Surface Modification of Dentin Powder With Alginate and Evaluation of Its Effects on the Viability and Proliferation of Dental Pulp Stem Cells (In Vitro), Its Biocompatibility (In Vivo).

INTRODUCTION: This study aimed to synthesize dentin powder surface modified with alginate, a potential substance for dental pulp regeneration, and evaluate its effects on the viability and proliferation of human dental pulp stem cells in vitro and its biocompatibility in vivo. METHODS: In the in vitro phase, dentin powder was synthesized in 3 size groups (150-250 µm, 250-500 µm, and 500-1000 µm) after demineralization and atelopeptidization which is used to remove dentin collagen telopeptides and eliminate host immune response. Surface modification with alginate was performed and followed by field-emission scanning electron microscopy, energy dispersive X-ray spectroscopy, and cell viability and proliferation testing for 14 days with human dental pulp stem cells studied. In the in vivo phase, dentin powders were implanted in rat calvarial defects for 8 weeks, and histologic analysis was conducted. All nonparametric data were analyzed with the Kruskal-Wallis test, and all the quantitative data were analyzed by 1-way analysis of variance using SPSS, and P < .05 was considered statistically significant. RESULTS: Demineralization and atelopeptidization were successful in all groups. Cell viability was optimal and equal (P > .05) in all groups. The 500- to 1000-µm group exhibited significantly higher cell proliferation (P < .05). Histologic assessment shows acceptable biocompatibility in all groups; the angiogenesis score was significantly greater in both 250-500 and 500-1000, and minimal inflammatory response was noted in the 500- to 1000-µm group, and the amount of newly formed bone in this group was higher than other groups. CONCLUSIONS: Surface modification of demineralized and atelopeptidized dentin powder with alginate enhanced surface physical properties and cell proliferation while showing great biocompatibility within tissue and reducing the host immune response. These findings hold promise for dentin-pulp complex regeneration.

Ultrasensitive Quantification of MUC16 Antigen/Amine-Terminated Aptamer Interaction by Surface Plasmon Resonance: Kinetic and Thermodynamic Studies.

Purpose: MUC16 is a commonly employed biomarker to identify and predict ovarian cancer (OC). Precise measurement of MUC16 levels is essential for the accurate diagnosis, prediction, and management of OC. This research seeks to introduce a new surface plasmon resonance (SPR) biosensor design that utilizes aptamer-based technology to enable the sensitive and real-time detection of MUC16. Methods: In this study, the sensor chip was immobilized with an anti-MUC16 aptamer (Ap) by utilizing 11-mercaptoundecanoic acid (MUA) as a linker to attach the amine-terminated Ap to the chip using EDC/NHS chemistry. Results: The results indicated that the newly created aptasensor had a detection limit of 0.03 U/mL for MUC16 concentration, with a linear range of 0.09 to 0.27 U/mL. The findings demonstrate good precision and accuracy (<15%) for each MUC16 concentration, with recoveries ranging from 93% to 96%. Additionally, the aptasensor exhibited high selectivity, good repeatability, stability, and applicability in real human serum samples, indicating its potential as a valuable tool for the diagnosis and treatment of OC. Conclusion: According to the outcomes, the designed aptasensor exhibited acceptable specificity to detect the CA125 antigen and could be utilized for the serum detection of target antigen by SPR method.

Functional characterization of the dimeric form of PDGF-derived fusion peptide fabricated based on theoretical arguments.

