In Silico Design of CT26 Polytope and its Surface Display by ClearColi™-Derived Outer Membrane Vesicles as a Cancer Vaccine Candidate Against Colon Carcinoma.

Simultaneous targeting of several mutations can be useful in colorectal cancer (CRC) due to its heterogeneity and presence of somatic mutations. As CT26 mutations and expression profiles resemble those of human CRC, we focused on designing a polyepitope vaccine based on CT26 neoepitopes. Due to its low immunogenicity, outer membrane vesicles (rOMV) as an antigen delivery system and adjuvant was applied. Herein, based on previous experimental and our in silico studies four CT26 neoepitopes with the ability to bind MHC-I and MHC-II, TCR, and induce IFN-α production were selected. To increase their immunogenicity, the gp70 and PADRE epitopes were added. The order of the neoepitopes was determined through 3D structure analysis using ProSA, Verify 3D, ERRAT, and Ramachandran servers. The stable peptide-protein docking between the selected epitopes and MHC alleles strengthen our prediction. The CT26 polytope vaccine sequence was fused to the C-terminal of cytolysin A (ClyA) anchor protein and rOMVs were isolated from endotoxin-free ClearColi™ strain. The results of the C-ImmSim server showed that the ClyA-CT26 polytope vaccine could induce T and B cells immunity.The ClyA-CT26 polytope was characterized as a soluble, stable, immunogen, and non-allergen vaccine and optimized for expression in ClearColi™ 24 h after induction with 1 mM IPTG at 25 °C. Western blot analysis confirmed the expression of ClyA-CT26 polytope by ClearColi™ and also on ClearColi™-derived rOMVs. In conclusion, we found that ClearColi™-derived rOMVs with CT26 polytope can deliver CRC neoantigens and induce antitumor immunity, but in vivo immunological studies are needed to confirm vaccine efficacy.

Design and expression of a chimeric recombinant antigen (SsIR-Ss1a) for the serodiagnosis of human strongyloidiasis: Evaluation of performance, sensitivity, and specificity.

BACKGROUND: The sensitivity of parasitological and molecular methods is unsatisfactory for the diagnosis of strongyloidiasis, and serological techniques are remaining as the most effective diagnostic approach. The present study aimed to design and produce a chimeric recombinant antigen from Strongyloides stercoralis immunoreactive antigen (SsIR) and Ss1a antigens, using immune-informatics approaches, and evaluated its diagnostic performance in an ELISA system for the diagnosis of human strongyloidiasis. METHODOLOGY/PRINCIPAL FINDINGS: The coding sequences for SsIR and Ss1a were selected from GenBank and were gene-optimized. Using bioinformatics analysis, the regions with the highest antigenicity that did not overlap with other parasite antigens were selected. The chimeric recombinant antigen SsIR- Ss1a, was constructed. The solubility and physicochemical properties of the designed construct were analyzed and its tertiary structures were built and evaluated. The construct was expressed into the pET-23a (+) expression vector and the optimized DNA sequences of SsIR-Ss1a (873 bp) were cloned into competent E. coli DH5α cells. Diagnostic performances of the produced recombinant antigen, along with a commercial kit were evaluated in an indirect ELISA system, using a panel of sera from strongyloidiasis patients and controls. The physicochemical and bioinformatics evaluations revealed that the designed chimeric construct is soluble, has a molecular with of 35 KDa, and is antigenic. Western blotting confirmed the immunoreactivity of the produced chimeric recombinant antigen with the sera of strongyloidiasis patients. The sensitivity and specificity of the indirect ELISA system, using the produced SsIR-Ss1a chimeric antigen, were found to be 93.94% (95% CI, 0.803 to 0.989) and 97.22% (95% CI, 0.921 to 0.992) respectively. CONCLUSIONS/SIGNIFICANCE: The preliminary findings of this study suggest that the produced SsIR-Ss1a chimeric antigen shows promise in the diagnosis of human strongyloidiasis. However, these results are based on a limited panel of samples, and further research with a larger sample size is necessary to confirm its accuracy. The construct has potential as an antigen in the ELISA system for the serological diagnosis of this neglected parasitic infection, but additional validation is required.

Designing a Secretory form of RTX-A as an Anticancer Toxin: An In Silico Approach.

