Immunological evaluation of a novel multi-antigenic DNA vaccine encoding SAG1, SAG3, MIC4, GRA5, GRA7, AMA1and BAG1 against Toxoplasma gondii in BALB/c mice.

Many recent studies have been conducted to find new DNA vaccines based on Toxoplasma gondii antigens. DNA vaccines encoding complex of different antigens showed better immune responses compared to single antigen vaccine. In this study, we constructed a DNA vaccine encoding SAG1, SAG3, MIC4, GRA5, GRA7, AMA1 and BAG1 against T. gondii, and evaluated the immune response it induced in BALB/c mice. For this purposes, thirty BALB/c mice were randomly divided into three groups containing tenmice each. There were two negative control groups (PBSand pVAX1 vector) and one vaccination group (pVAX1-MAF, Multantigenic Fragment). On days 0, 14 and 28, the mice were immunized intramuscularly, and 5 weeks later they were challenged with T. gondii RH strain. The immune responses were evaluated using lymphocyte proliferation assay, T-cell subsets detection, and measurement of antibody and cytokine levels. The results showed that mice immunized with pVAX1-MAF developed high levels of IL-2, IL-12, IgG and IFN- γ as well as CD3+CD4+ T cells. In addition, the survival time of mice immunized by pVAX1-MAF was longer than that control mice. In conclusion, our results show that the multiple DNA vaccine encodingSAG1, SAG3, mic4, GRA5, GRA7, AMA and BAG1effectively enhanced humoral and cellular immune responses, and prolonged the survival time. Together this would suggest that further investigation may result in a promising candidate vaccine to treat toxoplasmosis.

Corrigendum to "Functional analysis of recombinant codon-optimized bovine neutrophil ß-defensin" [J. Adv. Res. 7(5) (2016) 815-821].

[This corrects the article DOI: 10.1016/j.jare.2015.12.003.].

RNA interference targeting UL25 gene as a gene therapy approach against BHV-1.

BACKGROUND: Infectious bovine rhinotracheitis (IBR) is an important economic viral disease, which is caused by bovine herpesvirus-1. BHV1-UL25 plays an important role in the encapsidation process and the stabilization of the packaged DNA into the capsid. The application of lentiviral mediated shRNAs for knocking down the target genes is a powerful antiviral approach. Thus, in the present study, an RNAi- based antiviral approach was designed and assayed by the targeting of BHV1-UL25. METHOD: The suitable shRNA molecules were purposed using online software. Three recombinant lentiviral vectors expressing shRNAs down regulating the UL25 gene of BHV-1 were constructed.The effectiveness of designed shRNAs was assayed by the observation of BHV-1 cytopathic effects, calculating TCID50 titers, and evaluating the changes in viral gene expression using real-time RT PCR. RESULTS: All the shRNAs sufficiently decreased BHV1 titers (more than 90%) in comparison with the control groups. We observed the reduction value of more than 99% in the expression of viral RNA in the cells treated with all the shRNAs in comparison with the control groups. The reduction rate of BHV1-UL25 expression with shRNAs was more than 93%, in comparison with that in the cells expressing the selected domain. The reduction values were more than 99% for all three shRNAs compared to the cells expressing the selected BHV1-UL25 domain infected with a scrambled vector. CONCLUSION: The results indicated that lentiviral mediated shRNAs targeting BHV1- UL25 had considerable antiviral attributes. In conclusion, RNAi may be considered as a strong treatment proposal against viruses such as BHV-1.

Thermally stable and acidic pH tolerant mutant phytases with high catalytic efficiency from Yersinia intermedia for potential application in feed industries.

Heat- and pH-stable phytase efficiently hydrolyzes phytic acid. In this research, heat- and pH-stable mutant phytases, T83R, L287R, and T83R/L287R were generated by site-directed mutagenesis from Yersinia intermedia. After the induction and expression of recombinant wild-type and mutant phytases in E. coli BL21, the enzymes were purified using nickel sepharose affinity chromatography, and characterized kinetically and thermodynamically using spectroscopy methods. The mutants showed optimum activity at pH 5.15 and 55-61 °C. The catalytic efficiencies of T83R, L287R, T83R/L287R, and wild-type phytases were calculated to be 2941, 29346, 4906, and 6917 mmol/L-1s-1, respectively. Moreover, after the incubation of T83R, L287R, wild-type, and T83R/ L287R phytases at 100 °C for 1 h, the enzymes retained 22, 5, 4, and 2% of their initial activities, respectively. In addition, T83R, T83R/L287R, L287R, and wild-type phytases retained 82, 44, 16 as well as 11% of their initial activities after 1 h at pH 5.15, respectively. Among these mutants, T83R mutant showed 18% increase in thermal stability, 71% increase in pH stability, and +0.103 KJ/mole increase in ΔΔG, while the catalytic efficiency and ΔΔG value of L287R mutant increased by 4 times and +0.0903 KJ/mole, respectively. Thus, the mutants have the potential to be used in feed industries to increase the bioavailability of minerals while decreasing soil and water pollution.

