Targeted drug delivery to the thrombus by fusing streptokinase with a fibrin-binding peptide (CREKA): an in silico study.

Aim: Streptokinase has poor selectivity and provokes the immune response. In this study, we used in silico studies to design a fusion protein to achieve targeted delivery to the thrombus. Materials & methods: Streptokinase was analyzed computationally for mapping. The fusion protein modeling and quality assessment were carried out on several servers. The enzymatic activity and the stability of the fusion protein and its complex with plasminogen were assessed through molecular docking analysis and molecular dynamics simulation respectively. Results: Physicochemical properties analysis, protein quality assessments, protein-protein docking and molecular dynamics simulations predicted that the designed fusion protein is functionally active. Conclusion: Our results showed that this fusion protein might be a prospective candidate as a novel thrombolytic agent with better selectivity.

[Box: see text].

Neurobiochemical characteristics of arginine-rich peptides explain their potential therapeutic efficacy in neurodegenerative diseases.

Neurodegenerative diseases, including Alzheimer̕ s disease (AD), Parkinson̕ s disease (PD), Huntington̕ s disease (HD), and Amyotrophic Lateral Sclerosis (ALS) require special attention to find new potential treatment methods. This review aims to summarize the current knowledge of the relationship between the biochemical properties of arginine-rich peptides (ARPs) and their neuroprotective effects to deal with the harmful effects of risk factors. It seems that ARPs have portrayed a promising and fantastic landscape for treating neurodegeneration-associated disorders. With multimodal mechanisms of action, ARPs play various unprecedented roles, including as the novel delivery platforms for entering the central nervous system (CNS), the potent antagonists for calcium influx, the invader molecules for targeting mitochondria, and the protein stabilizers. Interestingly, these peptides inhibit the proteolytic enzymes and block protein aggregation to induce pro-survival signaling pathways. ARPs also serve as the scavengers of toxic molecules and the reducers of oxidative stress agents. They also have anti-inflammatory, antimicrobial, and anti-cancer properties. Moreover, by providing an efficient nucleic acid delivery system, ARPs can play an essential role in developing various fields, including gene vaccines, gene therapy, gene editing, and imaging. ARP agents and ARP/cargo therapeutics can be raised as an emergent class of neurotherapeutics for neurodegeneration. Part of the aim of this review is to present recent advances in treating neurodegenerative diseases using ARPs as an emerging and powerful therapeutic tool. The applications and progress of ARPs-based nucleic acid delivery systems have also been discussed to highlight their usefulness as a broad-acting class of drugs.

Finding and engineering the newly found bacterial superoxide dismutase enzyme to increase its thermostability and decrease the immunogenicity: a computational and experimental research.

Superoxide dismutase (SOD) is one of the most important antioxidant enzymes that can reduce oxidative stress in the cell environment. Nowadays, bacterial sources of enzyme are commercially applicable in the cosmetics and pharmaceutical industries, but the allergenic effect of proteins from non-human sources has been mentioned as disadvantage of these kinds of enzymes. In this study, to find the suitable bacterial SOD candidate for decreasing immunogenicity, the sequences of five thermophilic bacteria were selected as reference species. Then, linear and conformational B-cell epitopes of the SOD were analyzed by different servers. The stability and immunogenicity of mutant positions were also evaluated. The mutant gene was inserted into the pET-23a expression vector and transformed into E. Coli BL21 (DE3) for expression of the recombinant enzyme. Afterward, the expression of the mutant enzyme was evaluated by SDS-PAGE analysis and the recombinant enzyme activity was assessed. Anoxybacillus gonensis was selected as a reasonable SOD source according to BLAST search, physicochemical properties analysis, and prediction of allergenic features. Regarding our results, five residues including E84, E142, K144, G147, and M148 were predicted as candidates for mutagenesis. Finally, the K144A was chosen as the final modification due to the increase in the stability of the enzyme and decreased immunogenicity of the enzyme as well. The enzyme activity was 240 U/ml at room temperature. Alternation in K144 to alanine caused increased stability of the enzyme. In silico studies confirmed non-antigenic protein after mutation.

