Zinc Oxide Nanoparticles and Cancer Chemotherapy: Helpful Tools for Enhancing Chemo-sensitivity and Reducing Side Effects?

Cancer chemotherapy is still a serious challenge. Chemo-resistance and destructive side effects of chemotherapy drugs are the most critical limitations of chemotherapy. Chemo-resistance is the leading cause of chemotherapy failure. Chemo-resistance, which refers to the resistance of cancer cells to the anticancer effects of chemotherapy drugs, is caused by various reasons. Among the most important of these reasons is the increase in the efflux of chemotherapy drugs due to the rise in the expression and activity of ABC transporters, the weakening of apoptosis, and the strengthening of stemness. In the last decade, a significant number of studies focused on the application of nanotechnology in cancer treatment. Considering the anti-cancer properties of zinc, zinc oxide nanoparticles have received much attention in recent years. Some studies have indicated that zinc oxide nanoparticles can target the critical mechanisms of cancer chemo-resistance and enhance the effectiveness of chemotherapy drugs. These studies have shown that zinc oxide nanoparticles can reduce the activity of ABC transporters, increase DNA damage and apoptosis, and attenuate stemness in cancer cells, leading to enhanced chemo-sensitivity. Some other studies have also shown that zinc oxide nanoparticles in low doses can be helpful in minimizing the harmful side effects of chemotherapy drugs. In this article, after a brief overview of the mechanisms of chemo-resistance and anticancer effects of zinc, we will review all these studies in detail.

Designing and Characterization of Tregitope-Based Multi-Epitope Vaccine Against Multiple Sclerosis: An Immunoinformatic Approach.

BACKGROUND: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system(CNS). It is widely accepted that the development and progression of MS result from aberrant activation of potentially encephalitogenic reactive-T cells against CNS antigens. The pathologic roles of both CD4+ (T helper; Th) and CD8+ T cells have been demonstrated in MS lesions. OBJECTIVE: In the present work, we applied a series of bioinformatics tools to design a dendritic cell (DC)-targeting Tregitope-based multi-epitope vaccine for MS to induce tolerance in pathogenic myelin-specific T cells. METHODS: The 3D structure of anti-DEC205 scFv and the remaining part of the vaccine were modeled by ROSIE Antibody server and ITASSER software, respectively. AIDA web server (ab initio domain assembly server) was applied to assemble two parts of the vaccine and build the full construct. Following modeled structure refinement and validation, physicochemical properties, and allergenicity of the vaccine were assessed. In the final step, in silico cloning was done to ensure high-level expression in the desired host. RESULTS: This vaccine consists of three main parts; 1) Anti-DEC205 scFv antibody, 2) multiepitope vaccine part composed of multiple pathogenic CD4+, and CD8+ T cell epitopes originated from multiple known antigens in MS patients, as well as T-regulatory (Treg)-inducing epitopes (Tregitopes), and 3) vasoactive intestinal peptide (VIP). All parts of the final vaccine were joined together with the help of proper linkers. After vaccine construction, the three-D structure, as well as different physicochemical and immunological features of the vaccine were predicted. Finally, in silico gene cloning was also carried out to assure efficient production of protein vaccine in Escherichia coli K12 expression strain. CONCLUSION: Computational study revealed that this vaccination can regulate MS disease progression and even relapse by harnessing pathogenic T cells.

Immunosenescence and inflamm-ageing in COVID-19.

The destructive effects of coronavirus disease 2019 (COVID-19) on the elderly and people with cardiovascular disease have been proven. New findings shed light on the role of aging pathways on life span and health age. New therapies that focus on aging-related pathways may positively impact the treatment of this acute respiratory infection. Using new therapies that boost the level of the immune system can support the elderly with co-morbidities against the acute form of COVID-19. This article discusses the effect of the aging immune system against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the pathways affecting this severity of infection.

In silico Prediction and Evaluation of Human Parainfluenza Virus-3 CD4+ T Cell Epitopes.

