Saeed Khalili

An in silico DNA vaccine against Listeria monocytogenes

Listeria monocytogenes causes listeriosis with mortality rate >20%. Listeriolysin-O (LLO), a pore-forming hemolysin, belongs to the family of cholesterol-dependent toxins (CDTX) and plays roles in the pathogenicity. In this study bioinformatic analyses were carried out on LLO sequence as a major immunodominant listerial antigen toward designing a DNA vaccine stimulating cytotoxic T-lymphocytes (CTLs). Mouse and human constructs were designed based on predicted T cell epitopes and MHC class I binders, which were then tandemly fused together. LLO-derived construct codons and a variety of critical gene expression efficiency parameters were optimized. Post-translational modifications such as glycosylation, phosphorylation were analysed. The constructs corresponded to LLO sequences of L. monocytogenes in BLAST search. Neither human nor mouse construct was allergen. Secretory pathway was location of the human construct that enhances immune induction and contribute to the efficacy of the vaccine candidate. mRNAs from optimized DNA sequences of both human and mouse constructs are more stable than the native and are suitable for initiation of translation. The constructs contain several sites for phosphorylation that could improve its degradation and subsequent entry into the MHC class I pathway. Addition of GPI anchor, myristoylation and ubiquitin signals or proline (P), glutamic acid (E), serine (S), threonine (T) (PEST)-like motifs at the N-terminal of constructs increase efficacy of the DNA vaccine. Close physical contact between the favorable immunogen and the suitable CpG oligodeoxynucleotides (CpG ODN) promotes immune response. Vectors for checking the expression of constructs in mammalian cells and for harboring the foreign genes as DNA vaccine are suggested.

Precise detection of L. monocytogenes hitting its highly conserved region possessing several specific antibody binding sites.

Listeria monocytogenes, a facultative intracellular fast-growing Gram-positive food-borne pathogen, can infect immunocompromised individuals leading to meningitis, meningoencephalitis and septicaemias. From the pool of virulence factors of the organism, ActA, a membrane protein, has a critical role in the life cycle of L. monocytogenes. High mortality rate of listeriosis necessitates a sensitive and rapid diagnostic test for precise identification of L. monocytogenes. We used bioinformatic tools to locate a specific conserved region of ActA for designing and developing an antibody-antigen based diagnostic test for the detection of L. monocytogenes. A number of databases were looked for ActA related sequences. Sequences were analyzed with several online software to find an appropriate region for our purpose. ActA protein was found specific to Listeria species with no homologs in other organisms. We finally introduced a highly conserved region within ActA sequence that possess several antibody binding sites specific to L. monocytogenes. This protein sequence can serve as an antigen for designing a relatively cheap, sensitive, and specific diagnostic test for detection of L. monocytogenes.

Key role of Dkk3 protein in inhibition of cancer cell proliferation: An in silico identification.

Dkk3 is a member of Dkk family proteins, regulating Wnt signaling. Dkk3 plays different roles in human and mouse tumors. Dkk3 predominantly act as a tumor suppressor, however several reports revealed that Dkk3 could accelerate cancer cell proliferation. Herein, we aimed at launching an in silico study to determine Dkk3 structure and its interactions with Kremen and LRP as Wnt signaling receptors as well as EGF receptor. Using various softwares a model was built for Dkk3 molecule. Different protein modeling approaches along with model refinement processes were employed to arrive at the final model. To achieve the final complex of Dkk3 with Kremen, LRP and EGFR molecules protein-protein docking servers were employed. Model assessment softwares indicated the high quality of the finally refined Dkk3 3D structure, indicating the accuracy of modeling and refinement process. Our results revealed that Dkk3 is capable of interacting with Kremen, LRP and EGFR with comparable binding energies. Dkk3 efficiently interacts with LRP, Kremen and EGF receptor and may be a promising protein in cancer therapy by blocking Wnt and EGFR downstream signaling.

Computational vaccinology and epitope vaccine design by immunoinformatics.