In silico design and evaluation of a novel mRNA vaccine against BK virus: a reverse vaccinology approach.

Human polyomavirus type 1, or BK virus (BKV), is a ubiquitous pathogen belonging to the polyomaviridae family mostly known for causing BKV-associated nephropathy (BKVN) and allograft rejection in kidney transplant recipients (KTRs) following the immunosuppression regimens recommended in these patients. Reduction of the immunosuppression level and anti-viral agents are the usual approaches for BKV clearance, which have not met a desired outcome yet. There are also debating matters such as the effect of this pathogen on emerging various comorbidities and the related malignancies in the human population. In this study, a reverse vaccinology approach was implemented to design a mRNA vaccine against BKV by identifying the most antigenic proteins of this pathogen. Potential immunogenic T and B lymphocyte epitopes were predicted through various immunoinformatic tools. The final epitopes were selected according to antigenicity, toxicity, allergenicity, and cytokine inducibility scores. According to the obtained results, the designed vaccine was antigenic, neutral at the physiological pH, non-toxic, and non-allergenic with a world population coverage of 93.77%. Since the mRNA codon optimization ensures the efficient expression of the vaccine in a host cell, evaluation of different parameters showed our designed mRNA vaccine has a stable structure. Moreover, it had strong interactions with toll-like receptor 4 (TLR4) according to the molecular dynamic simulation studies. The in silico immune simulation analyses revealed an overall increase in the immune responses following repeated exposure to the designed vaccine. Based on our findings, the vaccine candidate is ready to be tested as a promising novel mRNA therapeutic vaccine against BKV.

Design of a multi-epitope protein vaccine against herpes simplex virus, human papillomavirus and Chlamydia trachomatis as the main causes of sexually transmitted diseases.

Sexually transmitted diseases (STDs) have a profound effect on reproductivity and sexual health worldwide. According to world health organization (WHO) 375 million new case of STD, including chlamydia trachomatis (chlamydia), Neisseria gonorrhoeae, HSV, HPV has been reported in 2016. More than 30 diverse pathogenesis have identified to be transmitted through sexual intercourse. Of these, viral infections (hepatitis B, herpes simplex virus (HSV or herpes), HIV, and human papillomavirus (HPV) are incurable. However, symptoms caused by the incurable viral infections can be alleviated through treatment. Antimicrobial resistance (AMR) of sexually transmitted infections (STIs) to antibiotics has increased recent years, in this regard, vaccination is proposed as an important strategy for prevention or treatment of STDs. Vaccine against HPV 16 and 18 suggests a new approach for controlling STDs but until now, there is no prophylactic or therapeutic vaccine have been approved for HSV-2 and Chlamydia trachomatis (CT); in this reason, developing an efficient vaccine is inevitable. Recently, different combinatorial forms of subunit vaccines against two or three type of bacteria have been designed. In this study, to design a combinatorial vaccine against HSV, CT, and HPV, the E7 and L2 from HPV, glycoprotein D from HSV-2 and ompA from CT were selected as final antigens. Afterward, the immunodominant helper T lymphocytes (HTLs) and cytolytic T lymphocytes (CTLs) epitopes were chosen from aforesaid antigens. P30 (tetanus toxoid epitope) as universal T-helper were also added to the vaccine. Moreover, flagellin D1/D0 as TLR5 agonist and the RS09 as a TLR4 ligand were incorporated to N and C-terminals of peptide vaccine, respectively. Finally, all selected parts were fused together by appropriate linkers to enhance vaccine efficiency. The physicochemical, structural, and immunological properties of the designed vaccine protein were assessed. To achieve the best 3D model of the protein vaccine, modeling, refinement, and validation of modeled structures were also done. Docking evaluation demonstrated suitable interaction between the vaccine and TLR5. Moreover, molecular dynamics (MD) studies showed an appropriate and stable structure of protein and TLR5. Based on immunoinformatic analysis, our vaccine candidate could potentially incite humoral and cellular immunities, which are critical for protection against HPV, HSV-2, and chlamydia trachomatis. It should be noted that, experimental studies are needed to confirm the efficacy of the designed vaccine.

