Comparative study of impact of animal source on physical, structural, and biological properties of bone xenograft.

BACKGROUND: Due to the unique features of xenografts including large supply from donors, minimal risk of human disease transmission, and the lower cost of preparation and production compared to autografts and allografts, they are considered as attractive alternatives to traditional bone grafts. The animal source accessibility and production process have a direct correlation with the cost and quality of the final product. To evaluate whether the animal source of the bone has any effect on the physicochemical and histological properties of the final xenograft, three deproteinized bone grafts were prepared from sources that are easily available in Iran, including the bovine (DBB), camel (DCB), and ostrich (DOB). METHODS: In the current study, three bone substitute materials intended to serve as bone xenografts were derived from the cow, camel, and ostrich using the thermochemical processing procedure. The physicochemical properties, in vitro cytocompatibility and in vivo bone regeneration capability of the prepared deproteinized bone grafts, were assessed and compared with OCS-B as an approved product in the global market. RESULTS: The physical tests confirmed the hydroxyapatite nature of the final products. SEM and BET analysis showed morphological and structural differences between the products due to differences in the animal sources. In vitro studies showed the prepared deproteinized bone was free of processing chemicals and was biocompatible with mouse fibroblast and myoblast cell lines. In vivo studies revealed that the bone formation capability of the DBB, DCB, and DOB has no significant difference with one another and with OCS-B despite their structural differences. The DCB showed the highest graft residue after two month. No signs of immunogenicity were observed in the study groups compared to the blank group. CONCLUSION: DBB, DCB, and DOB may offer a favorable cell response and bone regeneration similar to those of commercial bovine bone material.

A new triple chimeric protein as a high immunogenic antigen against anthrax toxins: theoretical and experimental analyses.

Background: Anthrax is a zoonotic disease caused by Bacillus anthracis and it can be deadly in 6 days. Considerable efforts have been conducted toward developing more effective veterinary and human anthrax vaccines because these common vaccines have several limitations. B. anthracis secretes a tripartite toxin, comprising protective antigen (PA), edema factor (EF), and lethal factor (LF). Several studies have shown important role of PA in protection of anthrax. LF and EF induce production of toxin neutralizing antibodies too. PA in fusion form with LF/EF has synergistic effects as a potential subunit vaccine. Methods: In this study, for the first time, a triple chimeric protein called ELP was modeled by fusing three different domains of anthrax toxic antigens, the N-terminal domains of EF and LF, and the C-terminal domain of PA as a high immunogenic antigen using Modeller 9.19 software. Immunogenicity of the ELP was assessed in guinea pigs using enzyme-linked immunosorbent assay (ELISA) test and MTT assay. Results: Theoretical studies and molecular dynamics (MD) simulation results suggest that the ELP model had acceptable quality and stability. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified ELP, its domains, and PA were matched with their molecular size and confirmed by western blotting analysis. In the immune guinea pigs, antibody was produced against all of the ELP domains. It was observed that ELP induced strong humoral response and could protect murine macrophage cell line (RAW 264.7 cells) against anthrax lethal toxin (LeTx). Conclusions: ELP chimeric antigen could be considered as a high immunogenic antigen.

Generation and screening of efficient neutralizing single domain antibodies (VHHs) against the critical functional domain of anthrax protective antigen (PA).

Since anthrax is an acute infectious disease, detection and neutralization of the toxins of pathogenic Bacillus anthracis are of great importance. The critical role of protective antigen (PA) component of tripartite anthrax toxin in toxin entry into the host cell cytosol provided a great deal of effort to generate monoclonal antibodies against this constitute. Regarding the importance of anthrax detection/neutralization and unique physicochemical and pharmacological features of VHHs as single domain antibodies, the present study aimed to generate VHHs against the receptor binding domain of PA, termed PAD4. After camel immunization, a gene repertoire of VHH fragments with a diversity of 4.7×108 clones was produced, followed by constructing a VHH phage display library. A stringent successive biopanning was then carried out to isolate the phages displaying high affinity VHHs against PAD4.Polyclonal and monoclonal Enzyme-linked immunosorbent assay (ELISA) verified binding specificity of phages to the target protein. Modeling of VHHs together with the docking simulation studies, illustrated the binding site of antibodies on antigen. Docking analysis revealed that all selected VHHs potently cover the key functional residues of PAD4. Since the selected VHHs could cover and block the receptor binding loops of PA, they could be proposed as hopeful anti-Anthrax candidates.

Display of B. pumilus chitinase on the surface of B. subtilis spore as a potential biopesticide.

BACKGROUND: Chitinases can inhibit the growth of many fungal diseases which are a great threat for global agricultural production. Biological control of pathogens like fungi, is believed to be one of the best ways to eliminate the adverse effects of plant pathogens. To this end, we expressed and displayed a chitinase from Bacillus pumilus (ChiS) on the surface of Bacillus subtilis spores, as a biocontrol agent. RESULT: ChiS enzyme from B. pumilus was expressed on the spores of B. subtilis using CotG as a carrier protein. Immunofluorescence microscopy confirmed the expression of ChiS on the surface of the spores. Enzyme activity assay showed that the surface displayed ChiS was active and was also able to inhibit the growth of Rhizoctonia solani and Trichoderma harzianum fungi. Western blot analysis also indicated that CotG-ChiS is partially processed after display. Molecular dynamics simulation showed that the stability of the heterologous protein was decreased after fusion. CONCLUSION: ChiS was successfully displayed on the surface of Bacillus spores by fusion to the CotG, one of the main spore coat proteins. In-vitro experiments showed that the displayed enzyme was effective in growth inhibition of R. solani and T. harzianum fungi.