BACKGROUND: Cancer is a leading cause of death and a significant public health issue worldwide. Standard treatment methods such as chemotherapy, radiotherapy, and surgery are only sometimes effective. Therefore, new therapeutic approaches are needed for cancer treatment. Sea anemone actinoporins are pore-forming toxins (PFTs) with membranolytic activities. RTX-A is a type of PFT that interacts with membrane phospholipids, resulting in pore formation. The synthesis of recombinant proteins in a secretory form has several advantages, including protein solubility and easy purification. In this study, we aimed to discover suitable signal peptides for producing RTX-A in Bacillus subtilis in a secretory form. METHODS: Signal peptides were selected from the Signal Peptide Web Server. The probability and secretion pathways of the selected signal peptides were evaluated using the SignalP server. ProtParam and Protein-sol were used to predict the physico-chemical properties and solubility. AlgPred was used to predict the allergenicity of RTX-A linked to suitable signal peptides. Non-allergenic, stable, and soluble signal peptides fused to proteins were chosen, and their secondary and tertiary structures were predicted using GOR IV and I-TASSER, respectively. The PROCHECK server performed the validation of 3D structures. RESULTS: According to bioinformatics analysis, the fusion forms of OSMY_ECOLI and MALE_ECOLI linked to RTX-A were identified as suitable signal peptides. The final proteins with signal peptides were stable, soluble, and non-allergenic for the human body. Moreover, they had appropriate secondary and tertiary structures. CONCLUSION: The signal above peptides appears ideal for rationalizing secretory and soluble RTX-A. Therefore, the signal peptides found in this study should be further investigated through experimental researches and patents.

Optimization of Spore Production in Bacillus coagulans Using Response Surface Methodology Approach.

Due to its spore-forming ability, Bacillus coagulans has advantages over the other non-spore-forming probiotics. Among them, survival and stability during food processing and storage, resistance to acid pH, and digestive enzymes are important. However, there are few studies on the quality and amount of sporulation in B. coagulans. This study investigated the spore densities and formation efficiency of B. coagulans. The optimal medium formulation consisted of yeast extract (1.00 g L-1), potassium acetate (20.00 g L-1), and MnSO4 (0.01 g L-1 and 0.03 g L-1). After reaching the optimal medium, a response surface regression equation was established based on the results of central composite design (CCD) experimental designs to optimize time, temperature, and pH parameters. The predicted results thus obtained were in good agreement (R2 = 95.19%) with the results obtained by performing experiments. Multiple regression analysis and analysis of variance (ANOVA) showed that pH is negative, and temperature and time dose are positive factors. The maximum spore cell densities by optimization plots have obtained 9.80 log at temperature 83.77 °C, pH 3.05, and time 111.19 h, considering that B. coagulans needs special environmental and cellular conditions to enter the sporulation stage. In this study, the composition of the culture medium and factors such as temperature, time, and pH were considered influencing factors in B. coagulans sporulation.

A versatile theranostic magnetic polydopamine iron oxide NIR laser-responsive nanosystem containing doxorubicin for chemo-photothermal therapy of melanoma.

Theranostics nanoparticles (NPs) have recently received much attention in cancer imaging and treatment. This study aimed to develop a multifunctional nanosystem for the targeted delivery of photothermal and chemotherapy agents. Fe3O4 NPs were modified with polydopamine, bovine serum albumin, and loaded with DOX via a thermal-cleavable Azo linker (Fe3O4@PDA@BSA-DOX). The size of Fe3O4@PDA@BSA NPs was approximately 98 nm under the desired conditions. Because of the ability of Fe3O4 and PDA to convert light into heat, the temperature of Fe3O4@PDA@BSA NPs increased to approximately 47 °C within 10 min when exposed to an 808 nm NIR laser with a power density of 1.5 W/cm2. The heat generated by the NIR laser leads to the breaking of AZO linker and drug release. In vivo and in vitro results demonstrated that prepared NPs under laser irradiation successfully eradicated tumor cells without any significant toxicity effect. Moreover, the Fe3O4@PDA@BSA NPs exhibited the potential to function as a contrasting agent. These NPs could accumulate in tumors with the help of an external magnet, resulting in a significant enhancement in the quality of magnetic resonance imaging (MRI). The prepared novel multifunctional NPs seem to be an efficient system for imaging and combination therapy in melanoma.

