogenous anti-IL-10 antibody treatment. The relative results showed that these interventions have differential effect on the expression levels of cellular and cytokines entities of the immune response. Notably, IL-21 enhances the expression of NK cells, Cytotoxic T lymphocytes and CD4+ T cells and hence restore the host immune potential. The models presented here provide a starting point for cost-effective analysis and more comprehensive modeling of biological phenomenon.

MicroRNA pharmacogenomics based integrated model of miR-17-92 cluster in sorafenib resistant HCC cells reveals a strategy to forestall drug resistance.

Among solid tumors, hepatocellular carcinoma (HCC) emerges as a prototypical therapy-resistant tumor. Considering the emerging sorafenib resistance crisis in HCC, future studies are urgently required to overcome resistance. Recently noncoding RNAs (ncRNAs) have emerged as significant regulators in signalling pathways involved in cancer drug resistance and pharmacologically targeting these ncRNAs might be a novel stratagem to reverse drug resistance. In the current study, using a hybrid Petri net based computational model, we have investigated the harmonious effect of miR-17-92 cluster inhibitors/mimics and circular RNAs on sorafenib resistant HCC cells in order to explore potential resistance mechanisms and to identify putative targets for sorafenib-resistant HCC cells. An integrated model was developed that incorporates seven miRNAs belonging to miR-17-92 cluster (hsa-miR-17-5p, hsa-miR-17-3p, hsa-miR-19a, hsa-miR-19b, hsa-miR-18a, hsa-miR-20a and hsa-miR-92) and crosstalk of two signaling pathways (EGFR and IL-6) that are differentially regulated by these miRNAs. The mechanistic connection was proposed by the correlation between members belonging to miR-17-92 cluster and corresponding changes in the protein levels of their targets in HCC, specifically those targets that have verified importance in sorafenib resistance. Current findings uncovered potential pathway features, underlining the significance of developing modulators of this cluster to combat drug resistance in HCC.

Petri Net-Based Model of Helicobacter pylori Mediated Disruption of Tight Junction Proteins in Stomach Lining during Gastric Carcinoma.

Tight junctions help prevent the passage of digestive enzymes and microorganisms through the space between adjacent epithelial cells lining. However, Helicobacter pylori encoded virulence factors negatively regulate these tight junctions and contribute to dysfunction of gastric mucosa. Here, we have predicted the regulation of important tight junction proteins, such as Zonula occludens-1, Claudin-2 and Connexin32 in the presence of pathogenic proteins. Molecular events such as post translational modifications and crosstalk between phosphorylation, O-glycosylation, palmitoylation and methylation are explored which may compromise the integrity of these tight junction proteins. Furthermore, the signaling pathways disrupted by dysregulated kinases, proteins and post-translational modifications are reviewed to design an abstracted computational model showing the situation-dependent dynamic behaviors of these biological processes and entities. A qualitative hybrid Petri Net model is therefore constructed showing the altered host pathways in the presence of virulence factor cytotoxin-associated gene A, leading to the disruption of tight junction proteins. The model is qualitative logic-based, which does not depend on any kinetic parameter and quantitative data and depends on knowledge derived from experiments. The designed model provides insights into the tight junction disruption and disease progression. Model is then verified by the available experimental data, nevertheless formal in vitro experimentation is a promising way to ensure its validation. The major findings propose that H. pylori activated kinases are responsible to trigger specific post translational modifications within tight junction proteins, at specific sites. These modifications may favor alterations in gastric barrier and provide a route to bacterial invasion into host cells.

VacSol: a high throughput in silico pipeline to predict potential therapeutic targets in prokaryotic pathogens using subtractive reverse vaccinology.

