Optimization and One-Step Purification of Recombinant V Antigen Production from Yersinia pestis

The purpose of this study was to develop an efficient and inexpensive method for the useful production of recombinant protein V antigen, an important virulence factor for Yersinia pestis. To this end, the synthetic gene encoding the V antigen was subcloned into the downstream of the intein (INT) and chitin-binding domain (CBD) from the pTXB1 vector using specific primers. In the following, the produced new plasmid, pTX-V, was transformed into E. coli ER2566 strain, and the expression accuracy was confirmed using electrophoresis and Western blotting. In addition, the effects of medium, inducer, and temperature on the enhancement of protein production were studied using the Taguchi method. Finally, the V antigen was purified by a chitin affinity column using INT and CBD tag. The expression was induced by 0.05 mM IPTG at 25 °C under optimal conditions including TB medium. It was observed that the expression of the V-INT�CBD fusion protein was successfully increased to more than 40 of the total protein. The purity of V antigen was as high as 90. This result indicates that V antigen can be produced at low cost and subjected to one-step purification using a self-cleaving INT tag. © 2020, Springer Science+Business Media, LLC, part of Springer Nature

Optimal predictive neuro-navigator design for mobile robot navigation with moving obstacles

Introduction: The challenge of navigating a Mobile robot in dynamic environments has grasped significant attention in recent years. Despite the available techniques, there is still a need for efficient and reliable approaches that can address the challenges of real-time near optimal navigation and collision avoidance.Methods: This paper proposes a novel Log-concave Model Predictive Controller (MPC) algorithm that addresses these challenges by utilizing a unique formulation of cost functions and dynamic constraints, as well as a convergence criterion based on Lyapunov stability theory. The proposed approach is mapped onto a novel recurrent neural network (RNN) structure and compared with the CVXOPT optimization tool. The key contribution of this study is the combination of neural networks with model predictive controller to solve optimal control problems locally near the robot, which offers several advantages, including computational efficiency and the ability to handle nonlinear and complex systems.Results: The major findings of this study include the successful implementation and evaluation of the proposed algorithm, which outperforms other methods such as RRT, A-Star, and LQ-MPC in terms of reliability and speed. This approach has the potential to facilitate real-time navigation of mobile robots in dynamic environments and ensure a feasible solution for the proposed constrained-optimization problem

A review on the heat and mass transfer phenomena in nanofluid coolants with special focus on automotive applications

Engineered suspensions of nanosized particles (nanofluids) are characterized by superior thermal properties. Due to the increasing need for ultrahigh performance cooling in many industries, nanofluids have been widely investigated as next-generation coolants. However, the multiscale nature of nanofluids implies nontrivial relations between their design characteristics and the resulting thermo-physical properties, which are far from being fully understood. This pronounced sensitivity is the main reason for some contradictory results among both experimental evidence and theoretical considerations presented in the literature. In this Review, the role of fundamental heat and mass transfer mechanisms governing thermo-physical properties of nanofluids is assessed, from both experimental and theoretical point of view. Starting from the characteristic nanoscale transport phenomena occurring at the particle-fluid interface, a comprehensive review of the influence of geometrical (particle shape, size and volume concentration), physical (temperature) and chemical (particle material, pH and surfactant concentration in the base fluid) parameters on the nanofluid properties was carried out. Particular focus was devoted to highlight the advantages of using nanofluids as coolants for automotive heat exchangers, and a number of design guidelines was suggested for balancing thermal conductivity and viscosity enhancement in nanofluids. This Review may contribute to a more rational design of the thermo-physical properties of particle suspensions, therefore easing the translation of nanofluid technology from small-scale research laboratories to large-scale industrial applications

Overview of Optimization Techniques in Geometric Path Planning for Mobile Robots with a new smooth PSO-IPF design

In this paper, we explore the practical implications of our research, offering significant advantages to the autonomous and robotics sector. Our focus revolves around enhancing geometric path planning for mobile robots, a pivotal aspect of automation. Notably, we not only delve into how to formulate optimal navigation problems while considering practical constraints and applications, but we also introduce a novel Smoothed PSO-IPF algorithm. This algorithm serves as an illustrative example of an innovative and context-specific approach to addressing navigation challenges. It furnishes engineers and practitioners in the field with a comprehensive framework for designing navigational solutions. By presenting the PSO-IPF method as a hybrid approach, we effectively bridge the gap between classical and reactive methods. Consequently, it leads to enhanced navigation efficiency, reduced collisions, and heightened mobile robot reliability. This innovation not only optimizes navigation issues but also underscores its potential for diverse applications across various industries.</p

Cloning and Expression of Recombinant Immunotoxin using Diphtheria Toxin and Granulocyte Colony Stimulating Factor (G-CSF)