A skin wound leads to the loss of skin integrity and the influx of pathogens into the tissue. Platelet-derived growth factors (PDGFs) are cytokines released from alpha granules during wound healing and interact with their cell surface receptors and activate signals involved in chemotaxis, growth, proliferation, and differentiation pathways. Due to the low stability of growth factors (GFs), a new peptide-derived PDGF-BB was designed, expressed in the Shuffle strain of E. coli, and purified by Ni-NTA agarose affinity column chromatography. The effect of fusion peptide was then evaluated on L929 fibroblast cells and animal models with skin lesions. In vitro, studies showed that the peptide led to an increase in the migration of fibroblast cells in the scratch assay. Its positive effect on wound healing was also observed in the skin-injured rats after 3, 7, and 12 days. A significant rise in neutrophils and granular tissue formation, re-epithelialization, angiogenesis, and collagen formation was exhibited on the third day of treatment when compared to the control group. The results showed that, despite reducing PDGF size, the fusion peptide was able to maintain at least some of the known functions attributed to full-length PDGF and showed positive results in wound healing.

Additive manufacture of PLLA scaffolds reinforced with graphene oxide nano-particles via digital light processing (DLP).

In this study, 3D printing of poly-l-lactic acid (PLLA) scaffolds reinforced with graphene oxide (GO) nanoparticles via Digital Light Processing (DLP) was investigated to mimic bone tissue. Stereolithography is one of the most accurate additive manufacturing methods, but the dominant available materials used in this method are toxic. In this research, a biocompatible resin (PLLA) was synthetized and functionalized to serve the purpose. Due to the low mechanical properties of the printed product with the neat resin, graphene oxide nanoparticles in three levels (0.5, 1, and 1.5 wt%) were added with the aim of enhancing the mechanical properties. At first, the optimum post cure time of the neat resin was investigated. Consequently, all the parts were post-cured for 3 h after printing. Due to the temperature-dependent structure of GO, all samples were placed in an oven at 85°C for different time periods of 0, 6, 12, and 18 h to increase mechanical properties. The compression test of heat-treated samples reveals that the compressive strength of the printed parts containing 0.5,1, and 1.5% of GO increased by 151,162 ad 235%, respectively. Scaffolds with the designed pore sizes of 750 microns and a porosity of 40% were printed. Surface hydrophilicity test was performed for all samples showing that the hydrophilicity of the samples increased with increasing GO percentage. The degradation behavior of the samples was evaluated in a PBS environment, and it revealed that by increasing GO, the rate of component degradation increased, but the heat treatment had the opposite effect and decreased the degradation rate. Finally, besides improving biological properties, a significant increase in mechanical properties under compression can introduce the printed scaffolds as a suitable option for bone implants.

Drug repositioning based on gene expression data for human HER2-positive breast cancer.

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer represents approximately 15-30% of all invasive breast cancers. Despite the recent advances in therapeutic practices of HER2 subtype, drug resistance and tumor recurrence still have remained as major problems. Drug discovery is a long and difficult process, so the aim of this study is to find potential new application for existing therapeutic agents. Gene expression data for breast invasive carcinoma were retrieved from The Cancer Genome Atlas (TCGA) database. The normal and tumor samples were analyzed using Linear Models for Microarray Data (LIMMA) R package in order to find the differentially expressed genes (DEGs). These genes were used as entry for the library of integrated network-based cellular signatures (LINCS) L1000CDS2 software and suggested 24 repurposed drugs. According to the obtained results, some of these drugs including vorinostat, mocetinostat, alvocidib, CGP-60474, BMS-387032, AT-7519, and curcumin have significant functional similarity and structural correlation with FDA-approved breast cancer drugs. Based on the drug-target network, which consisted of the repurposed drugs and their target genes, the aforementioned drugs had the highest degrees. Moreover, the experimental approach verified curcumin as an effective therapeutic agent for HER2 positive breast cancer. Hence, our work suggested that some repurposed drugs based on gene expression data can be noticed as potential drugs for the treatment of HER2-positive breast cancer.

Efficacy assessment of a triple anthrax chimeric antigen as a vaccine candidate in guinea pigs: challenge test with Bacillus anthracis 17 JB strain spores.