BACKGROUND: With advances in reverse vaccinology approaches, a progressive improvement has been observed in the prediction of putative vaccine candidates. Reverse vaccinology has changed the way of discovery and provides a mean to propose target identification in reduced time and labour. In this regard, high throughput genomic sequencing technologies and supporting bioinformatics tools have greatly facilitated the prompt analysis of pathogens, where various predicted candidates have been found effective against certain infections and diseases. A pipeline, VacSol, is designed here based on a similar approach to predict putative vaccine candidates both rapidly and efficiently. RESULTS: VacSol, a new pipeline introduced here, is a highly scalable, multi-mode, and configurable software designed to automate the high throughput in silico vaccine candidate prediction process for the identification of putative vaccine candidates against the proteome of bacterial pathogens. Vaccine candidates are screened using integrated, well-known and robust algorithms/tools for proteome analysis, and the results from the VacSol software are presented in five different formats by taking proteome sequence as input in FASTA file format. The utility of VacSol is tested and compared with published data and using the Helicobacter pylori 26695 reference strain as a benchmark. CONCLUSION: VacSol rapidly and efficiently screens the whole bacterial pathogen proteome to identify a few predicted putative vaccine candidate proteins. This pipeline has the potential to save computational costs and time by efficiently reducing false positive candidate hits. VacSol results do not depend on any universal set of rules and may vary based on the provided input. It is freely available to download from: https://sourceforge.net/projects/vacsol/ .

Mutation-Structure-Function Relationship Based Integrated Strategy Reveals the Potential Impact of Deleterious Missense Mutations in Autophagy Related Proteins on Hepatocellular Carcinoma (HCC): A Comprehensive Informatics Approach.

Autophagy, an evolutionary conserved multifaceted lysosome-mediated bulk degradation system, plays a vital role in liver pathologies including hepatocellular carcinoma (HCC). Post-translational modifications (PTMs) and genetic variations in autophagy components have emerged as significant determinants of autophagy related proteins. Identification of a comprehensive spectrum of genetic variations and PTMs of autophagy related proteins and their impact at molecular level will greatly expand our understanding of autophagy based regulation. In this study, we attempted to identify high risk missense mutations that are highly damaging to the structure as well as function of autophagy related proteins including LC3A, LC3B, BECN1 and SCD1. Number of putative structural and functional residues, including several sites that undergo PTMs were also identified. In total, 16 high-risk SNPs in LC3A, 18 in LC3B, 40 in BECN1 and 43 in SCD1 were prioritized. Out of these, 2 in LC3A (K49A, K51A), 1 in LC3B (S92C), 6 in BECN1 (S113R, R292C, R292H, Y338C, S346Y, Y352H) and 6 in SCD1 (Y41C, Y55D, R131W, R135Q, R135W, Y151C) coincide with potential PTM sites. Our integrated analysis found LC3B Y113C, BECN1 I403T, SCD1 R126S and SCD1 Y218C as highly deleterious HCC-associated mutations. This study is the first extensive in silico mutational analysis of the LC3A, LC3B, BECN1 and SCD1 proteins. We hope that the observed results will be a valuable resource for in-depth mechanistic insight into future investigations of pathological missense SNPs using an integrated computational platform.

Identification of drug resistance and immune-driven variations in hepatitis C virus (HCV) NS3/4A, NS5A and NS5B regions reveals a new approach toward personalized medicine.

Cellular immune responses (T cell responses) during hepatitis C virus (HCV) infection are significant factors for determining the outcome of infection. HCV adapts to host immune responses by inducing mutations in its genome at specific sites that are important for HLA processing/presentation. Moreover, HCV also adapts to resist potential drugs that are used to restrict its replication, such as direct-acting antivirals (DAAs). Although DAAs have significantly reduced disease burden, resistance to these drugs is still a challenge for the treatment of HCV infection. Recently, drug resistance mutations (DRMs) observed in HCV proteins (NS3/4A, NS5A and NS5B) have heightened concern that the emergence of drug resistance may compromise the effectiveness of DAAs. Therefore, the NS3/4A, NS5A and NS5B drug resistance variations were investigated in this study, and their prevalence was examined in a large number of protein sequences from all HCV genotypes. Furthermore, potential CD4+ and CD8+ T cell epitopes were predicted and their overlap with genetic variations was explored. The findings revealed that many reported DRMs within NS3/4A, NS5A and NS5B are not drug-induced; rather, they are already present in HCV strains, as they were also detected in HCV-naïve patients. This study highlights several hot spots in which HLA and drug selective pressure overlap. Interestingly, these overlapping mutations were frequently observed among many HCV genotypes. This study implicates that knowledge of the host HLA type and HCV subtype/genotype can provide important information in defining personalized therapy.