Targeting Common Signaling Pathways for the Treatment of Stroke and Alzheimer's: a Comprehensive Review.

Neurodegenerative diseases such as stroke and Alzheimer's disease (AD) are two inter-related disorders that affect the neurons in the brain and central nervous system. Alzheimer's is a disease by undefined origin and causes. Stroke and its most common type, ischemic stroke (IS), occurs due to the blockade of cerebral blood vessels. As an important feature, both of disorders are associated with irreversible damages to the brain and nervous system. In this regard, finding common signaling pathways and the same molecular origin between these two diseases may be a promising way for their solution. On the basis of literature appraisal, the most common signaling cascades implicated in the pathogenesis of AD and stroke including notch, autophagy, inflammatory, and insulin signaling pathways were reviewed. Furthermore, current therapeutic strategies including natural and synthetic pharmaceuticals aiming modulation of respective signaling factors were scrutinized to ameliorate neural deficits in AD and stroke. Taken together, digging deeper in the common connections and signal targeting can be greatly helpful in understanding and unified treating of these disorders.

An Overview of Aptamer: The Prominent Applications and Different Computational Tools for its Design.

BACKGROUND: Aptamers are short single-stranded oligonucleotides; due to their 3D structure, they can specifically bind to various targets. They can target several molecules ranging from metal ions, organic components to proteins, and large cells. METHODS: According to the high affinity of aptamers, they can be used for diagnosis, therapeutic, vaccine development, and gene silencing applications. The conventional method for aptamer selection is known as the Systematic Evolution of Ligands by Exponential (SELEX). However, despite the efficiency of SELEX as a screening procedure, it is beneficial to develop more rational procedures for aptamer selection. RESULTS & DISCUSSION: Herein, in silico approaches can play an effective role given their potential in representing an efficient, cost-effective, parallelizable, and rapid strategy. In recent years, several attempts have been applied to develop algorithms and software for the rational selection of aptamers. However, there is still a need for more efforts to achieve the most efficient techniques in this area. CONCLUSION: In this review, we aim to overview different computational approaches that are used for aptamer selection.

Small extracellular vesicles (sEVs): discovery, functions, applications, detection methods and various engineered forms.

INTRODUCTION: Extracellular vesicles (EVs) are cell-created delivery systems of proteins, lipids, or nucleic acids, and means of extracellular communication. Though sEVs were initially considered to be the waste disposal mechanism, today they are at the forefront of research with different biological and pathological functions. Such EVs play a key role in the immunoregulation, CNS development, nervous system physiology, mammary gland development, induction of immunosuppression in pregnancy, the developmental signaling pathways, regeneration of different tissues, inflammation, angiogenesis, coagulation, apoptosis, stem cell differentiation, and extracellular matrix turnover. AREAS COVERED: SEVs contribute to the pathogenesis of different cancers and the progression of various neurodegenerative diseases, infections, as well as metabolic and cardiovascular diseases. Expert Opinion: There is no exact classification for EVs; however, according to size, density, morphological features, content, and biogenesis, they can be categorized into three major classes: microvesicles (ectosomes or microparticles), apoptotic bodies, and sEVs. SEVs, as an important class of EVs, have a crucial role in distinct biological functions. Moreover, shedding light on different structural and molecular aspects of sEV has led to their application in various therapeutic, diagnostic, and drug delivery fields. In this review, we have endeavored to elaborate on different aspects of EVs, especially sEVs.

Overexpression of Hes1 is involved in sensitization of K562 cells to Imatinib.