In vitro Inhibition of Border Disease Virus Replication With Lentivirus-Mediated shRNAs.

Background: Border disease is believed to be one of the most important diseases in the animal husbandry industry, which has not yet been eradicated in Iran. The development of approaches based on the application of interfering RNA (RNAi) for antiviral therapy has attracted a great deal of attention over the recent years. The present research was conducted to design, construct, and apply shRNA against the NS3 gene of BDV to evaluate the prevention of BDV proliferation in the cell culture system. For this purpose, the suitable oligonucleotide sequence of NS3 gene coding was selected utilizing BDV- X818 strain. Afterwards, using shRNA design software, shRNA molecules were designed and synthesized. These shRNAs were cloned into the desired vectors and were finally transfected in HEK293T cells employing the third generation of lentiviral packaging system. Subsequently, these shRNA expressing lentiviruses were transduced to the MDBK cell line to challenge to border virus. In order to evaluate the efficacy of shRNAs, the viral infectious titer and RNA copy number were calculated with TCID50 and Real-time RT-PCR tests, respectively. Results: The results revealed that shRNAs 1, 2, and 3 decreased viral RNA by more than 90% compared to the control groups. BDV titer noticeably decreased after the challenge with shRNAs 1, 2, and 3 from ~88% up to 99% in comparison with the control groups. Conclusions: Overall, it could be concluded that RNAi may be considered as a strong treatment proposal against viruses, such as BDV.

Fabrication of an ultrasensitive aptasensor for precise electrochemical detection of the trace amounts of streptomycin in milk.

Designing a sensitive method for the detection of streptomycin residues in animal products is essential for controlling consumer health risk. In this study, a high-purity pencil lead graphite electrode coated with inner graphene layers and outer surface-adsorbed gold nanoparticles attached to streptomycin-specific thiolated aptamer was used as an electrochemical aptasensor. The aptasensor electrode fabrication steps were investigated by scanning electron microscope (SEM) and Fourier-transform infrared spectrophotometer (FTIR). Moreover, aptasensor performance during fabrication and binding of aptamer to streptomycin were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) methods. After the binding of sreptomycin to it's specific aptamer as a component of the aptasensor a decrease in the current and an increase in the charge transfer resistance (Rct) were recorded using the above-mentioned techniques. Under optimal conditions, the novel ultra-sensetive designed aptansensor detects streptomycin in the range of 10-8 to 10-16 M with a LOD of 0.8×10-18 M. The aptansensor demonstrates a high selectivity, good reproducibility and acceptable stability for the specific detection of streptomycin. According to the results, the manufactured aptansensor is a fast, low-cost, highly sensitive and selective device and thus the aptasensor can detect the trace amounts of streptomycin in milk in dairy industries.

Improved thermal stability of phytase from Yersinia intermedia by physical adsorption immobilization on amino-multiwalled carbon nanotubes.

Phytase is used in poultry diets to hydrolyze and release of phytate-bound phosphorus. Immobilization on nanomaterials optimizes enzyme's thermal stability and reusability. This study aimed to immobilize the recombinant phytase from Yersinia intermedia on the surface of amino-multi-walled carbon nanotubes (amino-MWCNTs) by physical adsorption. For this, zeta potential measurement, FTIR spectroscopic analysis, scanning electron microscope (SEM), kinetic as well as thermodynamic parameters were used to characterize immobilized phytase on amino-MWCNTs. According to results, the optimum temperature of the immobilized phytase increased from 50 to 70 °C and also thermal and pH stability improved considerably. Moreover, immobilization led to an increase in the value of Km and kcat from 0.13 to 0.33 mM and 2220 to 2776 s-1, respectively. In addition, the changes in activation energy of thermal inactivation (ΔE#a (D)), the free energy of thermal inactivation (ΔG#D) and the enthalpy of thermal inactivation (ΔH#D) for immobilized phytase increased by +11.05, +24.7 and +11.4 kj/mole, respectively, while the value of the change in the entropy of thermal inactivation (ΔS#D) decreased by - 0.04 kj/mole.K. Overall, our results showed that adsorption immobilization of phytase on amino-MWCNTs increases thermal, pH and storage stability as well as some of kinetic parameters.