Pangenome and immuno-proteomics analysis of Acinetobacter baumannii strains revealed the core peptide vaccine targets.

BACKGROUND: Acinetobacter baumannii has emerged as a significant nosocomial pathogen during the last few years, exhibiting resistance to almost all major classes of antibiotics. Alternative treatment options such as vaccines tend to be most promising and cost effective approaches against this resistant pathogen. In the current study, we have explored the pan-genome of A. baumannii followed by immune-proteomics and reverse vaccinology approaches to identify potential core vaccine targets. RESULTS: The pan-genome of all available A. baumannii strains (30 complete genomes) is estimated to contain 7,606 gene families and the core genome consists of 2,445 gene families (~32 % of the pan-genome). Phylogenetic tree, comparative genomic and proteomic analysis revealed both intra- and inter genomic similarities and evolutionary relationships. Among the conserved core genome, thirteen proteins, including P pilus assembly protein, pili assembly chaperone, AdeK, PonA, OmpA, general secretion pathway protein D, FhuE receptor, Type VI secretion system OmpA/MotB, TonB dependent siderophore receptor, general secretion pathway protein D, outer membrane protein, peptidoglycan associated lipoprotein and peptidyl-prolyl cis-trans isomerase are identified as highly antigenic. Epitope mapping of the target proteins revealed the presence of antigenic surface exposed 9-mer T-cell epitopes. Protein-protein interaction and functional annotation have shown their involvement in significant biological and molecular processes. The pipeline is validated by predicting already known immunogenic targets against Gram negative pathogen Helicobacter pylori as a positive control. CONCLUSION: The study, based upon combinatorial approach of pan-genomics, core genomics, proteomics and reverse vaccinology led us to find out potential vaccine candidates against A. baumannii. The comprehensive analysis of all the completely sequenced genomes revealed thirteen putative antigens which could elicit substantial immune response. The integration of computational vaccinology strategies would facilitate in tackling the rapid dissemination of resistant A.baumannii strains. The scarcity of effective antibiotics and the global expansion of sequencing data making this approach desirable in the development of effective vaccines against A. baumannii and other bacterial pathogens.

Identification of Circulating Biomarker Candidates for Hepatocellular Carcinoma (HCC): An Integrated Prioritization Approach.

Hepatocellular carcinoma (HCC) is the world's third most widespread cancer. Currently available circulating biomarkers for this silently progressing malignancy are not sufficiently specific and sensitive to meet all clinical needs. There is an imminent and pressing need for the identification of novel circulating biomarkers to increase disease-free survival rate. In order to facilitate the selection of the most promising circulating protein biomarkers, we attempted to define an objective method likely to have a significant impact on the analysis of vast data generated from cutting-edge technologies. Current study exploits data available in seven publicly accessible gene and protein databases, unveiling 731 liver-specific proteins through initial enrichment analysis. Verification of expression profiles followed by integration of proteomic datasets, enriched for the cancer secretome, filtered out 20 proteins including 6 previously characterized circulating HCC biomarkers. Finally, interactome analysis of these proteins with midkine (MDK), dickkopf-1 (DKK-1), current standard HCC biomarker alpha-fetoprotein (AFP), its interacting partners in conjunction with HCC-specific circulating and liver deregulated miRNAs target filtration highlighted seven novel statistically significant putative biomarkers including complement component 8, alpha (C8A), mannose binding lectin (MBL2), antithrombin III (SERPINC1), 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1), alcohol dehydrogenase 6 (ADH6), beta-ureidopropionase (UPB1) and cytochrome P450, family 2, subfamily A, polypeptide 6 (CYP2A6). Our proposed methodology provides a swift assortment process for biomarker prioritization that eventually reduces the economic burden of experimental evaluation. Further dedicated validation studies of potential putative biomarkers on HCC patient blood samples are warranted. We hope that the use of such integrative secretome, interactome and miRNAs target filtration approach will accelerate the selection of high-priority biomarkers for other diseases as well, that are more amenable to downstream clinical validation experiments.

