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Hilsa species are broadly described as largely anadromous fish with a large valuable population size. It is well-known as one of the most critical commercial and occurs in marine, estuarine and riverine environments Hilsa shad, Tenualosa ilisha locally known as Sbour, migrates to the freshwater environment of the Shatt Al-Arab River systems for breeding. It was determined the genetic variation in 70 specimens of T. ilisha from four sites at the Shatt Al-Arab River, the mtDNA cytochrome-b gene was selected as a molecular marker for detecting genetic diversity, origin, and divergence of the population through comparing them with 6 samples from different locations at Indo-Pacific. The AMOVA analysis showed that the variation between groups is 60.97% and 39.02% within population indicating statistically significant P value (P

Computational prediction of signal peptides is one of the most important steps in genetic engineering experiments. The periplasmic expression cause the reducing in the inherent destructive behavior of Bofurin I against its host and also reducing its susceptibility to proteolytic degradation. In order to predict the best signal peptides for expression of Buforin I in E. coli, 103 signal sequences were retired from signal peptide databases. Since the purpose of this study was to introduce the optimal signal peptides for periplasmic expression, first, sub-cellular localization site of signal peptides was analyzed. Then, n, h, and c regions of signal peptide, signal peptide probability and physico-chemical features were investigated. Base on the results, MalE, hofQ, papK, ugpB, zraP, and sfmC were introduced as the best signal peptides. For increasing the half-life of mRNA and the increasing the stability of the mRNA against exonuclease activity, secondary structures of mRNA including Shine-Dalgarno, untranslated region of ompA, start codon, signal peptide and sequences of Buforin I were analyzed. Based on the total free energy pilot evaluated and mRNA conformations, papK seemed more appropriate than the rest of the signal peptides. The obtained result of this study can be used for design the periplasmic expression constructs.

Background:Thanatin is the smallest member of Beta-hairpin class of cationic peptide derived from insects with vast activities against various pathogens.Objective:n this study, the antimicrobial activity of this peptide against some species of human bacterial pathogens as well as its toxicity on NIH cells were evaluated.Method:Thanatin DNA sequence was cloned into pcDNA3.1+ vector and transformed into a DH5α bacterial strain. Then the recombinant plasmids were transfected into HEK-293 cells by calcium phosphate co-precipitation. After applying antibiotic treatment, the supernatant medium containing thanatin was collected. The peptide quantity was estimated by SDS-PAGE and GelQuant software. The antimicrobial activity of this peptide was performed with Minimum Inhibitory Concentration (MIC) method. In addition, its toxicity on NIH cells were evaluated by MTT assay.Results:The peptide quantity was estimated approximately 164.21 µmolL-1. The antibacterial activity of thanatin was estimated between 0.99 and 31.58 µmolL-1 using MIC method. The result of cytotoxicity test on NIH cell line showed that the peptide toxicity up to the concentration of 394.10 µmolL-1 and for 48 hours, was not statistically significant from negative control cells (P>0.05). The antimicrobial assay demonstrated that thanatin had an antibacterial effect on some tested microorganisms. The results obtained in this study also showed that thanatin had no toxicity on mammalian cell lines including HEK293 and NIH.Conclusion:Antimicrobial peptides such as thanatin are considered to be appropriate alternatives to conventional antibiotics in treating various human pathological diseases bacteria.