Human immune system includes variety of different cells and molecules correlating with other body systems. These instances complicate the analysis of the system; particularly in postgenomic era by introducing more amount of data, the complexity is increased and necessity of using computational approaches to process and interpret them is more tangible.Immunoinformatics as a subset of bioinformatics is a new approach with variety of tools and databases that facilitate analysis of enormous amount of immunologic data obtained from experimental researches. In addition to directing the insight regarding experiment selections, it helps new thesis design which was not feasible with conventional methods due to the complexity of data. Considering this features immunoinformatics appears to be one of the fields that accelerate the immunological research progression.In this study we discuss advances in genomics and vaccine design and their relevance to the development of effective vaccines furthermore several division of this field and available tools in each item are introduced.

Structural pierce into molecular mechanism underlying Clostridium perfringens Epsilon toxin function

ABSTRACT Epsilon toxin of the Clostridium perfringens garnered a lot of attention due to its potential for toxicity in humans, extreme potency for cytotoxicity in mice and lack of any approved therapeutics prescribed for human. However, the intricacies of the Epsilon toxin action mechanism are yet to be understood. In this regard, various in silico tools have been exploited to model and refine the 3D structure of the toxin and its two receptors. The receptor proteins were embedded into designed lipid membranes within an aqueous and ionized environment. Thereafter, the modeled structures subjected to series of consecutive molecular dynamics runs to achieve the most natural like coordination for each model. Ultimately, protein‐protein interaction analyses were performed to understand the probable action mechanism. The obtained results successfully confirmed the accuracy of employed methods to achieve high quality models for the toxin and its receptors within their lipid bilayers. Molecular dynamics analyses lead the structures to a more native like coordination. Moreover, the results of previous empirical studies were confirmed, while new insights for action mechanisms including the detailed roles of Hepatitis A virus cellular receptor 1 (HAVCR1) and Myelin and lymphocyte protein (MAL) proteins were achieved. In light of previous and our observations, we suggested novel models which elucidated the existing interplay between potential players of Epsilon toxin action mechanism with detailed structural evidences. These models would pave the way to have more robust understanding of the Epsilon toxin biology, more precise vaccine construction and more successful drug (inhibitor) design. HIGHLIGHTSMAL and HAVCR1 are the key receptors of the E‐toxin.E‐toxin could convey its function through 3 possible models.MAL seems to be the key receptor which influences the fate of the toxin action mechanism.MAL could guide the toxin through a prepore stage or even independent from a pore.

In silico analyses of Wilms׳ tumor protein to designing a novel multi-epitope DNA vaccine against cancer

Predefined and pre-weighted objective criteria and essential role of Wilms׳ tumor wild type gene (WT1) for maintaining transformed features of cancer cells confirm the high potency of WT1 as a valuable cancer antigen. The antigen was at the top of the ranking among 75 representative cancer antigens. In the present study, an in silico approach was launched to characterized novel CTL epitopes and design a novel multi-epitope DNA vaccine to elicit a desirable immune response against cancers over expressing WT1. Forty-four novel epitopes were described. A multi-epitope construct was designed based on predicted epitopes which is 310 residues in length. The vaccine candidate designed here displays acceptable population coverage (>65%) in different ethnicities as well as high probability of eliciting WT1 antibodies which both are pertinent goals in the context of appropriate multi-epitope vaccines. Various in silico analyses indicate that final vaccine is a qualified immunotherapy candidate capable of eliciting both CD4+ and CD8+ T cell responses.