BACKGROUND: Human parainfluenza viruses type 3 (HPIV-3) through bronchiolitis and pneumonia is a common cause of lower respiratory tract infections. It is the main cause of hospitalization of infants and young children and also one of the main causes of morbidity and mortality in immuno-compromised and transplant patients. Despite many efforts, there is currently no specific anti-HPIV-3 drug or approved vaccine to prevent and control the virus. Identification of HPIV-3 epitopes with the capability of binding to human leukocyte antigen (HLA) class II molecules can be helpful in designing new vaccine candidates against HPIV-3 infection, and also can be useful for the in vitro stimulation and proliferation of HPIV-3-specific T cells for transplant and immunocompromised patients. OBJECTIVE: To predict and comprehensively evaluate CD4+T cell epitope (HLA-II binders) from four main HPIV-3 antigens. METHODS: In the present work, we predicted and comprehensively evaluated CD4+T cell epitope (HLA-II binders) from four main HPIV-3 antigens, including fusion protein (F), hemagglutininneuraminidase (HN), nucleocapsid (N) and matrix (M) proteins using bio- and immunoinformatics software. The toxicity, allergenicity, Blast screening and population coverage of the predicted epitopes were evaluated. The binding ability of the final selected epitopes was evaluated via a docking study. RESULTS: After several filtering steps, including blast screening, toxicity and allergenicity assay, population coverage and docking study, 9 epitopes were selected as candidate epitopes. The selected epitopes showed high population coverage and docking studies revealed a significantly higher binding affinity for the final epitopes in comparison with the negative control peptides. CONCLUSION: The final selected epitopes could be useful in designing vaccine candidates and for the treatment of immune-compromised individuals and patients with transplantation.

Utilization of SUMO Tag and Freeze-thawing Method for a High-level Expression and Solubilization of Recombinant Human Angiotensinconverting Enzyme 2 (rhACE2) Protein in E. coli.

BACKGROUND: SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as a receptor for entering the host cells. Production of the ACE2 molecule is important because of its potency to use as a blocker and therapeutic agent against SARS-CoV-2 for the prophylaxis and treatment of COVID-19. OBJECTIVE: The recombinant human ACE2 (rhACE2) is prone to form an inclusion body when expressed in the bacterial cells. METHODS: We used the SUMO tag fused to the rhACE2 molecule to increase the expression level and solubility of the fusion protein. Afterward, the freeze-thawing method plus 2 M urea solubilized aggregated proteins. Subsequently, the affinity of solubilized rhACE2 to the receptor binding domain (RBD) of the SARS-CoV-2 spike was assayed by ELISA and SPR methods. RESULTS: SUMO protein succeeded in increasing the expression level but not solubilization of the fusion protein. The freeze-thawing method could solubilize and recover the aggregated fusion proteins significantly. Also, ELISA and SPR assays confirmed the interaction between solubilized rhACE2 and RBD with high affinity. CONCLUSION: The SUMO tag and freeze- Conclusion: The SUMO tag and freeze-thawing method would be utilized for high-level expression and solubilization of recombinant rhACE2 protein.

A highly potential cleavable linker for tumor targeting antibody-chemokines.

Chemokines are the large family of chemotactic cytokines that play an important role in leukocyte movement and migration stimulation. Until now, several antibody-cytokine (chemokine) fusion proteins have been investigated in clinical trials because of their ability to evoke the circulating leukocytes far from the tumor site. In this case, creating the concentration gradient regarding the chemokine is very important to recruit the circulating leukocytes with maximum performance to the tumor environment. To achieve a proper gradient, the chemokine separation from the tumor antigen-bounded antibody can be very crucial. Thus, we designed a novel linker that can be cleaved by enzymes presented around the tumor site including cathepsin B, urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs). Also, it can inhibit tumor progression by competing with the native substrate of key proteases in the tumor microenvironment. The proposed linker was evaluated using some bioinformatics approaches. In silico results showed that the linker is structurally stable and could be detected and cleaved using the mentioned enzymes.Communicated by Ramaswamy H. Sarma.

Improving the specificity of organophosphorus hydrolase to acephate by mutagenesis at its binding site: a computational study.

Organophosphorus hydrolase (OPH) is one of the most important enzymes in order to bioremediation of organophosphorus (OP) pesticides. OPH is capable of degrading a wide variety of OPs, but it has poor specificity to OPs with P-S bond, including acephate. Given that the binding site residues of OPH determine its substrate specificity, this study was carried out to find the best OPH mutants containing a single point mutation in the binding site that possess improved specificity to acephate. Hence, we generated all possible mutant models and performed molecular docking of acephate with wild-type OPH (OPH-WT) and its mutants. After that, molecular dynamic (MD) simulations of OPH-WT and the best mutants, according to the docking results, were performed in both apo- and complex with acephate forms. Docking results signified that 51 out of 228 mutants possessed increased binding affinities to acephate, as compared to OPH-WT. Of them, W131N, W131G, and H254Y were the best mutants considering the high binding affinities and proper orientation of the ligand at their active sites. MD simulations confirmed the stability of the three mutants in both apo- and complex with acephate forms and also showed that these formed more stable complexes with acephate, as compared to OPH-WT. MD results also suggested that W131N and W131G, in addition to enhanced specificity, could keep the necessary configuration for acephate hydrolysis during the simulations.