Gamma amino butyric acid (GABA) is used as drugs, food ingredients, and dietary supplements. l-glutamate is converted to GABA by the decarboxylation reaction, which is catalyzed by the glutamate decarboxylase (GAD). Escherichia coli is widely being used to express proteins. However, without appropriate signal peptide, it cannot be applied for secretory proteins. Selecting a suitable signal peptide (SP) is a critical step in the secretory production of different proteins. In silico identification of suitable SP is a reliable and cost-effective alternative to experimental approaches. In previous studies, the localization of proteins was not considered and the SPs of periplasmic, membranes and extracellular were compared. Therefore, this study aimed to predict the best SP for the expression of recombinant GAD in the outer membrane of E. coli only. Also, we compared twelve servers to evaluate protein localization, solubility, and secretory pathway. In the present study, 127 SPs were taken from the Signal Peptide database. The localization site, physico-chemical properties, location of cleavage sites, regions and D-score of them were determined by ProtComp, ProtParam, and SignalP 3.0 and 4.1 servers, respectively. To rank SPs based on the secretion properties, PRED-TAT and SignalP 5.0 webservers were used. Based on the results, the localization site of 13 SPs was in the outer membrane of E. coli. Among them, the most suitable candidates seemed to be torT with a reasonably high D-score, aliphatic index, and GRAVY, followed by ccmH and then pspE. TorT accelerates GAD scale-up production and might be useful in future experimental research.

In the present study, a novel chimeric peptide was derived from camel lactoferrin designed with a considerable anti-HCV activity and its neutralization mechanism was predicted by molecular modelling tools. A novel anti-HCV peptide derived from camel lactoferrin (cLF36) was designed and expressed it recombinantly in HEK-293-T cells. Anti-viral activity of this peptide was evaluated against hepatitis C virus by Real-time PCR assay in vitro. Finally, to have a better insight into the mode of action of peptide on HCV entry inhibition, we examined the interaction of cLF36 with envelope glycoprotein E2 by molecular dynamic simulation. This chimeric peptide had significant inhibitory effects on both HCV entry (44 µg/mL) and viral replication (88 µg/mL) under in vitro (p > 0.01). Moreover, cLF36 peptide was not toxic to HEK cells as a normal cell at twofold of its anti-viral concentrations for HCV entry and even at concentrations as high as 250 µg/mL exhibited minimal hemolysis (2.5%) against human RBCs (red blood cells). The results of in silico analysis showed that cLF36 interacted with β-sandwich and front layer of E2 protein as two potential CD81 binding sites. We generated and characterized a new camel lactoferrin derived HCV inhibitors. This peptide blocked HCV entry and also intracellular HCV replication in cell culture experiment.

The production of polysaccharides derived from lactic acid bacteria (LAB) can be a valuable alternative to current polysaccharides. In this study, eight samples of Kishk (traditional dairy product) were collected in sterile conditions and directly cultured on MRS agar medium. Following purification and examination of microscopic, macroscopic characteristics and doing biochemical tests, 143 isolates were selected, and the production of exopolysaccharide (EPS) was investigated by the ruthenium red and disc methods. The EPS production of 79 isolates was confirmed. Total carbohydrate of EPS was determined by the phenol sulfuric acid method. Finally, Enterococcus durans K48, Enterococcus faecium R114, and Enterococcus faecium T52 strains were selected as the best EPS producers. The optimization of EPS production was then performed using Central Composite Design (CCD) and Response Surface Methodology (RSM) approaches. Optimization plots showed the highest EPS yield for E. durans K48, E. faecium R114, and E. faecium T52 occurred at 38.4, 37.4 and 36.7 °C and pH of 5.9, 5.6 and 5.8, respectively, cultured in a Sucrose-MRS medium. Under optimal conditions, the maximum predicted and actual production of EPSs for the examined isolates were 3.18, 3.21, 2.99 and 3.02, 3.15, 3.15, g L, respectively. As determined by Gel Permeation Chromatography, the EPSs molecular weights were in the range of 2.93 × 10 to 3.52 × 10 Da. Analysis of the component monosaccharides by HPLC showed that all three tested EPSs were heteropolysaccharides. After appropriate evaluation, the EPSs produced by native species of LAB isolated from Iranian Kishk could be of interest for industrial applications or as functional food ingredients., This work was financially supported by a grant (No. 3/47333) from the Ferdowsi University of Mashhad (Research Affairs), Iran.