ILP-2 modeling and virtual screening of an FDA-approved library: a possible anticancer therapy

BACKGROUND/AIM The members of the inhibitors of apoptosis protein (IAP) family inhibit diverse components of the caspase signaling pathway, notably caspase 3, 7, and 9. ILP-2 (BIRC-8) is the most recently identified member of the IAPs, mainly interacting with caspase 9. This interaction would eventually lead to death resistance in the case of cancerous cells. Therefore, structural modeling of ILP-2 and finding applicable inhibitors of its interaction with caspase 9 are a compelling challenge. MATERIALS AND METHODS Three main protein modeling approaches along with various model refinement measures were harnessed to achieve a reliable 3D model, using state-of-the-art software. Thereafter, the selected model was employed to perform virtual screening of an FDA approved library. RESULTS A model built by a combinatorial approach (homology and ab initio approaches) was chosen as the best model. Model refinement processes successfully bolstered the model quality. Virtual screening of the compound library introduced several high affinity inhibitor candidates that interact with functional residues of ILP2. CONCLUSION Given the 3D structure of the ILP2 molecule, we found promising inhibitory molecules. In addition to high affinity towards the ILP2 molecule, these molecules interact with residues that play pivotal rules in ILP2-caspase interaction. These molecules would inhibit ILP2-caspase interaction and consequently would lead to reactivated cell apoptosis through the caspases pathway.

3D structure of DKK1 indicates its involvement in both canonical and non-canonical Wnt pathways

Dikkoppf-1 (DKK1) is an antagonist of the canonical Wnt signaling pathway. The importance of DKK1 as a diagnostic and therapeutic agent in a wide range of diseases along with its significance in a variety of biological processes accentuate the necessity to decipher its 3D structure that would pave the way towards the development of relevant selective inhibitors. A DKK1 structure model predicted by the Robetta server with structural refinements including a 10 ns molecular dynamics run was subjected to functional and docking analyses. We hypothesize that the N-terminal region of the DKK1 molecule could be functionally important for both canonical and noncanonical Wnt pathways. Moreover, it seems that DKK1 could be involved in interactions with the Frizzled receptors, leading to the activation of the Planar Cell Polarity (PCP) pathway (activation of Jun N-terminal kinase (JNK) Pathway) and Wnt/Ca2+ pathway (activation of CamKII).

Lung cancer and miRNAs: a possible remedy for anti-metastatic, therapeutic and diagnostic applications

ABSTRACT Introduction: Lung cancer still accounts for the leading cause of cancer-related deaths worldwide and despite the emerging advances in diagnostic and therapeutic techniques it remains to be a serious global public health concern. Micro-ribonucleic acids (microRNAs) are responsible for invasion and metastasis of various tumors including lung cancer which underscores the necessity of understanding their functions. Areas covered: Herein, we aim to summarize the recent advances made in our understanding of the miRNAs with special reference to lung cancer. Moreover, the role of miRNAs in crucial cellular processes will be elucidated. Various applications of the miRNAs would be explained and different kinds of them would be discussed to delineate their significance in lung cancer biology, therapy and diagnosis. Expert commentary: the miRNA study in the field of respiratory disease and specially lung cancer has emerged lately. Given the several miRNAs, which are in the clinical trials, this field is passing through its maturation phase which ultimately could rise to a robust tool for lung cancer therapy, diagnosis and prevention.

Structural analyses of the interactions between the thyme active ingredients and human serum albumin

Abstract Objective: Therapeutic effects of thyme and the mechanism underlying the function of its active ingredients are the areas of active investigation. In this regard, understanding the potential interactions between the active ingredients of the thyme leaf and the serum albumin would bring about new insight on the bio-distribution, circulatory half-life and consequently their pharmacodynamics and pharmacokinetic properties. Methods: The 3D structures of carvacrol, linalool, p-cymene and thymol molecules as the thyme active ingredients and the 3D structure of albumin were harnessed from the structural databases. Then, these structures were prepared for molecular docking analyses by Autodock vina software. Ultimately, the binding energies between docked albumin and thyme active ingredients were calculated and their interactions were predicted. Results: Our results indicated that all active ingredients of thyme can interact with albumin molecule at drug binding site 3 and fatty acid binding site 5. The structural properties of the ingredients effect their interaction sites and binding energies. Conclusion: It could be concluded that albumin, as the most abundant protein of the serum, could act as the bio-distributor of thyme active ingredients. This property would be of great significance to exert the desired therapeutic effects.