CONTEXT: Bacillus anthracis secretes a tripartite toxin comprising protective antigen (PA), edema factor (EF), and lethal factor (LF). The human anthrax vaccine is mainly composed of the anthrax protective antigen (PA). Considerable efforts are being directed towards improving the efficacy of vaccines because the use of commercial anthrax vaccines (human/veterinary) is associated with several limitations. OBJECTIVE: In this study, a triple chimeric antigen referred to as ELP (gene accession no: MT590758) comprising highly immunogenic domains of PA, LF, and EF was designed, constructed, and assessed for the immunization capacity against anthrax in a guinea pig model. MATERIALS AND METHODS: Immunization was carried out considering antigen titration and immunization protocol. The immunoprotective efficacy of the ELP was evaluated in guinea pigs and compared with the potency of veterinary anthrax vaccine using a challenge test with B. anthracis 17JB strain spores. RESULTS: The results demonstrated that the ELP antigen induced strong humoral responses. The T-cell response of the ELP was found to be similar to PA, and showed that the ELP could protect 100%, 100%, 100%, 80% and 60% of the animals from 50, 70, 90, 100 and 120 times the minimum lethal dose (MLD, equal 5 × 105 spore/ml), respectively, which killed control animals within 48 h. DISCUSSION AND CONCLUSIONS: It is concluded that the ELP antigen has the necessary requirement for proper immunization against anthrax and it can be used to develop an effective recombinant vaccine candidate against anthrax.

Development of a phage display-mediated immunoassay for the detection of vascular endothelial growth factor.

Because of the critical role of vascular endothelial growth factor (VEGF) in angiogenesis and its significantly increased serum levels in early stages of cancer, VEGF is considered an important prognostic biomarker in different cancers. Herein, the amplification power of PCR combined with phage displaying anti-VEGF VHH, a sensitive real-time immunoassay, was precisely designed based on phage display-mediated immuno-PCR (PD-IPCR) for the detection of VEGF. This system benefits from strong and specific binding of antigen and antibody in a sandwich immunosorbent assay platform using avastin (anti-VEGF monoclonal antibody) as the capture antibody. The anti-VEGF phage particles were used as both anti-VEGF agent and DNA template in the PD-IPCR. Anti-VEGF phage ELISA showed a linear range of 3-250 ng/ml and a limit of detection (LOD) of 1.1 ng/ml. Using the PD-IPCR method, the linear range of VEGF detection was found to be 0.06-700 ng/ml, with a detection limit of 3 pg/ml. The recovery rate in serum ranged from 83% to 99%, with a relative standard deviation of 1.2-4.9%. These values indicate that the method has good sensitivity for use in clinical analysis. The proposed method was successfully applied to the clinical determination of VEGF in human serum samples, and the results showed excellent correlation with conventional ELISA (R2 = 0.995). The novel immunoassay provides a specific and sensitive immunoassay protocol for VEGF detection at very low levels. Graphical abstract.

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 multifunctional fusion peptide for tethering to hydroxyapatite and selective capture of bone morphogenetic protein from extracellular milieu.

PURPOSE: The present study sought to design a multi-functional fusion peptide with hydroxyapatite (HA) binding domain (HABD) and heparin binding domain (HBD). METHODS: The 74 amino acid fusion peptide contained N-terminus of the fibrinogen ß chain (ß 15-66), double G4S-linker and 12 residues with HA affinity. This construct was designed, synthesized and cloned into pET21a(+) vector and expressed in E. coli. RESULTS: HABD facilitated purification of the fusion peptide by HA affinity chromatography. Kinetic peptide binding and release on HA scaffold showed sustained release of peptide for up to 16 days. Competitive ELISA and intrinsic fluorescence assays were applied to determine HBD affinity to bone morphogenetic protein-2 (BMP-2). The disassociation rate constant (Kd ) for HBD and rhBMP-2 was approximately 9.2-12 nM. CONCLUSION: The fusion peptide developed in the present study, allowed for streamlined purification on HA affinity chromatography, as well as sustained release from HA scaffold, attributed to its HABD. HBD mediated binding to BMP-2, which may be potentially useful for bone repair. Additional studies, including in vivo investigation will be required to assess the efficacy of the fusion peptide in bone tissue engineering.