Peptide nanovaccine in melanoma immunotherapy.

Melanoma is an especially fatal neoplasm resistant to traditional treatment. The advancement of novel therapeutical approaches has gained attention in recent years by shedding light on the molecular mechanisms of melanoma tumorigenesis and their powerful interplay with the immune system. The presence of many mutations in melanoma cells results in the production of a varied array of antigens. These antigens can be recognized by the immune system, thereby enabling it to distinguish between tumors and healthy cells. In the context of peptide cancer vaccines, generally, they are designed based on tumor antigens that stimulate immunity through antigen-presenting cells (APCs). As naked peptides often have low potential in eliciting a desirable immune reaction, immunization with such compounds usually necessitates adjuvants and nanocarriers. Actually, nanoparticles (NPs) can provide a robust immune response to peptide-based melanoma vaccines. They improve the directing of peptide vaccines to APCs and induce the secretion of cytokines to get maximum immune response. This review provides an overview of the current knowledge of the utilization of nanotechnology in peptide vaccines emphasizing melanoma, as well as highlights the significance of physicochemical properties in determining the fate of these nanovaccines in vivo, including their drainage to lymph nodes, cellular uptake, and influence on immune responses.

Targeting Efficient Features of Urate Oxidase to Increase Its Solubility.

With the demand for mass production of protein drugs, solubility has become a serious issue. Extrinsic and intrinsic factors both affect this property. A homotetrameric cofactor-free urate oxidase (UOX) is not sufficiently soluble. To engineer UOX for optimum solubility, it is important to identify the most effective factor that influences solubility. The most effective feature to target for protein engineering was determined by measuring various solubility-related factors of UOX. A large library of homologous sequences was obtained from the databases. The data was reduced to six enzymes from different organisms. On the basis of various sequence- and structure-derived elements, the most and the least soluble enzymes were defined. To determine the best protein engineering target for modification, features of the most and least soluble enzymes were compared. Metabacillus fastidiosus UOX was the most soluble enzyme, while Agrobacterium globiformis UOX was the least soluble. According to the comparison-constant method, positive surface patches caused by arginine residue distribution are appropriate targets for modification. Two Arg to Ala mutations were introduced to the least soluble enzyme to test this hypothesis. These mutations significantly enhanced the mutant's solubility. While different algorithms produced conflicting results, it was difficult to determine which proteins were most and least soluble. Solubility prediction requires multiple algorithms based on these controversies. Protein surfaces should be investigated regionally rather than globally, and both sequence and structural data should be considered. Several other biotechnological products could be engineered using the data reduction and comparison-constant methods used in this study.

Graphene oxide as novel vaccine adjuvant.

To improve antigen immunogenicity and promote long-lasting immunity, vaccine formulations have been appropriately supplemented with adjuvants. Graphene has been found to enhance the presentation of antigens to CD8+ T cells, as well as stimulating innate immune responses and inflammatory factors. Its properties, such as large surface area, water stability, and high aspect ratio, make it a suitable candidate for delivering biological substances. Graphene-based nanomaterials have recently attracted significant attention as a new type of vaccine adjuvants due to their potential role in the activation of immune responses. Due to the limited functionality of some approved human adjuvants for use, the development of new all-purpose adjuvants is urgently required. Research on the immunological and biomedical use of graphene oxide (GO) indicates that these nanocarriers possess excellent physicochemical properties, acceptable biocompatibility, and a high capacity for drug loading. Graphene-based nanocarriers also could improve the function of some immune cells such as dendritic cells and macrophages through specific signaling pathways. However, GO injection can lead to significant oxidative stress and inflammation. Various surface functionalization protocols have been employed to reduce possible adverse effects of GO, such as aggregation of GO in biological liquids and induce cell death. Furthermore, these modifications enhance the properties of functionalized-GO's qualities, making it an excellent carrier and adjuvant. Shedding light on different physicochemical and structural properties of GO and its derivatives has led to their application in various therapeutic and drug delivery fields. In this review, we have endeavored to elaborate on different aspects of GO.

Identification of potential RapJ hits as sporulation pathway inducer candidates in Bacillus coagulans via structure-based virtual screening and molecular dynamics simulation studies.