Lentivirus expressing shRNAs inhibit the replication of contagious ecthyma virus by targeting DNA polymerase gene.

BACKGROUND: Contagious ecthyma or Orf is known as a zoonotic disease remains prevalently worldwide despite the application of some control strategies against it. RNAi particularly shRNA provides us with the chance to tackle this obstacle by an encouraging new approach. The current study indicates the design and experiment of third-generation lentivirus packaging systems delivering shRNAs to inhibit Orf virus (ORFV) replication and infection. Given the importance of DNA-pol gene in virus replication, in this study, three shRNAs against this gene were designed and cloned into lentiviral vectors to stabilize the expression of shRNAs. After producing lentivectors expressing ORFV-DNA- pol in HEK293T cells, the synthesized shRNAs were applied to downregulate viral replication and gene expression. The reduction in viral titer and RNA was evaluated by TCID50 test as well as real-time RT-PCR. The results were then analyzed in comparison with the control group. RESULTS: Designed shRNAs significantly reduced virus yield approximately 90 to 97% and 96.8 to 99.4%, respectively compared to the control groups (cells infected with ORFV and infected with ORFV and scrambled vector) by TCID50 test. Real-time RT-PCR revealed a dramatic reduction in the expression of viral RNA approximately 99% compared to cells infected with ORFV and from 92.6 to 99%, respectively compared to cells infected with ORFV and scrambled vector. CONCLUSIONS: Therefore, it can be stated that RNAi is capable of being used as a potent therapeutically option against viruses like ORFV.

Multi-functionPlantDefensin,AntimicrobialandHeavyMetal Adsorbent Peptide.

BACKGROUND: Defensin peptide isolated from plants are often heterogeneous in length, sequence and structure, but they are mostly small, cationic and amphipathic. Plant defensins exhibit broad-spectrum antibacterial and antifungal activities against Gram-positive and Gram-negative bacteria, fungi and etc. Plant defensins also play an important role in innate immunity, such as heavy metal and some abiotic stresses tolerance. OBJECTIVES: In this paper, in vitro broad-spectrum activities, antimicrobial and heavy metal absorption, of a recombinant plant defensin were studied. MATERIAL AND METHODS: SDmod gene, a modified plant defensin gene, was cloned in pBISN1-IN (EU886197) plasmid, recombinant protein was produced by transient expression via Agroinfiltration method in common bean. The recombinant protein was tested for antibacterial activity against Gram-negative, Gram-positive bacteria and Fusarium sp. the effects of different treatments on heavy metal zinc absorption by this peptide were tested. RESULTS: We confirmed the antibacterial activities of this peptide against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Bacillus cereus) bacteria, and antifungal activities of this peptide against Fusarium spp. (Fusarium oxysporum and Fusarium solani). High metal absorption coefficient for this peptide was also observed. RESULTS: Out of six actinobacterial isolates, VITVAMB 1 possessed the most efficient RO-16 decolorization property. It decolorized 85.6% of RO-16 (250 mg L-1) within 24hrs. Isolate VITVAMB 1 was identified to be Nocardiopsis sp. Maximum dye decolorization occurred at pH 8, temperature 35oC, 3% salt concentration and a dye concentration of 50 mg L-1. CONCLUSIONS: Results suggesting that modified defensin peptide facilitates a broader range of defense activities. dedefensins are an important part of the innate immune system in eukaryotes. These molecules have multidimensional properties that making them promising agents for therapeutic drugs.

Y-chromosomal Status of Six Indo-European-speaking Arab Subpopulations in Chaharmahal and Bakhtiari Province, Iran.