Expression, Solubilization, Refolding and Final Purification of Recombinant Proteins as Expressed in the form of "Classical Inclusion Bodies" in E. coli.

Escherichia coli has been most widely used for production of the recombinant proteins. Over-expression of the recombinant proteins is the mainspring of the inclusion bodies formation. The refolding of these proteins into bioactive forms is cumbersome and partly time-consuming. In the present study, we reviewed and discussed most issues regarding the recovery of "classical inclusion bodies" by focusing on our previous experiences. Performing proper methods of expression, solubilization, refolding and final purification of these proteins, would make it possible to recover higher amounts of proteins into the native form with appropriate conformation. Generally, providing mild conditions and proper refolding buffers, would lead to recover more than 40% of inclusion bodies into bioactive and native conformation.

Withdrawal Notice: EnzyPha, an Engineered Helper Phage Developed to Overcome Most of the Limitations Regarding Filamentous Phage Titration and ELISA Tests

The article has been withdrawn at the request of the authors of the journal Protein & Peptide Letters. Bentham Science apologizes to the readers of the journal for any inconvenience this may have caused. The Bentham Editorial Policy on Article Withdrawal can be found at https://benthamscience.com/editorial-policiesmain. php. BENTHAM SCIENCE DISCLAIMER: It is a condition of publication that manuscripts submitted to this journal have not been published and will not be simultaneously submitted or published elsewhere. Furthermore, any data, illustration, structure or table that has been published elsewhere must be reported, and copyright permission for reproduction must be obtained. Plagiarism is strictly forbidden, and by submitting the article for publication the authors agree that the publishers have the legal right to take appropriate action against the authors, if plagiarism or fabricated information is discovered. By submitting a manuscript, the authors agree that the copyright of their article is transferred to the publishers if and when the article is accepted for publication.

Designing a multi-epitope vaccine against blood-stage of Plasmodium falciparum by in silico approaches.

Plasmodium falciparum causes the most severe form of malaria disease and is the major cause of infection-related mortalities in the world. Due to increasing in P. falciparum resistance to the first-line antimalarial drugs, an effective vaccine for the control and elimination of malaria infection is urgent. Because the pathogenesis of malaria disease results from blood-stage infection, and all of the symptoms and clinical illness of malaria occur during this stage, there is a strong rationale to develop vaccine against this stage. In the present study, different structural-vaccinology and immuno informatics tools were applied to design an effective antibody-inducing multi-epitope vaccine against the blood-stage of P. falciparum. The designed multi-epitope vaccine was composed of three main parts including B cell epitopes, T helper (Th) cell epitopes, and two adjuvant motives (HP91 and RS09), which were linked to each other via proper linkers. B cell and T cell epitopes were derived from four protective antigens expressed on the surface of merozoites, which are critical to invade the erythrocytes. HP91 and RS09 adjuvants and Th cell epitopes were used to induce, enhance and direct the best form of humoral immune-response against P. falciparum surface merozoite antigens. The vaccine construct was modeled, and after model quality evaluation and refinement by different software, the high-quality 3D-structure model of the vaccine was achieved. Analysis of immunological and physicochemical features of the vaccine showed acceptable results. We believe that this multi-epitope vaccine can be effective for preventing malaria disease caused by P. falciparum.

Catalytic and structural effects of flexible loop deletion in organophosphorus hydrolase enzyme: A thermostability improvement mechanism.

Thermostability improvement of enzymes used industrially or commercially would develop their capacity and commercial potential due to increased enzymatic competence and cost-effectiveness. Several stabilizing factors have been suggested to be the base of thermal stability, like proline replacements, disulfide bonds, surface loop truncation and ionic pair networks creation. This research evaluated the mechanism of increasing the rigidity of organophosphorus hydrolase enzyme by flexible loop truncation. Bioinformatics analysis revealed that the mutated protein retains its stability after loop truncation (five amino acids deleted). The thermostability of the wild-type (OPH-wt) and mutated (OPH-D5) enzymes were investigated by half-life, Delta Gi, and fluorescence and far-UV CD analysis. Results demonstrated an increase half-life and Delta Gi in OPH-D5 compared to OPH-wt. These results were confirmed by extrinsic fluorescence and circular dichroism (CD) spectrometry experiments, therefore, as rigidity increased in OPHD5 after loop truncation, half-life and Delta Gi also increased. Based on these findings, a strong case is presented for thermostability improvement of OPH enzyme by flexible loop truncation after bioinformatics analysis.