BACKGROUND: The bacterium Bacillus coagulans has attracted interest because of its ability to produce spores and advantageous probiotic traits, such as facilitating food digestion in the intestine, managing some disorders, and controlling the symbiotic microbiota. Spore-forming probiotic bacteria are especially important in the probiotic industry compared to non-spore-forming bacteria due to their stability during production and high resistance to adverse factors such as stomach acid. When spore-forming bacteria are exposed to environmental stresses, they enter the sporulation pathway to survive. This pathway is activated by the final phosphorylation of the master regulator of spore response, Spo0A, and upon achieving the phosphorylation threshold. Spo0A is indirectly inhibited by some enzymes of the aspartate response regulator phosphatase (Rap) family, such as RapJ. RapJ is one of the most important Rap enzymes in the sporogenesis pathway, which is naturally inhibited by the pentapeptides. METHODS: This study used structure-based virtual screening and molecular dynamics (MD) simulation studies to find potential RapJ hits that could induce the sporulation pathway. The crystal structures of RapJ complexed with pentapeptide clearly elucidated their interactions with the enzyme active site. RESULTS: Based on the binding compartment, through molecular docking, MD simulation, hydrogen bonds, and binding-free energy calculations, a series of novel hits against RapJ named tandutinib, infigratinib, sitravatinib, linifanib, epertinib, surufatinib, and acarbose were identified. Among these compounds, acarbose obtained the highest score, especially in terms of the number of hydrogen bonds, which plays a major role in stabilizing RapJ-ligand complexes, and also according to the occupancy percentages of hydrogen bonds, its hydrogen bonds were more stable during the simulation time. Consequently, acarbose is probably the most suitable hit for RapJ enzyme. Notably, experimental validation is crucial to confirm the effectiveness of the selected ligands.

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.

Proteome Exploration of Human Coronaviruses for Identifying Novel Vaccine Candidate: A Hierarchical Subtractive Genomics and Reverse Vaccinology Approach.

BACKGROUND: The SARS-CoV-2 has been responsible for infecting more than 613,615,658 people in 222 countries by September 11, 2022, of which 6,516,076 have died. COVID-19 was introduced by World Health Organization as a global concern and a pandemic disease due to its prevalence. OBJECTIVE: Developing preventive or therapeutic medications against 2019-nCoV is an urgent need, and has been deemed as a high priority among scientific societies; in this regard, the production of effective vaccines is one of the most significant and high-priority requirements. Because of costly and time-consuming process of vaccine design, different immunoinformatics methods have been developed. METHODS: At the beginning of vaccine design, the proteome study is essential. In this investigation, the whole human coronavirus proteome was evaluated using the proteome subtraction strategy. Out of 5945 human coronavirus proteins, five new antigenic proteins were selected by analyzing the hierarchical proteome subtraction, and then their various physicochemical and immunological properties were investigated bioinformatically. RESULTS: All five protein sequences are antigenic and non-allergenic proteins; moreover, the spike protein group, including spike glycoprotein (E2) (Peplomer protein), spike fragment and spike glycoprotein fragment, showed acceptable stability, which can be used to design new vaccines against human coronaviruses. CONCLUSION: The selected peptides and the other proteins introduced in this study (HE, orf7a, SARS_X4 domain-containing protein and protein 8) can be employed as a suitable candidate for developing a novel prophylactic or therapeutic vaccine against human coronaviruses.

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.

Designing a humanized immunotoxin based on DELTA-stichotoxin-Hmg2a toxin: an in silico study.

Breast cancer remains the most frequently diagnosed cancer and the principal cause of mortality by malignancy in women. HER2 positive subtype includes 15-20% of breast cancer cases. This receptor could be an appropriate mark for targeting breast cancer cells. Immunotherapy methods compared to current cancer treatment methods have the lowest side effects. DELTA-stichotoxin-Hmg2a is isolated from the sea anemone and kills cells through pore formation. In the current study, we designed and evaluated an immunotoxin composed of pertuzumab and DELTA-stichotoxin-Hmg2a-derived scFv by bioinformatics tools. The designed immunotoxin was constructed using the amino acid sequences. Then, secondary structure and physico-chemical features were studied, and the tertiary structure of the immunotoxin was built according to the homology modeling methods. The validation and allergenicity of the model were assessed. The immunotoxin and receptor were docked and molecular dynamics simulation indicated the construct stability. The analysis results indicated that the construct is a stable protein that could have a natural-like structure and would not be an allergen, so this immunotoxin could effectively target HER2 receptors. Therefore, our designed immunotoxin could be an appropriate immunotoxin against HER2-positive breast cancer and could be a challenging topic for future in vitro and in vivo studies.