Modeling and analysis of innate immune responses induced by the host cells against hepatitis C virus infection.

An in-depth understanding of complex systems such as hepatitis C virus (HCV) infection and host immunomodulatory response is an open challenge for biologists. In order to understand the mechanisms involved in immune evasion by HCV, we present a simplified formalization of the highly dynamic system consisting of HCV, its replication cycle and host immune responses at the cellular level using hybrid Petri net (HPN). The approach followed in this study comprises of step wise simulation, model validation and analysis of host immune response. This study was performed with an objective of making correlations among viral RNA levels, interferon (IFN) production and interferon stimulated genes (ISGs) induction. The results correlate with the biological data verifying that the model is very useful in predicting the dynamic behavior of the signaling proteins in response to a stimulus. This study implicates that HCV infection is dependent upon several key factors of the host immune response. The effect of host proteins on limiting viral infection is effectively overruled by the viral pathogen. This study also analyzes activity levels of RNase L, miR-122, IFN, ISGs and PKR induction and inhibition of TLR3/RIG1 mediated pathways in response to targeted manipulation in the presence of HCV. The results are in complete agreement at the time of writing with the published expression studies and western blot experiments. Our model also provides some biological insights regarding the role of PKR in the acute infection of HCV. It might help to explain why many patients fail to clear acute HCV infection while others, with low ISG basal levels, clear HCV spontaneously. The described methodology can easily be reproduced, which suitably supports the study of other viral infections in a formal, automated and expressive manner. The Petri net-based modeling approach applied here may provide valuable insights for study design and analyses to evaluate other disease associated integrated pathways in biological systems.

Identification of putative vaccine candidates against Helicobacter pylori exploiting exoproteome and secretome: a reverse vaccinology based approach.

Helicobacter pylori (H. pylori) is an important pathogen associated with diverse gastric disorders ranging from peptic ulcer to malignancy. It has also been recognized by the World Health Organization (WHO) as class I carcinogen. Conventional treatment regimens for H. pylori seem to be ineffective, possibly due to antibiotic resistance mechanisms acquired by the pathogen. In this study we have successfully employed a reverse vaccinology approach to predict the potential vaccine candidates against H. pylori. The predicted potential vaccine candidates include vacA, babA, sabA, fecA and omp16. Host-pathogen interactions analysis elaborated their direct or indirect role in the specific signaling pathways including epithelial cell polarity, metabolism, secretion system and transport. Furthermore, surface-exposed antigenic epitopes were predicted and analyzed for conservation among 39 complete genomes of H. pylori (Genbank) for all the candidate proteins. These epitopes may serve as a base for the development of broad spectrum peptide or multi-component vaccines against H. pylori. We also believe that the proposed pipeline can be extended to other pathogens and for the identification of novel candidates for the development of effective vaccines.

In-silico analysis of claudin-5 reveals novel putative sites for post-translational modifications: Insights into potential molecular determinants of blood-brain barrier breach during HIV-1 infiltration.

The blood-brain barrier (BBB) poses a huge challenge and is a serious issue in deciphering the pathophysiology of central nervous system disorders. Endothelial tight junctions play an essential role in maintaining the integrity of the BBB. Post-translational modifications (PTMs) in endothelial tight junction proteins are known to cause deleterious functional impairment and possible disruptions in BBB integrity. PTMs in tight junction proteins play an important role in human immunodeficiency virus type 1 (HIV-1) entry through the BBB. Human claudin-5 is one of the highly expressed brain endothelial tight junction protein and various PTMs in claudin-5 are expected to aid HIV-1 in crossing the BBB. A precise characterization of PTMs in claudin-5 is important for understanding its role in HIV-1 brain infiltration. In this study, we have examined post-translational crosstalk between phosphorylation, O-glycosylation, palmitoylation and methylation sites in claudin-5, which could alter claudin-5's ability to maintain BBB integrity. To the best of our knowledge, this is the first report on claudin-5 protein that suggests a novel interplay between potential PTM sites. PTMs of predicted residues in claudin-5, suggested in this study, can serve as compelling targets for potential therapeutic agents against HIV-1 induced neuropathogenesis. Further site-specific experimental studies in this aspect are highly recommended.