Generation and characterization of a specific single-chain antibody against DSPP as a prostate cancer biomarker: Involvement of bioinformatics-based design of novel epitopes.

Isolation of specific single chain antibodies (scFvs) against key epitopes of cancer markers are applied for cancer immunotherapy and diagnosis. In this study following the prediction of the 3D structure of the DSP part of Dentin sialophosphoprotein (DSPP), the epitope was chosen using in silico programs. Panning process was applied to isolate specific human scFv against the epitope. PCR and DNA fingerprinting differentiated the specific clones, which were evaluated by phage ELISA. Following DNA sequencing, the 3D structure of isolated scFv was modeled and Docked on DSP. Results demonstrated the selection of a specific anti-DSPP scFv with 40% frequency, which reacted significantly with the predicted epitope and PCa patients' urines in ELISA tests (P-value < 0.05). The VH and VL of the isolated scFv were from VH1 and VL3 gene families with several amino acid changes in CDRs and FRs domains. The scFv tightly bound to the DSP epitope with the lowest energy level by hydrogen bonds, cation-pi, hydrophobic and ionic interactions demonstrating the specificity of Ag-Ab interactions. The anti-DSPP scFv selected in this study with significant specificity to DSPP antigen offers a promising new agent for both PCa early detection and treatment of cancers with DSPP expression.

Disulfide bridge formation to increase thermostability of DFPase enzyme: A computational study.

Organophosphate compounds bioremediation by use of organophosphorus degradation enzymes such as DFPase is a developing interest in industry and medicine. The most important problem with the bio-catalytic enzymes is their instability on high temperatures. This work carried out to find suitable locations for introducing disulfide bridges in DFPase enzyme. We employed some computational approaches to design the disulfide bridges and evaluate their roles in the enzyme structural thermostability. According to the in silico results, mutant 6 (V24C, C76) increased the enzyme thermostability relative to wild-type.

Application of molecular dynamics simulations to design a dual-purpose oligopeptide linker sequence for fusion proteins.

Proteins are often monitored by combining a fluorescent polypeptide tag with the target protein. However, due to the high molecular weight and immunogenicity of such tags, they are not suitable choices for combining with fusion proteins such as immunotoxins. In this study, we designed a polypeptide sequence with a dual role (it acts as both a linker and a fluorescent probe) to use with fusion proteins. Two common fluorescent tag sequences based on tetracysteine were compared to a commonly used rigid linker as well as our proposed dual-purpose sequence. Computational investigations showed that the dual-purpose sequence was structurally stable and may be a good choice to use as both a linker and a fluorescence marker between two moieties in a fusion protein.

Cell growth inhibition and apoptosis in breast cancer cells induced by anti-FZD7 scFvs: involvement of bioinformatics-based design of novel epitopes.

BACKGROUND: FZD7 has a critical role as a surface receptor of Wnt/ß-catenin signaling in cancer cells. Suppressing Wnt signaling through blocking FZD7 is shown to decrease cell viability, metastasis and invasion. Bioinformatic methods have been a powerful tool in epitope designing studies. Small size, high affinity and human origin of scFv antibodies have provided unique advantages for these recombinant antibodies. METHODS: Two epitopes from extracellular domain of FZD7 were designed using bioinformatic methods. Specific anti-FZD7 scFvs were selected against these epitopes through panning process. The specificity of the scFvs was assessed by phage ELISA and the ability to bind to FZD7 expressing cell line (MDA-MB-231) was determined by flowcytometry. Antiproliferative and apoptotic effects of the scFvs were evaluated by MTT and Annexin V/PI assays. The effects of selected scFvs on expression level of Surivin, c-Myc and Dvl genes were also evaluated by real-time PCR. RESULTS: Results demonstrated selection of two specific scFvs (scFv-I and scFv-II) with frequencies of 35 and 20%. Both antibodies bound to the corresponding peptides and cell surface receptors as shown by phage ELISA and flowcytometry, respectively. The scFvs inhibited cell growth of MDA-MB-231 cells significantly as compared to untreated cells. Growth inhibition of 58.6 and 53.1% were detected for scFv-I and scFv-II, respectively. No significant growth inhibition was detected for SKBR-3 negative control cells. The scFvs induced apoptotic effects in the MDA-MB-231 treated cells after 48 h, which were 81.6 and 74.9% for scFv-I and scFv-II, respectively. Downregulation of Surivin, c-Myc and Dvl genes were also shown after 48h treatment of cells with either of scFvs (59.3-93.8%). ScFv-I showed significant higher antiproliferative and apoptotic effects than scFv-II. CONCLUSIONS: Bioinformatic methods could effectively select potential epitopes of FZD7 protein and suggest that epitope designing by bioinformatic methods could contribute to the selection of key antigens for cancer immunotherapy. The selected scFvs, especially scFv-I, with high antiproliferative and apoptotic effects could be considered as effective agents for immunotherapy of cancers expressing FZD7 receptor including triple negative breast cancer.