Exosome-based vaccines and their position in next generation vaccines.

Extracellular vesicles (EVs), which include exosomes as a subset, are generated by most cell types and play crucial roles in intercellular communication. Exosomes offer intriguing tools as potential vaccines due to their ability to deliver a wide range of antigens and immunomodulatory properties. Exosome-based vaccines have demonstrated promising results against different types of infectious diseases as well as cancers, both in vitro and in vivo. In this review, a number of studies on exosome-based vaccines are highlighted and relevant clinical trials are discussed.

B-Cell Epitope Mapping from Eight Antigens of Candida albicans to Design a Novel Diagnostic Kit: An Immunoinformatics Approach.

Invasive candidiasis is an emerging fungal infection and a leading cause of morbidity in health care facilities. Despite advances in antifungal therapy, increased antifungal drug resistance in Candida albicans has enhanced patient fatality. The most common method for Candida albicans diagnosing is blood culture, which has low sensitivity. Therefore, there is an urgent need to establish a valid diagnostic method. Our study aimed to use the bioinformatics approach to design a diagnostic kit for detecting Candida albicans with high sensitivity and specificity. Eight antigenic proteins of Candida albicans (HYR1, HWP1, ECE1, ALS, EAP1, SAP1, BGL2, and MET6) were selected. Next, a construct containing different immunodominant B-cell epitopes was derived from the antigens and connected using a suitable linker. Different properties of the final construct, such as physicochemical properties, were evaluated. Moreover, the designed construct underwent 3D modeling, reverse translation, and codon optimization. The results confirmed that the designed construct could identify Candida albicans with high sensitivity and specificity in serum samples of patients with invasive candidiasis. However, experimental studies are needed for final confirmation. Supplementary Information: The online version contains supplementary material available at 10.1007/s10989-022-10413-1.

A structural vaccinology approach for in silico designing of a potential self-assembled nanovaccine against Leishmania infantum.

Visceral leishmaniasis (VL) remains a major public health problem across 98 countries. To date, VL has no effective drug. Vaccines, as the most successful breakthroughs in medicine, can promise an effective strategy to fight various diseases. More recently, self-assembled peptide nanoparticles (SAPNs) have attracted considerable attention in the field of vaccine design due to their multivalency. In this study, a SAPN nanovaccine was designed using various immunoinformatics methods. High-ranked epitopes were chosen from a number of antigens, including Leishmania-specific hypothetical protein (LiHy), Leishmania-specific antigenic protein (LSAP), histone H1, and sterol 24-c-methyltransferase (SMT). To facilitate the oligomerization process, pentameric and trimeric coiled-coil domains were employed. RpfE, a resuscitation-promoting factor of Mycobacterium tuberculosis, was added to induce strong immune responses. Pentameric and trimeric coiled-coil domains as well as eight immunodominant epitopes from antigenic proteins of Leishmania infantum, the causative agent of VL, were joined together using appropriate linkers. High-quality 3D structure of monomeric and oligomeric structures followed by refinement and validation processes demonstrated that the designed nanovaccine could be considered to be a promising medication against the parasite; however, experimental validation is essential to confirm the effectiveness of the nanovaccine.

Designing a Multi-Epitope Antigen for Serodiagnosis of Strongyloides stercoralis Based on L3Nie.01 and IgG Immunoreactive Epitopes.

Background: Serological diagnosis of Strongyloides stercoralis (S. stercoralis) is fre-quently challenging because of cross-reactivity with other parasitic nematodes. Therefore, it is necessary to introduce novel serological tests with high performance to properly diagnose this neglected parasitic infection. The purpose of the current study was to design a multi-epitope construct for the diagnosis of S. stercoralis. Methods: For the purpose of this study, first, highly antigenic segments and potential immunodominant epitopes of S. stercoralis were identified from two antigenic proteins, and then all of the selected parts were linked by an appropriate linker. Next, the physicochemical features of the designed construct were analyzed. Then, tertiary structures of the construct were built and evaluated to find out the best one. Lastly, the amino acid sequence was reverse-translated and optimized for over-expression in Escherchia coli (E. coli). Results: The bioinformatic evaluation indicated that the designed protein construct could be hydrophilic, thermostable, and acidic and the estimated half-life was more than 10 hr in E. coli. Conclusion: According to the results of the study, the designed construct could be used as an efficient antigen in the ELISA system for serological diagnosis of human strong-yloidiasis.