Expression and Secretion of Cyan Fluorescent Protein (CFP) in B. subtilis using the Chitinase Promoter from Bacillus pumilus SG2

Background: Improved cyan fluorescent protein (ICFP) is a monochromic, green fluorescent protein (GFP) derivative produced by Aequorea macrodactyla in a process similar to GFP. This protein has strong absorption spectra at wavelengths 426-446 nm. ICFP can be used in cell, organelle or intracellular protein labeling, investigating the protein-protein interactions as well as assessing the promoter activities. Methods: In our previous study, the promoters of two chitinases (ChiS and ChiL) from Bacillus pumilus SG2 were assessed in B. subtilis and their regulatory elements were characterized. In the present study, icfp was cloned downstream of several truncated promoters obtained in the former study, and ICFP expression was evaluated in B. subtilis. Results: Extracellular expression and secretion of ICFP were analyzed under the control of different truncated versions of ChiSL promoters grown on different media. Results from SDS-PAGE and fluorimetric analyses showed that there were different expression rates of CFP; however, the UPChi-ICFP3 construct exhibited a higher level of expression and secretion in the culture medium. Conclusion: Our presented results revealed that inserting this truncated form of Chi promoter upstream of the ICFP, as a reporter gene, in B. subtilis led to an approximately ten fold increase in ICFP expression.

A conformation-based phage-display panning to screen neutralizing anti-VEGF VHHs with VEGFR2 mimicry behavior.

The potency of VEGF-based anti-angiogenic strategies in cancer therapy and the brilliant characteristics of VHHs motivated us to directly block VEGF binding to its receptor with neutralizing single domain antibodies, thereby fading away the VEGF signaling pathway. Considering with high resolution crystal structure of VEGF-RBD/VEGFR2 complex, we could adopt a combinatorial screening strategy: stringent panning and competition ELISA, to direct the panning procedure to dominantly screen the favorable binders that bind and block the key functional regions of VEGF. Based on competition assay, the majority of the screened clones (82%) showed the VEGFR2 mimicry behavior for binding to VEGF molecule. The phage pool gets enriched in favor of sequences that bind the receptor binding sites of VEGF. Different immunoassays and molecular docking simulation verified that all selected VHHs could bind and cover the receptor binding sites of VEGF. Consequently, some modifications in panning procedure with considering the structural features and detailed information of functional regions of a protein antigen, led us to successfully trap the high-affinity specific binders against its hot functional regions. Since the selected VHHs could cover the receptor binding site of VEGF and block VEGF binding to the receptor, they might be promising candidates for anti-angiogenic therapies.

The design of a new truncated and engineered alpha1-antitrypsin based on theoretical studies: an antiprotease therapeutics for pulmonary diseases.

Alpha 1- antitrypsin (α1AT) a 54 kDa glycoprotein is a protease inhibitor. In the absence of α1AT, elastase released by lung macrophages, was not inhibited and lead to elastin breakdown and pulmonary problems such as emphysema or COPD. α1AT has three site of N-glycosylation and a characteristic reactive central loop (RCL). As small-scale medicines are preferred for pulmonary drug delivery, in this study α1ATs (1, 2, 3, 4 and 5) were engineered and shortened from the N-terminal region. In order to investigate the effect of different mutations and the deletion of 46 amino acids theoretical studies were performed. Homology modeling was performed to generate the 3D structure of α1ATs. The 10 ns Molecular Dynamic (MD) simulations were carried out to refine the models. Results from MD and protein docking showed that α1AT2 has the highest binding affinity for neutrophil elastase, provided the basis for the experimental phase in which sequences from the five α1AT constructs were inserted into the expression vector pGAPZα and expressed in the yeast Pichia pastoris. Although, the α1AT2 construct has the highest inhibitory activity even more that the native construct (α1AT5), results indicated the presence of protease inhibitory function of all the proteins' construct against elastase.

Efficient binding of nickel ions to recombinant Bacillus subtilis spores.

We report the use of recombinant spores of Bacillus subtilis as a potential bioremediation tool for adsorption of nickel ions. The spore surface protein CotB, previously used for the display of heterologous antigens, was engineered to express eighteen histidine residues within the spore coat. Wild type and recombinant spores were then analyzed to assess their efficiency in adsorbing nickel ions, and the latter proved to be significantly more efficient than wild type spores in metal-binding. The quantities of spores used in the adsorption reaction significantly affected nickel binding, while other factors such as pH and temperature did not show relevant effects. In addition, simple washing procedures were used to partially release spore-bound nickel ions by wild type and recombinant spores. The efficiency of nickel binding, together with the simple purification procedure, the high robustness and safety of B. subtilis spores and the possibility of recovering bound nickel, makes the recombinant spore a new and potentially powerful tool for the treatment of contaminated ecosystems.