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ABSTRACT
Background and purpose:
Enterotoxigenic Escherichia coli is one of the most common bacterial causes of diarrhea worldwide.
One of the most important ETEC colonization factors is B subunit of the Heat Labile Enterotoxin, which forms the connecting part of the toxin. In the present study, egg yolk immunoglobulin (IgY) was produced and its effectiveness against recombinant LTB protein was tested on the animal model.
Material and methods:
Hens were injected intramuscularly with 100 μg of recombinant LTB protein. The antibody titer was estimated and the eggs were collected. The water soluble part of the yolk was separated and the antibody titer was subsequently estimated by ELISA.Effecacy of different concentrations of IgY on nutralising the effect of LT toxin on Y1 cells was also studied. In order to investigate the effect of IgY-treated bacteria on intestinal epithelial cells, the standard Ileal loop  technique was used.
Results:
Protein expression led to the production of recombinant LTB showing molecular weight of 11 kDa on SDS PAGE. Immunization of hens induced serum antibody rise. The purified antibody was 144 mg per 12 ml of egg yolk. Purified IgY exhibited significant effect against recombinant LTB proteinAt the concentration of 125 μg per ml, the IgY could prevent the effects of Heat Labile Enterotoxin on Y1 cells. In the Ileal loop test, 1.5 mg / ml IgY neutralized the toxin effect of LTB   on the intestine. The accumulation of fluid in the test loops decreased by 74.8% compared to the untreated control loops.
Conclusion:
The results showed that specific egg yolk immunoglobulin was effective against recombinant LTB protein and can be used as a preventive antibody to inhibit the Heat Labile Enterotoxin function of ETEC bacteria.

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Background and Aim: Increasing prevalence of nosocomial infections, due to arbitrary use of antibiotics, has caused serious health problems. Natural compounds have been used for many years for the treatment of many infections. Rare actinobacteria are one of the main sources used for discovery and isolation of natural compounds with pharmaceutical properties. The main purpose of this review article was to study new metabolites derived from rare actinobacteria, and evaluate their medicinal and pharmaceutical applications.
Materials and Methods: This study included over 183 peer-reviewed papers from the databases of Scopus, PubMed, Web of Science and ScienceDirect from 2015 to 2021.
Results: In this study chemical structure and biological activity of 113 new compounds isolated from rare actinobacteria were evaluated. This study also showed that rare actinobacteria have the capability to produce a variety of compounds such as alkaloids, flavonoids, terpenes and polyketides with a diverse range of biological activities including antibacterial, antifungal, antiviral and anticancer properties.
Conclusion: The results of this study revealed that rare actinobacteria can be considered as an excellent source for the production of natural active compounds with high potentials for therapeutic and applied medical, pharmaceutical and agricultural biotechnology. It also showed the key role of these compounds in the development of necessary drugs for humans in the near future

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Background and Aim: Genome editing is an efficient and accurate method in biological and medical studies. Among the wide range of genome editing techniques, clustered regularly interspaced short palindromic repeats (CRISPR) is one of the simplest and yet promising methods. The CRISPR system consists of two key components; an endonuclease enzyme called Cas9 and guide RNA (gRNA), ensuring that Cas9 enzyme cuts at the right point in the genome. Although CRISPR genome editing is one of the useful methods to modify the genome, there are still multiple challenges in the technique steps.
Materials and Methods: sgRNAs were designed and ordered accordingly on the basis of the target site and vector of interest. Cloning procedures were performed and confirmed by sequencing as a gold standard. The list of high efficient sgRNAs for the LAMP-2 gene was prepared based on the available outstanding databases by analyzing and comparing the specificity and functionality as two main characteristics.
Results: In this study, we tried to evaluate the main challenges in the process of preparing CRISPR/Cas9 popular vectors (pX330/pX459). A list of high efficient sgRNAs for an autophagy gene is also prepared as an example for clarification of the sgRNA design procedures.
Conclusion: This study tried to depict problems that may be encountered during sgRNA design and plasmid preparation, followed by giving appropriate recommendations.
 

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Background and Aim: Evaluation of the alkaline phosphatase expression is known as one of the diagnostic markers to confirm the stem cells differentiation into osteoblasts. Several methods, including immunocytochemistry, are used to measure alkaline phosphatase in different studies. Due to limitations of using this method, we decided to optimize the calorimetric method as an alternative, low cost and efficient method for quantitative assessment of alkaline phosphatase.
Materials and Methods: Mesenchymal stem cells derived from human adipose tissue were used. Cells were differentiated into osteoblasts using an induction medium. Alkaline phosphatase levels were assessed on the days 0 and 21 of differentiation using both immunocytochemistry and calorimetric methods.
Results: Immunocytochemistry findings demonstrated a significant increase (approximately 5-fold) in alkaline phosphatase expression level on the 21^st day of differentiation compared to that on the day 0 (p <0.0001). Also, statistical analysis of the results of the calorimetric test showed a significant increase (approximately 9-fold) in alkaline phosphatase expression level on the 21^st day of differentiation compared to  that on the day 0 (p<0.0001).
Conclusion: Comparison of the data between calorimetric and immunocytochemistry showed similar results. These findings suggested that colorimetric assay can be used as an alternative, quantitative, fast, and low cost method for determining the levels of alkaline phosphatase in osteoblasts differentiated from mesenchymal stem cells.
 