Platelet-rich fibrin elicits an anti-inflammatory response in macrophages in vitro.

BACKGROUND: Platelet-rich fibrin (PRF) serves as a reservoir of bioactive molecules to support wound healing and bone regeneration. The beneficial action of PRF might involve macrophage polarization from proinflammatory M1 toward pro-resolving M2 phenotypes. This study aims to evaluate the effect of PRF on macrophage polarization. METHODS: Murine primary macrophages and RAW 264.7 cells were exposed to saliva and lipopolysaccharides (LPS) with and without PRF lysates obtained by repeated freeze-thawing or the secretome of PRF membranes, termed PRF conditioned medium. The expression of the M1 marker genes interleukin 1ß (IL1ß) and interleukin 6 (IL6) along with the M2 markers arginase-1 and chitinase-like 3 (Chil3 or YM1) were evaluated by real time polymerase chain reaction. Immunoassay and immunofluorescence staining were performed for IL6 and p65 translocation, a subunit nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), respectively. RESULTS: We report here that PRF lysates and PRF conditioned medium, the latter containing the secretome, greatly decreased the proinflammatory response of primary macrophages and RAW 264.7 cells as indicated by the expression of IL1ß and IL6. The anti-inflammatory activity of PRF lysates was further confirmed by IL6 immunoassay. Moreover, PRF lysates suppressed the translocation of p65 from the cytoplasm into the nucleus after incubation with saliva. In support of M2 polarization, PRF lysates and PRF conditioned medium enhanced the expression of arginase-1 and YM1 in primary macrophages. CONCLUSION: Our results indicate that PRF holds an anti-inflammatory activity and shifts the macrophage polarization from an M1 toward an M2 phenotype.

Platelet-rich fibrin suppresses in vitro osteoclastogenesis.

BACKGROUND: Platelet-rich fibrin (PRF) membranes can preserve alveolar ridge dimension after tooth extraction. Thus, it can be presumed that PRF suppresses the catabolic events that are caused by osteoclastic bone resorption. METHODS: To address this possibility, we investigated the impact of soluble extracts of PRF membranes on in vitro osteoclastogenesis in murine bone marrow cultures. Osteoclastogenesis was induced by exposing murine bone marrow cultures to receptor activator of nuclear factor kappa B ligand (RANKL), macrophage colony-stimulating factor (M-CSF) and transforming growth factor-beta 1 (TGF-ß1) in the presence or absence of PRF. Osteoclastogenesis was evaluated based on histochemical, gene expression, and resorption analysis. Viability was confirmed by formation of formazan crystals, live-dead staining and caspase-3 activity assay. RESULTS: We report here that in vitro osteoclastogenesis is greatly suppressed by soluble extracts of PRF membranes as indicated by tartrate-resistant acid phosphatase (TRAP) staining and pit formation. In support of the histochemical observations, soluble extracts of PRF membranes decreased expression levels of the osteoclast marker genes TRAP, Cathepsin K, dendritic cell-specific transmembrane protein (DCSTAMP), nuclear factor of activated T-cells (NFATc1), and osteoclast-associated receptor (OSCAR). PRF membranes, however, cannot reverse the process once osteoclastogenesis has evolved. CONCLUSION: These in vitro findings indicate that PRF membranes can inhibit the formation of osteoclasts from hematopoietic progenitors in bone marrow cultures. Overall, our results imply that the favorable effects of PRF membranes in alveolar ridge preservation may be attributed, at least in part, by the inhibition of osteoclastogenesis.

Potential of Treated Dentin Matrix Xenograft for Dentin-Pulp Tissue Engineering.

