Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR

Darbepoetin alfa is an engineered and hyperglycosylated analog of recombinant human erythropoietin (EPO) which is used as a drug in treating anemia in patients with chronic kidney failure and cancer. This study desribes the secretory expression of a codon-optimized recombinant form of darbepoetin alfa in Leishmania tarentolae T7-TR. Synthetic codon-optimized gene was amplified by PCR and cloned into the pLEXSY-I-blecherry3 vector. The resultant expression vector, pLEXSYDarbo, was purified, digested, and electroporated into the L. tarentolae. Expression of recombinant darbepoetin alfa was evaluated by ELISA, reverse-transcription PCR (RT-PCR), Western blotting, and biological activity. After codon optimization, codon adaptation index (CAI) of the gene raised from 0.50 to 0.99 and its GC content changed from 56 to 58. Expression analysis confirmed the presence of a protein band at 40 kDa. Furthermore, reticulocyte experiment results revealed that the activity of expressed darbepoetin alfa was similar to that of its equivalent expressed in Chinese hamster ovary (CHO) cells. These data suggested that the codon optimization and expression in L. tarentolae host provided an efficient approach for high level expression of darbepoetin alfa. © 2015 Elsevier Ltd. All rights reserved

Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae

Darbepoetin alfa is a biopharmaceutical glycoprotein that stimulates erythropoiesis and is used to treat anemia, which associated with renal failure and cancer chemotherapy. We herein describe the structural characterization of recombinant darbepoetin alfa produced by Leishmania tarentolae T7-TR host. The DNA expression cassette was integrated into the L. tarentolae genome through homologous recombination. Transformed clones were selected by antibiotic resistance, diagnostic PCRs, and protein expression analysis. The structure of recombinant darbepoetin alfa was analyzed by isoelectric focusing, ultraviolet–visible spectrum, and circular dichroism (CD) spectroscopy. Expression analysis showed the presence of a protein band at 40 kDa, and its expression level was 51.2 mg/ml of culture medium. Darbepoetin alfa have 5 isoforms with varying degree of sialylation. The UV absorption and CD spectra were analogous to original drug (Aranesp), which confirmed that the produced protein was darbepoetin alfa. Potency test results revealed that the purified protein was biologically active. In brief, the structural and biological characteristics of expressed darbepoetin alfa were very similar to Aranesp which has been normally expressed in CHO. Our data also suggest that produced protein has potential to be developed for clinical use. © 2016, Springer Science+Business Media New York

Algorithms for crime prediction in smart cities through data mining

The concentration of police resources in conflict zones contributes to the reduction of crime in the region and the optimization of those resources. This paper presents the use of regression techniques to predict the number of criminal acts in Colombian municipalities. To this end, a set of data was generated merging the data from the Guardia Civil with public data on the demographic structure and voting trends in the municipalities. The best regressor obtained (Random Forests) achieves a RRSE (Root Relative Squared Error) of 40.12% and opens the way to keep incorporating public data of another type with greater predictive power. In addition, M5Rules were used to interpret the results

Cloning and expression of human recombinant erythropoietin in Leishmania tarentolae

Background and Objective: Human erythropoietin (EPO) is a glycosylated hormone with molecular weight of about 40 KDa which is synthesized in kidneys and plays an important role in proliferation and differentiation of erythrocytes.This study was done to assess and analyze the expression of recombinant EPO in Leishmania tarentolae host. Method: In this descriptive study, the EPO gene was codoned , optimized with bioinformatics database prior to be synthesized. It was cleaved by KpnIandXbaI enzymes and cloned into pLEXSY expression vector. The constructed expression cassette was transfected into Leishmania tarentolae through electroporaton method. Identification and confirmation of transfected colonies was performed using PCR expression diagnostic primers and EPO specific primers. Induction of the cloned gene was done with tetracycline. The expression in induced strains was analyzed by SDS-PAGE and western blotting techniques. The amount of recombinant protein was quantified by ELISA method. Confirmation of cloning and EPO expression cassette was carried out through genetic engineering procedures. Results: Expression analysis of transfected parasitic strain with SDS-PAGE and western blotting confirmed gene integration into chromosomal of host as well as expression. The optimal conditions for expression were found to be 10μg of tetracycline and 72h induction time. Molecular weight of expressed protein estimated to be 40 KDa and expression level was determined to be 12.4 mg/l which was equal to 1% of total protein mass. Conclusion: EPO expression cassette for cloning and expression in Leishmania tarentolae was designed and protein of interest was successfully induced and identified Leishmania tarentolae can be used as a suitable host for production of recombinant EPO and this technology has a potential for localization

A Resilience-Based Architecture for Joint Distributed Energy Resources Allocation and Hourly Network Reconfiguration

As a result of the recent innovations in the deployment of plug-in electric vehicles (PEVs), this technology can play an important role as a distributed energy resource in supplying the system demand of the power systems of the future. This paper introduces a methodology for optimal coordinated allocation of wind farms (WFs), energy storage systems, and PEV's parking lots considering demand response programs and hourly distribution network reconfiguration in normal and severe contingency conditions. In the proposed methodology, the participation of different types of loads is also examined. The objective function is to minimize the total costs of purchased power from the upstream network and WFs, along with the costs of commercial/industrial loads flexibility and residential loads curtailment. To validate the performance of the proposed methodology, it is implemented on the well-known IEEE 33-bus distribution test system. The simulation results validate the feasibility and effectiveness of the proposed approach

Outcomes of Phaco-viscocanalostomy in Primary Open Angle Glaucoma Versus Pseudoexfoliation Glaucoma

Purpose: Viscocanalostomy represents an alternative to standard penetrating glaucoma surgery. The aim of this study is to compare the outcomes of combined phacoemulsification and viscocanalostomy in eyes with primary open-angle glaucoma (POAG) versus eyes with pseudoexfoliation glaucoma (PEXG). Methods: In this prospective non-randomized comparative study, eyes with Cataract and POAG or PEXG were enrolled. Pre- and postoperative data including best corrected visual acuity (BCVA), intraocular pressure (IOP), and the number of antiglaucoma medications administered were recorded at each visit. All patients underwent phacoviscocanalostomy. Complete success was defined as the IOP of 21 mmHg or less without the administration of medication while a qualified success reported the same IOP parameters either with or without the administration of medication. Results: Fifty-four eyes with POAG and fifty-four with PEXG underwent phacoviscocanalostomy. The mean follow-up time was 23.36 ± 8.8 months (range, 6–40 months). The mean postoperative IOP reduced significantly in both groups, although the mean IOP reduction was significantly greater in PEXG eyes (14.7 ± 8.9 vs 10.1 ± 7.7 mmHg) (P = 0.05). At the final follow-up visit, the mean postoperative IOP was 14.1 ± 2.1 and 16.6 ± 3.5 mmHg in the PEXG and POAG eyes, respectively (P = 0.001). A complete success rate of 88.9% and 75.9% was achieved in PEXG and POAG eyes, respectively (P = 0.07). The qualified success rate was 100% in the PEXG and 85.2% in POAG groups (P = 0.03). Conclusion: Phacoviscocanalostomy achieved significant IOP reduction and visual improvement in both POAG and PEXG patients. Our results indicated that in terms of IOP reduction, this procedure was more effective in treating PEXG