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Background and Aim: Caspase-9 is a key enzyme in the intrinsic pathway of apoptosis that its activity is regulated by various mechanisms such as phosphorylation. It has been reported that phosphorylation of serine 310 in murine caspase-9 prevents enzyme processing. The role of this residue in human caspase-9 activity in not clear. In this study we investigated the effect of negative charge on serine 310 in caspase-9 activity.
Materials and Methods: Considering that phosphorylation leads to negative charge on caspase-9, the codon of serine 310 in human capsase-9 was mutated to aspartate via quick change site-directed mutagenesis. Recombinant wild type and mutant caspase-9 were expressed in BL21(DE3) and purified by affinity chromatography. The temperature profile and activity of the mutant caspase-9 were assessed by chromogenic substrate of Ac-LEHD-pNA in vitro, and compared to those of wild enzyme. Student’s t test was used for data analysis.
Results: The results showed that kinetics parameters of S310D mutant and wild type caspase-9 were similar, but their temperature profiles were different. Comparison of S310D mutant enzyme and wild type caspase-9 showed that S310D mutant enzyme had higher activity at 37 ^oC and lower activity at 4, 15, 45 and 60 ^oC.
Conclusion: In our study, the negative charge on serine 310 in caspase-9 led to change in the profile temperature of the enzyme with no effect on kinetic parameters.

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Background and Aim: The current treatment for hemophilia B is replacement therapy, which involves the intravenous infusion of human coagulation factor IX (hFIX) purified from plasma or a recombinant form produced in mammalian cells. In this study, using a bicistronic expression system, the stable expression of the hFIX in a serum-free and suspension-adapted Chinese hamster ovary cell line (CHO-s) was investigated.
Materials and Methods: A DNA fragment consisting of hFIX, Internal Ribosome Entry Site (IRES) and Enhanced Green Fluorescent Protein (EGFP) nucleotide sequences was cloned into pcDNA3.1 expression plasmid under the control of Cytomegalovirus (CMV) promoter. The bicistronic plasmid was then linearized using BglII restriction enzyme and transfected into CHO-s cells. The transfected cells were treated with geneticin for 14 days. The culture medium of the stable cells was then collected and the expression level of the hFIX were examined using western blotting and ELISA. The coagulation activity was also evaluated by the chromogenic method.
Results: The recombinant CHO-s cells resistant to geneticin were observed under a fluorescence microscope in green color, which indicated the expression and accumulation of the EGFP in the cytoplasm of the cells. The results of Western blotting confirmed the expression and secretion of the hFIX into the culture medium. The amount of the secreted hFIX was 150 ng/mL/10⁶cells with a coagulation activity of 5.6 ±0.2 mU/mL.
Conclusion: Our findings demonstrated that this bicistronic expression system could simultaneously produce EGFP and hFIX in CHO-s cells. This expression system facilitates selection and isolation of hFIX-expressing cells.

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Background and Aim: Botulism, a syndrome caused by food poisoning, results from use of food contaminated with the botulinum toxin, which is very dangerous and deadly. Botulism is caused by the effects of bacterial toxins on the terminals of the motor nerves. Botulinum neurotoxins are among the most potent toxins. The aim of this study was to investigate expression, purification and, evaluation of the immunogenicity of the recombinant BoNT/B-HcC protein as an immunogen candidate in mice.
Materials and Methods: The C-terminus of the receptor-binding domain of  botulinum neurotoxin type B(BoNT/B-HcC) was selected as the antigen for bioinformatics evaluations. The pET17b-BoNT/B-HcC plasmid was transferred to E. coli BL21(DE3) by heat shock. The recombinant protein was purified and analyzed by SDS-PAGE. After verification of the recombinant protein by western blot, immunization of mice was performed. Antibody titers of recombinant proteins were evaluated by indirect ELISA and the results were compared using t-test.
Results: The codon adaptation index (CAI) of the optimized gene was 0.99. The percentage of codons having high-frequency distribution was improved to 78%. Restriction analysis confirmed the 1119 bp construct gene and the recombinant protein with a molecular weight of 43.8 kDa was expressed in the prokaryotic host. The total yield of purified protein was 23 mg of protein per liter of culture. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer was significantly different compared to that of the control sample.
Conclusion: The designed recombinant antigen showed high antigenicity that could be considered as an immunogen against botulinum type B neurotoxin in future studies.


