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Avian influenza subtype H9N2 is the most prevalent influenza virus in poultry worldwide. It imposes economic losses on the poultry industry and has zoonotic potential. Currently, there are two main groups of anti-influenza drugs in use; Adamantanes and Neuraminidase inhibitors. In recent years, there has been increasing resistance to existing anti-influenza drugs. Antimicrobial peptides are a group of potential drug candidates with broad-spectrum activity. cLF-chimera is an antimicrobial peptide synthesized from camel milk lactoferrin. This study's objective is to evaluate the inhibitory effects of cLF-chimera on avian influenza, subtype H9N2. For this purpose, one hundred and seventy 11-day-old embryonated eggs were randomly distributed in 17 groups. Different virus and peptide concentrations were injected into the eggs. The eggs were incubated for four days with daily candling for viability assessment. On the 4th day of incubation, each group's alive or dead embryos were sorted and evaluated for gross anomalies. Next, the chick embryos were fixed with 10% neutral buffered formalin for one week for histopathological studies. The MTT assay was also performed for the peptide and virus concentrations. Overall, the embryo viability results and macroscopic and histopathologic findings showed that the peptide has inhibitory effects against the virus. These findings agree with the MTT assay. Moreover, the peptide has proven effects against pathogenic bacteria that can be advantageous compared to common anti-influenza medications.

Background: Immunomodulatory peptides are capable of stimulating or suppressing the immune system. Hence, deregulation of them can be considered as an immunotherapy approach. These peptides may have dual behavior in response to different infections. For instance, an antimicrobial peptide may act as an anticancer, tumor marker or even cause cancer progression. Methods: In this study, we used single-cell RNA sequencing analysis to investigate the deregulation of immunomodulatory peptides in malignant versus normal human breast epithelial cells. We validated the obtained results in chromatin accessibility level. Furthermore, we used a drug repositioning approach to change the expression of these peptides based on their role in cancer biology. Results: As a result, ten immunomodulatory peptides were upregulated in breast cancer versus normal. Chromatin was more accessible for these peptides in cancer cell lines versus normal. Among these ten peptides, five of them were tumor drivers (i.e., BST2, GAPDH, S100A8, S100A9 and HMGB1), three of them were anticancer (i.e., H2AFJ, SCGB2A1 and HMGN2), S100A7 had dual behavior in different cancers and ZG16B was a tumor marker. Using the LINCS L1000 database, we proposed a list of drugs that can deregulate the candidate peptides according to their role in the progression of malignancy. Conclusions: In conclusion, immunomodulatory peptides can be considered as drug targets based on their role in cancer biology.

BackgroundImmunomodulatory peptides are capable of stimulating or suppressing the immune system. Hence, deregulation of them can be considered as an immunotherapy approach. These peptides may have dual behavior in response to different infections. For instance, an antimicrobial peptide may act as an anticancer, tumor marker or even cause cancer progression.MethodsIn this study, we used single-cell RNA sequencing and spatial transcriptomiocs analysis to investigate the deregulation of immunomodulatory peptides in malignant versus normal human breast epithelial cells. We validated the obtained results in chromatin accessibility level. Furthermore, we used a drug repositioning approach to change the expression of these peptides based on their role in cancer biology.ResultsTen immunomodulatory peptides were upregulated in breast cancer versus normal. Chromatin was more accessible for these peptides in cancer cell lines versus normal. Among these ten peptides, five of them were tumor drivers (i.e., BST2, GAPDH, S100A8, S100A9 and HMGB1), three of them were anticancer (i.e., H2AFJ, SCGB2A1 and HMGN2), S100A7 had dual behavior in different cancers and ZG16B was a tumor marker. Using the LINCS L1000 database, we proposed a list of drugs that can deregulate the candidate peptides according to their role in the progression of malignancy.ConclusionsImmunomodulatory peptides can be considered as drug targets based on their role in cancer biology.

