Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin

Reza Ahangari Cohan1, Armin Madadkar-Sobhani2,3, Hossein Khanahmad1, Farzin Roohvand4, Mohammad Reza Aghasadeghi4, Mohammad Hossein Hedayati5, Zahra Barghi5, Mehdi Shafiee Ardestani4, Davoud Nouri Inanlou1, Dariush Norouzian11Research and Development Department, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran; 2Department of Bioinformatics, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; 3Department of Life Sciences, Barcelona Supercomputing Center, Barcelona, Spain; 4Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran; 5Quality Control Department, Production and Research Complex, Pasteur Institute of Iran, Tehran, IranBackground: Recombinant human erythropoietin (rhEPO) is considered to be one of the most pivotal pharmaceutical drugs in the market because of its clinical application in the treatment of anemia-associated disorders worldwide. However, like other therapeutic proteins, it does not have suitable pharmacokinetic properties for it to be administrated at least two to three times per week. Chemoselective cysteine PEGylation, employing molecular dynamics and graphics in in silico studies, can be considered to overcome such a problem.Methods: A special kind of EPO analog was elicited based on a literature review, homology modeling, molecular dynamic simulation, and factors affecting the PEGylation reaction. Then, cDNA of the selected analog was generated by site-directed mutagenesis and subsequently cloned into the expression vector. The construct was transfected to Chinese hamster ovary/dhfr- cells, and highly expressed clones were selected via methotrexate amplification. Ion-immobilized affinity and size exclusion (SE) chromatography techniques were used to purify the expressed analog. Thereafter, chemoselective PEGylation was performed and a nanosize PEGylated EPO was obtained through dialysis. The in vitro biologic assay and in vivo pharmacokinetic parameters were studied. Finally, E31C analog Fourier transform infrared, analytical SE-high-performance liquid chromatography, zeta potential, and size before and after PEGylation were characterized.Results: The findings indicate that a novel nanosize EPO31-PEG has a five-fold longer terminal half-life in rats with similar biologic activity compared with unmodified rhEPO in proliferation cell assay. The results also show that EPO31-PEG size and charge versus unmodified protein was increased in a nanospectrum, and this may be one criterion of EPO biologic potency enhancement.Discussion: This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.Keywords: nanoPEGylated EPO, cysteine PEGylation, pharmacokinetic propert

Effects of Long-term Induced Hyperthyroidism on Egg Quality Traits in Cobb 500 Broiler Breeder Hens

Published data on 4-week-long administration of exogenous thyroxine in broiler breeder hens to decline the cold-induced ascites in their progeny suggest that the long-term maternal hyperthyroidism would affect egg quality characteristics traits in Cobb 500 broiler breeder hens. Seventy 47-w-old broiler breeder Cobb 500 hens (5 replicates and 7 hens each) were assigned in separate cages and allotted to two treatments, control and hyperthyroid. Thyroxine was orally administered to the hyperthyroid group (0.3 mg hen/day) for a period of 100 days consecutively. Simultaneously, distilled water was orally administered to the control group. The blood sampling was done every two weeks to analyze T3, T4, and estrogen assays, using commercially kits and the egg quality attributes were evaluated for weeks 0, 3, 6, 9, and 12. Thyroxine treatment resulted in an increase in plasma concentration of T4; however, the T3 level and estrogen were not affected. The results of this study showed that the long-term administration of thyroxine had adverse effect on the most of egg quality traits in broiler breeder hens; although the results may be distinct for treatments that using other birds, doses and duration of treatments, among the different egg quality traits of broiler breeder hens evaluated in this research. Therefore, further studies should be done to make a final conclusion to use of long-term maternal hyperthyroidism treatment to reduce the ascites incidence

Empirical investigation to explore potential gains from the amalgamation of Phase Changing Materials (PCMs) and wood shavings