 

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Background and Aim: Conventional treatments have shown little success in cancer treatment. Using antibody fragments conjugated with specific proteins and targeting specific receptors is a new strategy for producing anticancer drugs. Despite the advantages of prokaryotic hosts, protein expression in the form of inclusion bodies (cytoplasmic) has been challenging and leads to subsequent difficulties. Production of proteins as soluble forms will significantly help to solve this problem.
 The primary purpose of this study was to clone and express Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) conjugated with anti CD₂₀ single chain fragment variable (scFV) region of anti-CD₂₀ antibody as a soluble protein using a small ubiquitin-related modifier (SUMO) fusion protein system in E. coli as a new method for optimal production of anticancer drugs.
Materials and Methods: Appropriate primers for a previously designed DNA sequence encoding the fusion protein were used to amplify the fragment. The amplified fragment was sub-cloned downstream of the SUMO tag in pSUMO vector. Once confirmed by standard methods, the recombinant plasmid was transformed into E. coli strain BL21 (DE₃). Expression was induced by isopropyl β- d-1-thiogalactopyranoside. The expressed proteins were assessed by SDS-PAGE and then isolated by affinity chromatography. Western blotting was used to confirm protein expression.
Results: The results showed the DNA of the recombinant fusion protein was sub-cloned correctly, and the relevant analyses indicated that the expression process was successful. The recombinant fusion protein was confirmed by appropriate analysis.
Conclusion: The production of recombinant proteins in a soluble state in the prokaryotic host E. coli can reduce the costs of the downstream process. Production of soluble antiCD20 scFV-TRAIL fusion protein can be considered a proper method to produce recombinant protein in a soluble state in the E. coli system and can decrease the cost of downstream processes.

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Background and Aim: Polychlorinated biphenyls (PCB) industrial pollutants are one of the most important environmental pollutants whose removal is very important. PCBs are degraded biologically by several enzymes and in a multi-step process. One of these enzymes is called DHBD (2,3-dihydroxy biphenyl 1,2-dioxygenase) and is encoded by the BphC gene. Enhancing the function of the enzyme and reducing the binding affinity of the enzyme to the inhibitor (tert-butanol) will improve the function of the enzyme and increase its efficiency. This research has been carried out in bioinformatics to strengthen the enzyme and weaken the inhibitory effect through mutation in the amino acids of the active site.
Materials and Methods: The amino acid sequence of the enzyme was obtained from the UniPprot database and to check similar sequences with PSI-BLAST method, similar sequences were searched from close to distant protein species. By performing multiple alignments of PSI-BLAST sequences, 250 sequences were matched. The results of sequencing the amino acids of the active site showed that some sites have variable amino acids and were used as candidates for mutagenesis. The position of the T-Butanol inhibitor was simulated using DISCOVERY software.
Results: By molecular docking with PYRX software between the wild enzyme and the substrate, the binding energy -6.2 Kcalmol-1and for the candidates of mutations resulting from the alignment, Phenylalanine 201 to Threonine (6. 9 Kcalmol-1) and Threonine 280 to serine (6. 8 Kcalmol-1) Calculated.
Conclusion: The more negative binding energy indicates the greater stability of this interaction in the mutant enzyme. As a result, these mutations will be able to improve the strength of the enzyme function. The simulation of the position of the inhibitor and the starting material in the enzyme showed that the distance of the inhibitor from the active site and the starting material is likely to be favorable if the interaction of the inhibitor on the amino acids of the active site is reduced and as a result, the binding stability of the biphenyl starting material with the enzyme is increased. Decreasing the inhibitory power will increase the catalytic power of the enzyme in the destruction of PCBs.
 

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Background and Aim: Chitosan nanoparticles are becoming a popular alternative to deliver nucleic acids to tissues for gene transfer. The present study was carried out to design a combined multi-epitope gene construct based on the pathogenic genes of Salmonella typhimurium and Escherichia coli strain O157:H7 and to transfer it to BALB/c mice with the help of chitosan nanoparticles.
Materials and Methods: In this experimental study, pcDNA3.1(+) recombinant vector containing a multi-epitope gene construct was designed and synthesized. E. coli strain TOP10F was transformed with the mentioned vector. Using the plasmid extraction kit, the plasmid was extracted from the transformed bacteria and confirmed. In the next step, this recombinant vector was combined with chitosan nanoparticles, and the characteristics of the complex after production were investigated. Plasmid-containing complexes with or without nanoparticles were injected into the thigh muscle of animals at time intervals of 0, 7, and 14 days. The animal tissue was collected, and the expression of gene structure and cytokines (IFN-γ and IL-10) was analyzed by the reverse transcription method. The spleen tissue of the animals was also examined histopathologically.
Results: The correctness of the synthesis and extraction of the recombinant vector was successfully confirmed. Chitosan nanoparticles and recombinant vector were combined in the ratio (1:1). In animals treated with the recombinant vector, compared to the control group, the expression of the gene construct was successfully confirmed. Examination of cytokines showed that the cellular immune response increased in the challenge groups compared to the control group. However, the structure of the spleen tissue in the group that was transferred with the recombinant vector carrying the gene construct along with chitosan nanoparticles was healthy and normal.
Conclusion: The recombinant vector, after combining with chitosan nanoparticles, was correctly transferred and expressed in the animal tissue, and there was no damage to the spleen pulp. In the future, this multi-epitope gene structure can be used as a vaccine against Salmonella Typhimurium and Escherichia coli.