BACKGROUND: We analyzed the Y-chromosome haplogroups of six documented Arab subpopulations that accommodated separately in different counties of Chaharmahal and Bakhtiari Province but nowadays speak Indo-European language (Luri and Farsi). METHODS: This was an outcome study conducted in 2015 to test whether there was any genetic relatedness among some Indo-European-speaking Arab subpopulation accommodated in a geographically similar region, Chaharmahal and Bakhtiari Province, Iran. Seven main Y-chromosome bi-allelic markers were genotyped in six documented Arab subpopulations. Therefore, after DNA extraction from blood samples, PCR reaction carried out by designed primers for J1-M267, J2-M172, and J-M304, I-M170, IJ-M429, F-M89 and K-M9 markers. Then PCR products after quality control on agarose gel were sequenced. RESULTS: Most subjects (83.3%) belonged to F-M89 haplogroup. These subjects belonged to K-M9 (40%), J2-M172 (40%) and I-M170 (20%). Generally, there were at least three genetically distinct ancestors with a divergence date of about 22200 yr for I, 429000 for J and 47400 before present for K haplogroup and may show separate historical migrations of studied populations. As the most recent common ancestor (MRCA) of most of these populations, haplogroup F, lived about 40000-50000 yr ago, the data do not support a nearly close genetic relationship among all of these populations. However, there were populations with same haplogroups J2 (n=2), K (n=2), or with a closer MRCA, IJ haplogroups, among I and J2 haplogroups. Finding haplogroup I, a specific European haplogroup, among Arab populations was not expected. CONCLUSION: Identification of various haplogroups in Arab subpopulations despite its small area and geographically conserved region of this part of Iranian plateau was unexpected.

Experimental and theoretical studies of cadmium ions absorption by a new reduced recombinant defensin.

Heavy metal pollutants such as Cd, Hg, Pb, As, and Se are considered as both a global problem and a growing threat to the humanity. Being strongly poisonous to the metal-sensitive enzymes and leading to the growth inhibition and death of organisms, these metals have a toxic impact on the plants and animals. Inducing the metal-binding cysteine-rich peptides such as metallothioneins, phytochelatins, and defensins, higher organisms like plants and animals usually react to the heavy metal stress. In this study, a recombinant defensin protein was expressed in bean and its ability in the cadmium absorption was determined. Experimental studies revealed that this protein was able to absorb cadmium ions in reduced form more than oxide one. Molecular dynamics simulations were carried out in order to evaluation of experimental studies, using a model of Cd2+ or Na+ and Cl- ions enclosed in a fully hydrated simulation box with the recombinant defensin. The theoretical results also suggested that the reduced recombinant defensin was more powerful in the absorption of Cd2+ than its oxide form. The present study is the first report of Cd2+ absorption potential of this new reduced recombinant defensin. The results unraveled that this recombinant defensin can be adopted as a molecular switch in the cadmium pollution of the environment and also the important role of sulfur groups in the absorption of cadmium ions.

Isolation of Taxol-Producing Endophytic Fungi from Iranian Yew Through Novel Molecular Approach and Their Effects on Human Breast Cancer Cell Line.

Taxol or paclitaxel, an approved drug by the Food and Drug Administration, is being used for the treatment of human cancers. This study aimed to isolate and determine different species of native endophytic fungi from Iranian Taxus baccata (yew) plants located in the northern forests of Iran. To do so, a novel molecular screening approach was performed for 50 isolated endophytic fungi through amplification of exon No. 1 of taxadine synthase as a key gene in taxol production pathway. We used effective colony-polymerase chain reaction technique for rapid screening of potent taxol-producing fungi instead of genomic DNA extraction. Production of taxol was performed in batch culture by selected fungi individually and produced taxol was assayed quantitatively by high-performance liquid chromatography using standard taxanes. We found that only six fungi could produce taxol and baccatin III. Interestingly, after 7 days of incubation, the highest level of taxol was found to be 129 and that of baccatin 139.2 mg/kg dw for two native isolated Cladosporium sp. named F1 and F3. The fungal taxols could decrease cell viability in MTT assay same as commercial taxol. The fungal taxols semi-quantitatively showed antimitotic effects on MCF-7 cells as human breast cancer cell line. The expression of bcl-2 anti-apoptotic gene, in contrast to bax pro-apoptotic gene, significantly decreased after treatment by standard and fungal taxols. As fungal taxol was produced simpler than other methods and could significantly affect viability and specific genes expression profile, it is recommended that using of taxol-producing fungi from Iranian yew could be a safe and confident procedure to overcome challenges of using other methods.

Functional expression and purification of recombinant Hepcidin25 production in Escherichia coli using SUMO fusion technology.