Abstract Background: Immunotoxin (IT) is a directed toxin containing two distinct sections (immune and toxin parts) covalently bond using specific chemical or peptide linkers. The aim of this study is to produce a recombinant and hybrid protein containing diphtheria toxin (DT) and granulocyte colony stimulating factor (G-CSF). Materials and Methods: According to the structure of the first commercial recombinant immunotoxin (Ontak, hybrid protein containing DT fused interlukine2), gene encoding of DT and G-CSF was amplified using specific primers and plasmid template of pET-IDZ3 (pET21 harboring the gene encoding ontak immunotoxine) and pET-GCSF (pET23 harboring G-CSF), respectively. The DT-GCSF fusion protein produced using soeing PCR and specific primers. Finally, pET-DT-GCSF construction prepared using subcloning of DT-GCSF into pET21a(+) and confirmed by sequencing, SDS-PAGE and western blot technique. Results: Gene encoding of DT-GCSF inserted into NdeI/EcoRI site of pET21 and the construction of strain producing DT-GCSF recombinant immunotoxin was confirmed using customary methods. Conclusion: The cytokine fusion protein, DT-GCSF, could be used for the inhibition of G-CSF receptor bearing cancer cells

The Modified Recombinant Proinsulin: A Simple and Efficient Way to Produce Insulin Glargine in E. coli

Background: Recombinant insulin glargine, a long-acting analog of insulin, is expressed as proinsulin in the host cell and, after purification and refolding steps, is processed to mature insulin by using trypsin and carboxypeptidase B. Because of several internal residues of arginine and lysine in the proinsulin B and C chains, several unwanted products are formed after treatment with these enzymes. To overcome this problem, we introduced three thrombin recognition sites into the proinsulin encoding sequence. Materials and methods: After the design, the modified proinsulin encoding sequence containing the 5′ His-Tag tail and three thrombin recognition sites located between the His-Tag and B chain, B and C chains, and C and A chains, respectively, was synthesized by overlap extension Polymerase Chain Reaction (PCR) using seven specific primers in multiple sequential PCR reactions. The final amplified fragment was cloned in the pGEM-5zf vector by the EcoRV enzyme. After sequencing, the modified proinsulin encoding sequence was subcloned into the pET-26b(+) vector using NdeI and XhoI enzymes. Finally, the modified proinsulin was expressed in E. coli BL-21(DE3) by induction with Isopropyl β-d-1-thiogalactopyranoside (IPTG). Results: The accuracy of the synthesized modified proinsulin sequence was confirmed by DNA sequencing. The modified proinsulin cloning was evaluated by PCR with specific primers and digestion with specific restriction enzymes. In this study, the modified proinsulin protein was expressed up to 40%. The modified proinsulin protein expression was assessed using sodium dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) and western blotting. Conclusions: This modified proinsulin can be used to easily and efficiently produce insulin glargine without any impurities after processing with thrombin in one step in a nickel chromatographic column

Designing and Construction of the Suicide Vector pDS132-ΔkanR to Delete Kanamycin Resistance Sequence in Mutant Strain of Brucella melitensis Rev1 Mutant Strain in order to Generate a Candidate Vaccine Strain

Background and Aim: Brucellosis is a common disease between humans and animals. Vaccination is the best approach to prevent brucellosis. Existing vaccines such as Rev1 are associated with disadvantages such as abortion in pregnant animals. Therefore, it is necessary to generate an appropriate vaccine that is produced by molecular methods. The aim of this study was designing and construction of pDS132-Δkanr suicide vector to delete kanamycin resistance sequence (kanR) in a mutant strain of Brucella melitensis rev1 to generate a candidate vaccine strain. Materials and Methods: In this study, the plasmid pDS132 was used as a suicide vector to delete target gene. In order to construct the deletion cassette, two homologous fragments were separately designed and constructed by PCR, and tandemly cloned into the pBluescriptSK(-) vector using appropriate restriction enzymes. Then, the deletion cassette was digested from the recombinant vector by terminal restriction enzymes, and sub cloned into the pDS132 vector to construct the suicide vector pDS132-ΔkanR.  Results: The pDS132-ΔkanR contains 590bp upstream sequences and 421bp downstream sequences on the deletion cassette fragment; it can be used as specific suicide vector to disrupt the kanR sequence in genome of mutant strain of Brucella melitensis Rev1. Conclusions: The use of suicide pDS132-ΔkanR system facilitated mutant construction which is a more specific and effective system in comparison with the other positive marker-dependent suicide systems and primary techniques such as serial passages

Molecular Detection of Zonula Occludens Toxin (zot) Genes in Vibrio Cholerae O1 using PCR

Background: Zot (Zonula occludens toxin) is one of the secretion toxins of Vibrio cholerae in small intestine that binds to certain receptors in the epithelial cells and causes a change in the structure of tight junction. The purpose of this research is rapid detection of zot enterotoxin gene using PCR. Materials and Methods: The genomic DNA was extracted by DNA isolation kit and gene amplification was carried out by the zot gene-specific primers. Then, PCR products were investigated by electrophoresis on 1.2% agarose gel stained by ethidium bromide. Also, the specificity of primers was measured using bacterial samples other than V. cholerae, such as enterotoxigenic Escherichia coli&nbsp;)ETEC(, Salmonela. typhi and Aeromonas hydrophila. The sensitivity of the PCR reaction was also evaluated using serial dilutions of V. cholerae O1 concentration (cfu/ml). Results: The data showed that the designed primers specificity for zot gene was successful and the sensitivity of this method was determined about 142 cfu/ml. Conclusion: In conclusion, this molecular detection can be used as a simple diagnostic kit in clinical laboratories for identification of V. cholerae. &nbsp