There is a continuing need to prevent the increasing use of common antibiotic and find the replacement to combat the drug/antibiotic resistant bacteria such as antimicrobial peptides (AMPs) such as thanatin peptide. In this study, recombinant thanatin peptide was expressed in the HEK293 cell line. Then the antimicrobial properties of this peptide on some poultry and farm animal’s pathogen strains were assessed. The thermal-stability of thanatin was predicted in various temperatures through in silico analysis. Afterwards, according to Minimum Inhibitory Concentration (MIC) results, Escherichia coli and Pseudomonas aeruginosa were chosen to test the hypothesis of LptA/LptD–thanatin interaction, computationally. Relative amino acid sequences and crystallography structures were retrieved and missed tertiary structures were predicted. The interaction of thanatin with LptA and LptD of Escherichia coli and Pseudomonas aeruginosa were analyzed subsequently. The antibacterial activity of thanatin peptide was evaluated between 6.25 and 100 μg/mL using minimum inhibitory concentration. Also, the amounts of minimum bactericidal concentrations (MBC) were between 12.5 and 200 μg/mL. The bioinformatics analysis followed by the in vitro assessment, demonstrated that thanatin would be thermally stable in the body temperature of poultry and farm animals. Thanatin could penetrate to the outer membrane domain of LptD in Escherichia coli and it could block the transition path of this protein while the entrance of LptD in Pseudomonas aeruginosa was blocked for thanatin by extra residues in comparison with Escherichia coli LptD. In addition, the quality of interaction, with regard to the number and distance of interactions which leads to higher binding energy for thanatin and LptD of Escherichia coli was much better than Pseudomonas aeruginosa. But the site and quality of interaction for thanatin and LptA was almost the same for Escherichia coli and Pseudomonas aeruginosa. Accordingly, thanatin can prevent the assembly of LptA periplasmic bridge in both pathogens. The antibacterial and thermal stability of the thanatin peptide suggested that thanatin peptide might serve as a natural alternative instead of common antibiotics in the veterinary medicine. The outcome of this in silico study supports the MIC results. Therefore, a probable reason for different level of activity of thanatin against Escherichia coli and Pseudomonas aeruginosa might be the quality of LptA/LptD–thanatin interaction.

Nowadays, engineered mammalian cell lines such as CHO and HEK-293 are extensively used to express recombinant proteins which require post translation modifications. Post translation modifications completely depend on presence of an appropriate signal peptide in nascent proteins. Erythropoietin is a complex protein with post translation modifications that is widely used to treat anemia, hence production this protein as a recombinant protein needs an appropriate signal peptide. The objective of this study was prediction of the best signal peptide to express recombinant erythropoietin in eukaryote system. In order to predict the best signal peptide, first, 28 signal sequences were extracted from database, then, regions and signal peptide probability of them were evaluated by Signal P version 4.1. To investigate physical and chemical features of signal sequences such as isoelectric point, net positive charge, aliphatic index and amino acid length, Protparam was applied. The results revealed that among 28 signal sequences, only 15 of them can be applied as signal peptide to express erythropoietin. Eventually, among 15 signal peptides, SCGB1D1, APOB, ALB, ULBP2 and scrg1 (respectively) were theoretically introduced as the best signal peptides to employ.

This study was conducted to evaluate the effects of apple cider vinegar (ACV) administration on non-specific immunity of serum and skin mucus, growth indices, and activity of digestive enzymes (amylase, lipase, and protease) in Carassius auratus. For this purpose, 180 fish (weighing 7.35 ± 0.19 g) were allocated to 4 treatment groups with 3 replications in a completely randomized design. Fish were fed for 105 days using a basal diet supplemented with 0% (control), 1% (T 1), 2% (T 2), and 4% (T 3) ACV (contained 5% acetic acid). Results showed a significant increase in lysozyme activity, ACH50, and total immunoglobulin of skin mucus in fish fed with T2 diet (p