The reduction of gained heat, heat peak shifting and the mitigation of air temperature fluctuations are some desirable properties that are sought after in any thermal insulation system. It cannot be overstated that these factors, in addition to others, govern the performance of such systems thus their effect on indoor ambient conditions. The effect of such systems extends also to Heating, Ventilation and Air-conditioning (HVAC) systems that are set up to operate optimally in certain conditions. Where literature shows that PCMs and natural materials such as wood-shavings can provide efficient passive insulation for buildings, it is evident that such approaches utilise methods that are of a degree of intricacy which requires specialist knowledge and complex techniques, such as micro-encapsulation for instance. With technical and economic aspects in mind, an amalgam of PCM and wood-shavings has been created for the purpose of being utilised as a feasible thermal insulation. The amalgamation was performed in the simplest of methods, through submerging the wood shavings in PCM. An experimental procedure was devised to test the thermal performance of the amalgam and compare this to the performance of the same un-amalgamated materials. Comparative analysis revealed that no significant thermal gains would be expected from such amalgamation. However, significant reduction in the total weight of the insulation system would be achieved that, in this case, shown to be up to 20.94%. Thus, further reducing possible strains on structural elements due to the application of insulation on buildings. This can be especially beneficial in vernacular architectural approaches where considerably large amounts and thicknesses of insulations are used. In addition, cost reduction could be attained as wood shavings are significantly cheaper compared to the cost of PCMs

Design of a Constrained Nonlinear Controller using Firefly Algorithm for Active Suspension System

Active vehicle suspension system is designed to increase the ride comfort and road holding of vehicles. Due to limitations in the external force produced by actuator, the design problem encounters the constraint on the control input. In this paper, a novel nonlinear controller with the input constraint is designed for the active suspension system. In the proposed method, at first, a constrained multi-objective optimization problem is defined. In this problem, a performance index is defined as a weighted combination of the predicted responses of the nonlinear suspension system and control input. Then, this problem is solved by the modified firefly optimization algorithm to find the constrained optimal control input. To evaluate the performance of the proposed method, the results of the unconstrained and constrained controllers are provided and discussed for various road excitations. The results show a remarkable increase in the ride comfort with the limited force, while other suspension outputs including the suspension travel and tire deflection being in the acceptable ranges. In addition, these controllers are compared with Sliding Mode Control (SMC) and Nonlinear Model Predictive Control (NMPC) in the presence of model uncertainty

Sequencing and expression of Elastase II gene in Polymorphoneuclear leukocytes in healthy individuals for determination of single nucleotide polymorphism (SNP) by RT-PCR

Background: ELA2 gene responsible for coding human neutrophil elastase, a powerful serine protease carried by blood neutrophils and capable of destroying most connective tissue proteins. The NE gene consist of 5 exons and 4 introns. Methods: Peripheral blood obtained from healthy individuals. Total RNA was isolated using RNA standard techniques from fresh separated cells by polymorphoprep. RNA was analyzed by employing PCR amplification of reveres transcribed using a total of ten specific primers. We amplified five exon of ELA2 gene separately and sequenced each exon. Mutational analysis was performed by directed capillary sequencing method. Findings: We have found new single nucleotide polymorphism (SNP) in exon II codon 44. It was a silent mutation G to A substitution but no changes in amino acid sequences were seen. The codon sequence was GCG that has changed to GCA. Conclusion: The purpose of SNP detection is mainly recognition of susceptible people to certain diseases and providing suitable drugs based on each person genetic information. SNP information in Iranian patients can be used for analysis of drug response to human diseases

RT-PCR Based Mutation Detection of the Inflammatory Molecules Elastase II Gene Encoding Neutrophil Elastase in Cyclic Neutropenia Patients by Capillary Sequencing

Cyclic neutropenia is characterized by the maturation arrest of myeloid cells at the promyelocyte stage of hematopoiesis. In cyclic neutropenia, due to the disorder of neutrophil production, patients are predisposed to recurrent bacterial infections. Detected mutations in neutrophil elastase (ELA2) gene in genomic DNA of cyclic neutropenia were recently reported. Peripheral blood was obtained from 18 patients with cyclic neutropenia and 20 healthy individuals. Total RNA was isolated using RNA standard techniques from fresh separated cells by polymorphoprep. Elastase II mRNA expression was analyzed by employing reverse transcription PCR amplification using a total of ten specific primers. We amplified five exon of ELA2 gene separately and sequenced each exon. Mutational analysis was performed by directed capillary sequencing method. We found mutations in 15 out of 18 cyclic neutropenia patients (83%) and no mutation in 20 healthy individuals. Most of the mutations were in exon 4 and fewer mutation were found in exon 1