Anti-ROR1 scFv-EndoG as a Novel Anti-Cancer Therapeutic Drug

Aim: Immunotoxins are proteins that consist of an antibody fragment linked to a toxin, used as agents for targeted therapy of cancers. Although the most potent immunotoxins are made from bacterial and plant toxins, obstacles which contribute to poor responses are immunogenicity in patients and rapid development of neutralizing antibodies. In the present study we proposed a new therapeutic immunotoxin for targeted cancer therapy of ROR1 expressing cancers: an anti ROR1 single chain fragment variable antibody (scFv)-endonuclease G (anti ROR1 scFv-EndoG). Methods: The three-dimensional structure of anti ROR1 scFv-EndoG protein was modeled and structure validation tools were employed to confirm the accuracy and reliability of the developed model. In addition, stability and integrity of the model were assessed by molecular dynamic (MD) simulation. Results: All results suggested the protein model to be acceptable and of good quality. Conclusions: Anti-ROR1 scFv-EndoG would be expected to bind to the ROR1 extracellular domain by its scFv portion and selectively deliver non-immunogenic human endonuclease G enzyme as an end-stage apoptosis molecule into ROR1-expressing cancer cells and lead rapidly to apoptosis. We believe that anti ROR1 and other anti-tumor antigen scFv-EndoG forms may be helpful for cancer therapy.

ScFv Improvement Approaches.

The most common recombinant antibody format is the single chain fragment variable (scFv) which it contains the complete antigen-binding domains of an intact antibody. ScFv fragments have found vast medical and non-medical applications. Several approaches have been employed to increase the affinity, avidity and structural stability related to these antibody fragments. Most approaches related to scFv improvement have been included in this review.

Anti-Metastatic and Anti-Invasion Effects of a Specific Anti-MUC18 scFv Antibody on Breast Cancer Cells.

Breast cancer is the most common malignancy in women. Altered expression of MUC18, a cell surface receptor, and its interaction with Wnt-5a as its ligand, affects the motility and invasiveness of breast cancer cells. In this study, we explored the Wnt-5a binding site and designed an antigenic epitope on the MUC18 receptor using in silico methods. A specific single-chain variable fragment (scFv) was isolated against the epitope by several panning processes. The binding ability of the scFv to the related epitope was evaluated in ELISA and flow cytometry. The inhibitory effects of the selected scFv on MUC18 positive cell line, MDA-MB231, was assessed by migration and invasion assays. The results demonstrated isolation of specific scFv with frequency of 40 % which showed significant binding with the epitope in both ELISA and fluorescence-activated cell sorting (FACS) analyses. The antibody inhibited the migration (76 %) and invasion (67 %) of MUC18 positive cell line. The results suggest the specific anti-MUC18 scFv as an effective antibody for breast cancer immunotherapy.

Insilico analysis of three different tag polypeptides with dual roles in scFv antibodies.

Single chain fragment variable (scFv) antibodies are composed of variable heavy (VH) and variable light (VL) domains that are joined by a polypeptide linker. Typically, [(Gly4Ser) n] sequence is used as a linker to retain the integrity of the antigen-binding domain. Due to its low immunogenicity, this sequence cannot be used as a tag for scFv detection and purification. Several evidences have shown that the addition of an N or C-terminal tag for scFv detection and purification will result in the decreased expression and binding capacity of this antibody fragment. In this study, we substituted the traditional linker (GGGGS) with His-tag, C-myc or E-tag sequences through molecular modeling. Stability and integrity of all models were assessed by molecular dynamic (MD) simulation. Based on MD simulation analysis, the model containing E-tag sequence as a linker indicated more stability compared to other molecules. The results suggest that E-tag not only can be substituted for the traditional linker, also eliminates the necessity of using additional tag for scFv detection and purification.