The present study was performed to design an immunotoxin consisting of engineered RNase A and scFv of Cetuximab. To accomplish this study goal, at first to evade RNase A from its inhibitors in the cytoplasm, six amino acids of RNase A were substituted, then the physicochemical features of engineered RNase A were assessed. To investigate the interaction between the engineered RNase A and the ribonuclease inhibitor, protein-protein docking was performed. After engineering the RNase A, it was theoretically conjugated with scFv of Cetuximab using a cleavable linker to produce scFv-engineered RNase A. Then, wild-RNase A (14 kD), engineered RNase A (14 kD) and scFv-engineered RNase A (42 kDa) were expressed in the BL21 (DE3) strain of Escherichia coli and purified by Ni-NTA columns. To confirm the expressed proteins, western blot analysis was performed. The functioning of wild-RNase A and engineered RNase A were investigated by RNA fragmentation assay. Finally, to evaluate the cytotoxicity of scFv-engineered RNase A, a dose-response cytotoxicity assay was performed on Her1-positive and Her1-negative cell lines. The results showed that engineered RNase A could maintain its structure and disulfide bonds and evade its inhibitor. Expression and purification were successfully conducted and both enzymes could degrade yeast RNA. The result of cytotoxicity showed that the engineered immunotoxin could induce cell death to Her1-positive cell lines with an IC50 of 50 nM. It appears that scFv-engineered RNase A can be a promising molecule for use.

In most bacteria, Glutamate decarboxylase (GAD) activity significantly reduces at pH > 6. Since l -glutamate is neutral to slightly alkaline, a large amount of hydrochloric acid must be added to the reaction mixture. Acidic reaction conditions greatly increase the corrosion of production equipment and shorten the life of manufacturing equipment. Therefore, this study aims at studying the overexpression of recombinant GAD that is active at the neutral pH and high-level production of γ-aminobutyric acid. A gene sequence encoding GAD of Enterococcus faecium DO was cloned into PET21a (+) and was overexpressed in Escherichia coli BL21 (DE3). The construct was confirmed by colony PCR and double digested with BamHI and XhoI. The result from Western blot analysis showed a signal band at the molecular mass of 54 kDa. The specific activity of the recombinant GAD was 78.17 U mg−1 at 45 °C and pH = 6.6. It was reduced rapidly when pH > 7.8. The Km, Vmax, and kcat values of recombinant GAD were 1.8 mM, 12.9 μmol L−1 min−1, and 0.43 s−1, respectively. Therefore, recombinant GAD is a potent candidate for GABA production in food and pharmaceutical industries, because it increases the efficiency of GABA synthesis, especially in foods with a neutral pH range.

Genomic imprinting results in monoallelic expression of genes in mammals and flowering plants. Understanding the function of imprinted genes improves our knowledge of the regulatory processes in the genome. In this study, we have employed classification and clustering algorithms with attribute weighting to specify the unique attributes of both imprinted (monoallelic) and biallelic expressed genes. We have obtained characteristics of 22 known monoallelically expressed (imprinted) and 8 biallelic expressed genes that have been experimentally validated alongside 208 randomly selected genes in bovine (Bos taurus). Attribute weighting methods and various supervised and unsupervised algorithms in machine learning were applied. Unique characteristics were discovered and used to distinguish mono and biallelic expressed genes from each other in bovine. To obtain the accuracy of classification, 10-fold cross-validation with concerning each combination of attribute weighting (feature selection) and machine learning algorithms, was used. Our approach was able to accurately predict mono and biallelic genes using the genomics and proteomics attributes.

Protein degradation in the rumen often proceeds at a rate which exceeds the ability of the microbial population to utilize the resulting breakdown products. Bacteria are the most active proteolytic organisms in the rumen, and many species of rumen bacteria are known to be proteolytic (Wallaceet al., 1985). The commonly isolated proteolytic bacteria are also able to break down peptides and amino acids, and it was assumed that they were responsible for the ruminal degradation of intact protein through to ammonia. However, studies comparing the specific activities of ammonia production between mixed ruminal bacteria and the well-known proteolytic bacteria noted that no individual bacterium had an activity which could explain the activity of the mixed ruminal culture (Russellet al., 1988). Subsequently, three gram-positive, monensin-sensitive, ammonia hyper producing (HAP) bacteria were isolated from the rumen. The present study reports the sequence of 16sr RNA for ruminal ammoniabacters of Iranian Holstein cows.