Telmisartan anti-cancer activities mechanism through targeting N-cadherin by mimicking ADH-1 function.

This study aimed to investigate if Telmisartan as a novel N-cadherin antagonist, can overcome cell migration of cancer cells. We investigated the mechanism and influence of Docetaxel and Telmisartan (as an analogous to ADH-1, which is a well-known N-cadherin antagonist) on cancer cells. The effect of ADH-1 and Telmisartan on cell attachment in PC3, DU145, MDA-MB-468 cell lines using recombinant human N-cadherin was studied. Cell viability assay was performed to examine the anti-proliferative effects of Telmisartan, ADH-1 and Docetaxel. Migration was examined via wound healing assay, and apoptosis was determined by flow cytometry. The expression of AKT-1 as a downstream gene of N-cadherin signalling pathway was assayed by real-time PCR. Treatment of PC3, MDA-MB-468 and DU145 cells with Telmisartan (0.1 µM) and ADH-1 (40 µM) resulted in 50%, 58% and approximately 20% reduction in cell attachment to N-cadherin coated plate respectively. It shows reduction of cell attachment in PC3 and MDA-MB-468 cell lines appeared to be more sensitive than that of DU145 cells to the Telmisartan and ADH-1 treatments. Telmisartan (0.1 µM) and Docetaxel (0.01 nM) significantly reduced cell migration in PC3 and MDA-MB-468 cell lines compared with the control group. Using Real-time PCR, we found that Telmisartan, Docetaxel and ADH-1 had significant influence on the AKT-1 mRNA level. The results of the current study for the first time suggest that, Telmisartan, exerts anti-proliferation and anti-migration effects by targeting antagonistically N-cadherin. Also, these data suggest that Telmisartan as a less expensive alternative to ADH-1 could potentiate Docetaxel anticancer effects.

Different strategies for expression and purification of the CT26-poly-neoepitopes vaccine in Escherichia coli.

BACKGROUND: Due to the association of hypermutated colorectal cancer (CRC) with many neo-antigens, poly-neo-epitopes are attractive vaccines. The molecular features of murine CT26 are similar to those of aggressive human CRC. CT26 contains some antigenic mutations, which can provide specific immunotherapy targets. Herein, we aimed to express, and purify the previously designed hexatope containing CT26 neoepitopes, CT26-poly-neoepitopes. METHODS AND RESULTS: In the current study, expression of the CT26-poly-neoepitopes was optimized in three different Escherichia coli strains including BL21 (DE3), Origami (DE3), and SHuffle®. Furthermore, the effect of ethanol on the CT26-poly-neoepitopes expression was investigated. The highest amount of CT26-poly-neoepitopes, which included CT26-poly-neoepitopes with the uncleaved pelB signal sequence and the processed one, was achieved when BL21 containing pET-22 (CT26-poly-neoepitopes) was induced with 0.1 mM IPTG for 48 h at 22 ºC in the presence of 2% ethanol. However, 37 ºC was the optimized induction temperature for expression of the CT26-poly-neoepitopes in the absence of ethanol. To purify the CT26-poly-neoepitopes, Ni-NTA affinity chromatography under denaturing and hybrid conditions were applied. High and satisfactory CT26-poly-neoepitopes purity was achieved by the combined urea and imidazole method. CONCLUSION: The effect of ethanol on expression of the CT26-poly-neoepitopes was temperature-dependent. Furthermore, the pelB-mediated translocation of the CT26-poly-neoepitopes into the periplasm was inefficient. Moreover, higher concentration of imidazole in the washing buffer improved the CT26-poly-neoepitopes purification under hybrid condition. Overall, the immunogenicity of CT26-poly-neoepitopes expressed in BL21 under the optimum condition and purified under hybrid condition can be studied in our future in vivo study.