Hepcidin25 is a small cysteine-rich peptide hormone known as a new class of antimicrobial peptides. The purpose of the present study was to express, purify and investigate the antibacterial properties of recombinant human hepcidin25 protein production in Escherichia coli. Human hepcidin25 gene was optimized and fused to a small ubiquitin-related modifier (SUMO) gene for higher expression. Then SUMO-hepcidin25 was cloned into the pET-32a (+) vector and expressed in E. coli Origami. The fusion protein with a molecular weight of approximately 35kDa was analyzed on SDS-PAGE gel. The highest expression was observed after 6h induction and the fusion protein consisted approximately 47% of the total cellular protein. The purified SUMO-hepcidin25 purity was determined to be higher than 95%, with a final yield of 3.9mgl-1 of media. The recombinant hepcidin25 showed antibacterial activity against both Gram negative (Klebsiella pneumonia) and Gram positive (Staphylococcus aureus and Bacillus cereus) bacteria with minimum inhibitory concentrations (MICs) of 150µgml-1, 18.7µg/ml-1 and 37.5µg/ml-1, respectively. These results indicated that thioredoxin and SUMO dual fusion system is an efficient production system for synthesis functional human hepcidin25.

Super RLuc8: A novel engineered Renilla luciferase with a red-shifted spectrum and stable light emission.

Renilla luciferase is a bioluminescent enzyme which is broadly used as a reporter protein in molecular biosensors. In this study, a novel luciferase with desired light emission wavelength and thermostability is reported. The results indicated that the new luciferase, namely super RLuc8, had a red-shifted spectrum and showed stable light emission. Super RLuc8 showed a 10-fold (p-value=0.0084) increase in the thermostability at 37°C after 20min incubation, in comparison to the native enzyme. The optimum temperature of the mutant increased from 30 to 37°C. Molecular dynamics simulation analysis indicated that the increased thermostability was most probably caused by a better structural compactness and more local rigidity in the regions out of the emitter site.

Cloning, Codon Optimization, and Expression of Yersinia intermedia Phytase Gene in E. coli.

BACKGROUND: Phytate is an anti-nutritional factor in plants, which catches the most phosphorus contents and some vital minerals. Therefore, Phytase is added mainly as an additive to the monogastric animals' foods to hydrolyze phytate and increase absorption of phosphorus. OBJECTIVES: Y. intermedia phytase is a new phytase with special characteristics such as high specific activity, pH stability, and thermostability. Our aim was to clone, express, and characterizea codon optimized Y. intermedia phytase gene in E. coli . MATERIALS AND METHODS: The Y. intermedia phytase gene was optimized according to the codon usage in E. coli. The sequence was synthesized and sub-cloned in pET-22b (+) vector and transformed into E. coli Bl21 (DE3). The protein was expressed in the presence of IPTG at a final concentration of 1 mM at 30°C. The purification of recombinant protein was performed by Ni2+ affinity chromatography. Phytase activity and stability were determined in various pH and temperatures. RESULTS: The codon optimized Y. intermedia phytase gene was sub-cloned successfully.The expression was confirmed by SDS-PAGE and Western blot analysis. The recombinant enzyme (approximately 45 kDa) was purified. Specific activity of enzyme was 3849 (U.mg-1) with optimal pH 5 and optimal temperature of 55°C. Thermostability (80°C for 15 min) and pH stability (3-6) of the enzyme were 56 and more than 80%, respectively. CONCLUSIONS: The results of the expression and enzyme characterization revealed that the optimized Y. intermedia phytase gene has a good potential to be produced commercially andto be applied in animals' foodsindustry.

The construction of a short gene by a very fast, modified, and simplified gene synthesis and the analysis of various effects on this synthesis

We have constructed a short gene with a single step assembly PCR without any additional amplification primers with Taq and Pfu polymerases. Since the Taq polymerase is a common and conventional enzyme for PCR reactions, we have analyzed various effects on its efficiency. Eventually, we have been able to synthesize the gene in less than 40 minutes by Taq polymerase.

Translational selection on SHH genes.

Codon usage bias has been observed in various organisms. In this study, the correlation between SHH genes expression in some tissues and codon usage features was analyzed by bioinformatics. We found that translational selection may act on compositional features of this set of genes.

Translational selection on SHH genes

Codon usage bias has been observed in various organisms. In this study, the correlation between SHH genes expression in some tissues and codon usage features was analyzed by bioinformatics. We found that translational selection may act on compositional features of this set of genes.

Development of a bacterial surface display of hexahistidine peptide using CS3 pili for bioaccumulation of heavy metals.

A novel cell surface display system for metal uptake was developed using CS3 pili of enterotoxigenic Escherichia coli, which is a suitable system for display of heterologous peptides. The recombinant bacteria producing the hybrid pili containing the hexahistidine peptide accumulated high concentrations of Cd(2+) and Ni(2+) at 656.2 and 276.5 nmol per mg dry weight of bacterial cell, respectively. The recombinant bacteria may be useful in water and waste water treatment.