INTRODUCTION: This study aims to develop and characterize the regenerative potential of an atelopeptidized treated dentin matrix xenograft using in vitro and in vivo models. METHODS: Freshly extracted bovine dentin was pulverized into 250- to 500-µm particles and demineralized with 17% EDTA for 1, 7, and 13 days. The samples were atelopeptidized with pepsin. The degree of demineralization and the effect of atelopeptidization were assessed using field emission scanning electron microscopy combined with energy-dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy, respectively. The expression of dentin matrix acidic phosphoprotein 1, dentin sialophosphoprotein, and osteopontin was evaluated in dental pulp stem cells using quantitative real-time polymerase chain reaction. The samples were then implanted intramuscularly in rats for 30 days, and the inflammatory cells were quantified histologically. RESULTS: Field emission scanning electron microscopy combined with energy-dispersive X-ray spectroscopy revealed an exposed tubular structure of dentin after 1 and 7 days of demineralization. Fourier transform infrared spectroscopy confirmed the absence of amide peaks at 1260 to 1640/cm after atelopeptidization. The dental pulp stem cell expression of dentin matrix acidic phosphoprotein 1 and dentin sialophosphoprotein increased in all compared with the untreated control group (P < .05). The maximum expression rates were observed for the 1-day demineralized and atelopeptidized group. The 1-day demineralized group elicited the highest inflammatory response compared with the 7- or 13-day demineralized groups (P < .001). Atelopeptidization significantly decreased the inflammatory response only in the 1-day demineralized dentin group (P < .05). CONCLUSIONS: Atelopeptidization of 1-day demineralized dentin xenograft preserved the collagen structure, minimized the immune reaction, and provided sufficient regenerative potential.

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.

Exploring single-domain antibody thermostability by molecular dynamics simulation.

Single-domain antibodies also known as nanobodies are recombinant antigen-binding domains that correspond to the heavy-chain variable region of camelid antibodies. Previous experimental studies showed that the nanobodies have stable and active structures at high temperatures. In this study, the thermal stability and dynamics of nanobodies have been studied by employing molecular dynamics simulation at different temperatures. Variations in root mean square deviation, native contacts, and solvent-accessible surface area of the nanobodies during the simulation were calculated to analyze the effect of different temperatures on the overall conformation of the nanobody. Then, the thermostability mechanism of this protein was studied through calculation of dynamic cross-correlation matrix, principal component analyses, native contact analyses, and root mean square fluctuation. Our results manifest that the side chain conformation of some residues in the complementarity-determining region 3 (CDR3) and also the interaction between α-helix region of CDR3 and framework2 play a critical role to stabilize the protein at a high temperature. Communicated by Ramaswamy H. Sarma.

Rapid screening of drug candidates against EGFR/HER2 signaling pathway using fluorescence assay.

Over the recent decade, the calcium-based assays have gained much popularity in order to discover new drugs. Since breast cancer is the second cause of death in the female population, rapid and effective methods are needed to screen drug compounds with fewer side effects. Human epidermal growth factor receptor 2 (HER2) increases intracellular free Ca2+ on its signaling pathways. In the present study, BT474 cell line, which overexpresses HER2 receptor, was selected and using fura-2-AM, intracellular Ca2+ release was investigated. The changes in the concentration of intracellular Ca2+ were evaluated by variation in the amount of fluorescence intensity. In the presence of epidermal growth factor (EGF), an increase in fluorescence intensity was observed so that after 20 min it raised to the maximum level. After treatment of BT474 cells by lapatinib, as a tyrosine kinase inhibitor (TKI), the signaling pathway of EGFR/HER2 heterodimer was significantly inhibited, which resulted in a decrease in Ca2+ entry into the cytoplasm and fluorescence emission decreased. The IC50 value for the effect of lapatinib on BT474 cells was 113.2 nmol/L. Our results suggest this method is a simple, efficient and specific approach and can potentially be useful for screening new drug candidates against EGFR/HER2 heterodimer signaling pathways. Graphical abstract ᅟ.

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.

Zein nanoparticle as a novel BMP6 derived peptide carrier for enhanced osteogenic differentiation of C2C12 cells.

