Construction of an engineered alpha 1-antitrypsin with inhibitory activity based on theoretical studies

Background: The elastase inhibitor \u3b1-1-antitrypsin (AAT), is a member of the serpin superfamily of protease inhibitors. AAT has a characteristic secondary structure of three-\u3b2-sheets, nine-\u3b1-helices and a reactive central loop (RCL). This protein inhibits target proteases by forming a stable complex in which the cleaved RCL is inserted into \u3b2-sheet-A of the serpin, leading to a conformational change in the AAT protein. Spontaneous polymerization and instability of AAT are challenges with regard to producing drugs against AAT-deficient diseases. Therefore, the purpose of many investigations currently is to produce drugs with lower degrees of polymerization and higher stabilities. In order to investigate the effect of the N-terminal segment (residues 1-43) on AAT structure, molecular dynamic (MD) simulation was used to study structural properties including Root-mean-square deviation (RMSD), internal motions, intramolecular non-bonded interactions and the total accessible surface area (ASA) of native and reduced AAT. These properties were compared in native and truncated AAT. Results: Theoretical studies showed no noticeable differences in the dynamic and structural properties of the two structures. These findings provided the basis for the experimental phase of the study in which sequences from the two AAT constructs were inserted into the expression vector pGAPZ and transformed into Pichia pastoris. Results showed no differences in the activities and polymerization of the two AAT constructs. Conclusions: As small-scale medicines are preferred by lung drug delivery systems, in this study AAT was designed and constructed by decreasing the number of amino acids at the N-terminal region

Encapsulation of Alpha-1 antitrypsin in PLGA nanoparticles: In Vitro characterization as an effective aerosol formulation in pulmonary diseases

Abstract Background Alpha 1- antitrypsin (α1AT) belongs to the superfamily of serpins and inhibits different proteases. α1AT protects the lung from cellular inflammatory enzymes. In the absence of α1AT, the degradation of lung tissue results to pulmonary complications. The pulmonary route is a potent noninvasive route for systemic and local delivery. The aerosolized α1AT not only affects locally its main site of action but also avoids remaining in circulation for a long period of time in peripheral blood. Poly (D, L lactide-co glycolide) (PLGA) is a biodegradable and biocompatible polymer approved for sustained controlled release of peptides and proteins. The aim of this work was to prepare a wide range of particle size as a carrier of protein-loaded nanoparticles to deposit in different parts of the respiratory system especially in the deep lung. Various lactide to glycolide ratio of the copolymer was used to obtain different release profile of the drug which covers extended and rapid drug release in one formulation. Results Nonaqueous and double emulsion techniques were applied for the synthesis of nanoparticles. Nanoparticles were characterized in terms of surface morphology, size distribution, powder X-ray diffraction (XRD), encapsulation efficiency, in vitro drug release, FTIR spectroscopy and differential scanning calorimetry (DSC). To evaluate the nanoparticles cytotoxicity, cell cytotoxicity test was carried out on the Cor L105 human epithelial lung cancer cell line. Nanoparticles were spherical with an average size in the range of 100 nm to 1μ. The encapsulation efficiency was found to be higher when the double emulsion technique was applied. XRD and DSC results indicated that α1AT encapsulated in the nanoparticles existed in an amorphous or disordered-crystalline status in the polymer matrix. The lactic acid to glycolic acid ratio affects the release profile of α1AT. Hence, PLGA with a 50:50 ratios exhibited the ability to release %60 of the drug within 8, but the polymer with a ratio of 75:25 had a continuous and longer release profile. Cytotoxicity studies showed that nanoparticles do not affect cell growth and were not toxic to cells. Conclusion In summary, α1AT-loaded nanoparticles may be considered as a novel formulation for efficient treatment of many pulmonary diseases.</p

The Construction of Chimeric T-Cell Receptor with Spacer Base of Modeling Study of VHH and MUC1 Interaction

Adaptive cell immunotherapy with the use of chimeric receptors leads to the best and most specific response against tumors. Chimeric receptors consist of a signaling fragment, extracellular spacer, costimulating domain, and an antibody. Antibodies cause immunogenicity; therefore, VHH is a good replacement for ScFv in chimeric receptors. Since peptide sequences have an influence on chimeric receptors, the effect of peptide domains on each other's conformation were investigated. CD3Zeta, CD28, VHH and CD8α, and FcgIIα are used as signaling moieties, costimulating domain, antibody, and spacers, respectively. To investigate the influence of the ligation of spacers on the conformational structure of VHH, models of VHH were constructed. Molecular dynamics simulation was run to study the influence of the presence of spacers on the conformational changes in the binding sites of VHH. Root mean square deviation and root mean square fluctuation of critical segments in the binding site showed no noticeable differences with those in the native VHH. Results from molecular docking revealed that the presence of spacer FcgIIα causes an increasing effect on VHH with MUC1 interaction. Each of the constructs was transformed into the Jurkat E6.1. Expression analysis and evaluation of their functions were examined. The results showed good expression and function

sibling conflicts, gender and parenting patterns in students

Sibling conflicts are one of the most common forms of aggression in families. In this study, the method of child raising, as parents' controlling function and, gender, as one of the factors determining parents' behavioral patterns towards their children, were investigated in relation to sibling conflicts. The study is a correlational one and its population includes all secondary school girl and boy students in the city of Robat Karim during 1390-1391. From the population, a sample of 124 students and their families were selected through multi-stage cluster sampling. The data of the study were collected through the questionnaires of «IBR and RIS» Further, a questionnaire was used to gather the participants' demographic information. The results of correlations indicated that sibling conflicts had a negative relationship with the parents' authority and Stringent methods of child raising but it did not have a significant relationship with inconsiderate child raising. The results of stepwise regression analysis showed that the parents' authority child raisin g was the strongest predictor of the extent of sibling conflicts; girls reported more conflicts with both their sisters and their brothers than boys. In addition, the results of the study indicated that boys reported more conflicts with their younger brothers and girls reported more conflicts with their elder brothers and younger sisters. According to the above findings, parents' child birth patterns and children's' genders are significant in predicting the extent of sibling conflict and aggression

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-mu m 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