Tyrosine kinase inhibitor (TKI)-based therapy has created promising results among much chronic myeloid leukemia (CML) patients. Imatinib as a relatively specific inhibitor of Bcr-Abl is at present one of the undisputed therapeutic agent for newlydiagnosed patients with CML. However, the occurrence of imatinib-resistance enlightens the urgent need to identify other therapeutic agents against CML. Juglone (5-hydroxy-2-methyl-1, 4-naphthoquinone) exerts cytotoxic effects against various human cancer cell lines. However, the mechanisms through which Juglone induces anticancer effects in CML especially in comparison with imatinib treatment remain unknown. Our results revealed that Juglone-inhibited K562 cells growth through inducing apoptosis. Based on our Western blot analyses, Juglone significantly reduced p-Akt levels and increased the expression level of Forkhead box O1 (FoxO1) and FoxO3a proteins. Moreover, hairy/enhancer of split-1 (Hes1) protein, overexpressed under the influence of Juglone, is apparently involved in Juglone-induced apoptosis among K562 cells. Conversely, treatment with imatinib attenuated Hes1 protein expression. Considering the different functional mechanism of Juglone compared with imatinib, it seems that Juglone treatment could be a useful alternative strategy for the treatment of patients with imatinib-resistance.

Bcl6 gene-silencing facilitates PMA-induced megakaryocyte differentiation in K562 cells.

Targeted therapy via imatinib appears to be a promising approach for chronic myeloid leukemia (CML) therapy. However, refractory and resistance to imatinib therapy has encouraged many investigators to get involved in development of new therapeutic agents such as Phorbol 12-myrestrat 13-acetate (PMA) for patients with CML. In that line, we attempted to investigate the chemosensitizing effect of PMA on the imatinib-resistant cells. Based on our western blot analyses, resistant K562 cells (K562R) showed high levels of FoxO3a and Bcl6 expressions which were not modulated by imatinib treatment. However, upon PMA treatment, the levels of both FoxO3a and Bcl6 were up-regulated among both the sensitive and the resistant cells and this treatment was associated with initiation of megakaryocytic differentiation of the cells. SiRNA-silencing of FoxO3a led to augmentation of megakaryocytic differentiation of the cells. Similarly, siRNA gene silencing of Bcl6 enhanced the differentiation and induced cell apoptosis among both types of cells. Regarding these results, it might be concluded that Bcl6 knockdown combined with PMA therapy could present a new therapeutical strategy for refractory CML patients to imatinib.

Engineering of a disulfide loop instead of a Zn binding loop restores the anti-proliferative, anti-angiogenic and anti-tumor activities of the N-terminal fragment of endostatin: Mechanistic and therapeutic insights.

Although considerable effort has been devoted to understanding the molecular mechanism of endostatin's anti-cancer activity, the role of its Zn bound N-terminal loop has not been completely clarified. To investigate whether Zn binding or the N-terminal loop is involved in the anti-cancer properties of endostatin, we compared the structure and biological activity of a native Zn binding endostatin peptide (ES-Zn) with three variants: a Zn free variant (ES), a variant containing both a Zn binding site and a disulfide bond (ES-SSZn), and a variant including a disulfide loop but incapable of Zn binding (ES-SS). Spectroscopic studies indicated that ES-Zn and ES-SS consist of random coil and ß structures, whereas ES-SSZn and ES fold into random coils. Theoretical analysis proposed that ES-Zn and ES-SS have a similar binding site to αVß3 integrin. The anti-proliferative activity of endostatin was retained by all peptides except ES, and the in vitro anti-angiogenic property was preserved in ES-Zn and ES-SS. Remarkably, breast tumor growth and CD31 activity were inhibited more effectively by ES-SS than by ES-Zn. Therefore, a correlation exists between the N-terminal loop and anti-cancer properties of endostatin fragment and a disulfide loop may be more promising than a Zn binding loop for inhibiting tumor growth.