Zein nanoparticles as a carrier system for BMP6-derived peptide were prepared by liquid-liquid phase separation procedure and characterized with SEM, DLS, FTIR and thermogravimetric methods. After peptide encapsulation, nanoparticle size increased from 236.3 ± 92.2 nm to 379.4 ± 116.8 nm. The encapsulation efficiency of peptide was 72.6% and the release of peptide from Zein nanoparticles was partly sustained in trypsin containing phosphate buffered saline (pH 7.4) for up to 14 days. Peptide-loaded nanoparticles showed similar cell viability compared with blank ones. ALP activity of C2C12 cells treated with peptide-loaded nanoparticles (500 µg/mL) was evaluated 7, 14, 21 and 28 days after culture. In peptide-loaded nanoparticles, ALP activity was significantly higher (p < .05) compared with other groups at day 14. Alizarin Red S staining showed, C2C12 cells behind peptide-loaded nanoparticles had significantly (p < .05) higher calcium deposition at day 21. The results of RT-qPCR show that the BMP-6 peptide activated expression of RUNX2 as a transcription factor. In turn, RUNX2 regulates SPP1 and BGLAP gene expression, as osteogenic marker genes. The results confirm that the peptide-loaded Zein nanoparticles, as osteoinductive material, may be used to repair small area of bone defects, with low load bearing.

Co-expression of alpha-1 antitrypsin with cytoplasmic domain of v-SNARE in Pichia pastoris: Preserving biological activity of alpha-1 antitrypsin.

Alpha-1-antitrypsin (A1AT) is a major serum protein in human with protease inhibitory activity. Because of its extensive application in medicine, recombinant DNA technology has been considered for its production. The current study examines coexpression of A1AT and soluble domain of v-SNARE in Pichia pastoris, which can prevent the secretion of A1AT after thoroughly passing the secretory pathway. This was done mainly to preserve the biological activity of A1AT, which in the secretory mode might be impaired in the fermentation and early clarification conditions. SNARE proteins are the driving force for vesicle docking and membrane fusion in the exocytosis. Intracellular expression of the cytoplasmic domain of v-SNARE and its subsequent interaction to form SNARE complex can intensify the competition for A1AT secretory vesicles to be fused and released to the media. Our investigation shows successful coexpression of A1AT in the form of post-Golgi vesicles and the cytoplasmic domain of v-SNARE. Our findings confirmed the reduction of A1AT secretion by 45% caused accumulation of post-Golgi secretory vesicles filled with A1AT inside the yeast cell. A1AT trapped in secretory vesicles were biologically more active than secretory A1AT. These results indicate that the inhibition of A1AT secretion can protect its biological activity in fermentation and clarification processes.

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 investigation of epsilon toxin effects on different cancerous cell lines and its synergism effect with methotrexate.

BACKGROUND: The overall goal of this study is to use a bacterial toxin as drug delivery agents for chemotherapy drugs and overcome the development of resistance to these medicines. COR-L105 and MDA-MB 231 which are epithelial-like were used in this study. Cytotoxicity assays were performed by 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as metabolic indicator. The toxin was essential to kill 50% (CT50) and IC 50 value (inhibition growth value) for methotrexate were determined as optical density at 540 nm. Epsilon toxin-loaded PLGA nanoparticles were prepared using non-aqueous technique. Surface morphology, in vitro drug release, and encapsulation efficiency of the nanoparticles was determined. RESULTS: Results confirmed that using non-toxic concentration of epsilon toxin, resistance to cancerous cell decreased significantly, which could be an important result in cancer therapy. The synergistic effect of MTX and epsilon toxin showed that bio toxins can be used as supplement with chemical drugs and increase the effect of chemotherapy. The results illustrated that application of PLGA as drug delivery system due to its controlled release properties was beneficial. CONCLUSION: These finding proposed that due to the ease of local accessibility of lung tumors with aerosol drug delivery, biotoxins can directly be used with chemotherapy drugs in aerosol form.