Since Brucella infection mostly occurs through the mucosal surfaces, immune response induced by vaccine that is delivered by a way of mucosal route can be drastically enhanced to control the brucellosis. Omp31is the major outer membrane protein of Brucella, and is considered as a protective antigen against Brucella infection. Accordingly, Lactococcus lactis has been used as an antigen-delivering vector to develop a vaccine-induced mucosal response for having a safer vaccination against brucellosis. A designed omp31 gene fused to the usp45 signal peptide and M6 cell wall anchor was sub cloned in the pNZ7021 expression vector, and a recombinant L. lactis displaying Omp31 was constructed. Omp31 protein expression was confirmed using Western blotting and immunofluorescence analysis. Animals were orally and intraperitoneally immunized with live or killed L. lactis expressing Omp31, respectively. The humoral and cellular immune responses were evaluated by measuring the specific cytokines and antibodies. sIgA, serum IgA, IgM, and total IgG antibodies significantly increased in the mice immunized with live recombinant L. lactis expressing Omp31 and also serum IgM, and total IgG antibodies significantly increased in mice immunized with killed recombinant L. lactis expressing Omp31. Among IgG subtypes, IgG2a response was significantly higher in both groups compared to IgG1. In mice groups immunized with recombinant L. lactis, the IFN-γ and IL-10 level elevated; however, there was no change in the level of IL-4. These results indicated that recombinants L. lactis induce both humoral and cellular immune responses in mice, and also vaccines based on L. lactis-derived live carriers are promising interventions against Brucella melitensis infections.

BackgroundA cross-protective avian influenza vaccine candidate can be designed by using a preserved antigen against mutation in various subtypes of influenza. M2e peptide sequence has remained remarkably unchanged in influenza type A isolated since 1918.MethodsA consensus sequence of M2e peptide was obtained from 31 sequences of H5N8, H5N1, H9N2 and H7N9 subtypes of avian influenza virus isolated from 7 avian species in 5 Asian countries. A partial sequence of flagellin was considered as an adjuvant. Subsequently, two chimeric antigens were designed to be virtually cloned and expressed using PYD1 vector andEBY100yeast strain. The stability and conformational features of these two antigens were assessed through molecular dynamic (MD) simulations. The detectability of vaccine candidates by a specific monoclonal antibody (MAb148) were estimated through docking studies.ResultsIn spite of significant compactness and stability of the first candidate in comparison with the second design, it was less detectable by MAb148. Contrary to the first chimeric antigen, Van der Waals, electrostatic and binding energies of the interaction of the second antigen with MAb148 were significantly closer to the positive control. It is shown that epitopes of the second chimeric antigen could be correctly located in the specific pocket of CDR region of MAb148.ConclusionThe second chimeric antigen could be considered as a yeast-displayed avian influenza vaccine candidate due to the capability of provoking humoral immunity and innate immune system by M2e and flagellin respectively.

Antimicrobial peptides (AMPs) are native and safe short peptides with valuable biological effects. Nowadays, these components and their importance have attracted the attention of many researchers to determine their mode of action. Computational peptide engineering can donate a useful insight for investigating the stability and potency of AMPs. In the present study, to improve the effect of CLF36, a chimeric peptide derived camel lactoferrin, the atomic insight into peptide-DNA interaction was analyzed using MD simulation. Targeted mutants were performed in wild type amino acid sequences and obtained engineered peptides were homology modeled for peptide-DNA interaction analysis. SASA, Hydrogen binding and free binding energy analyses revealed that all changes in wild type of peptide in this study improved the peptide-DNA interaction. Our in silico results showed that simultaneous substitution of GLU12 with ALA and also removing SER36 in wild type had more substantial effects on complex formation with DNA. The obtained result of this study could be useful to improve the stability and potency of engineered peptides to use of in the experimental study.

Background and Objectives: There are many pertussis outbreaks which is mainly due to the reduction in the immunity of acellular pertussis (aP) vaccines. Therefore, there is a crucial necessity to develop a new generation of pertussis vaccine. Pre- ceding researches have shown that Bordetella pertussis outer membrane vesicles (OMVs) have appropriate specifications, making them a suitable vaccine candidate against pertussis. Materials and Methods: The OMVs were separated by a new serial ultra centrifugation technique. Transmission electron microscopy (TEM) examination, SDS-PAGE, Western blotting and ELISA assay were used to characterize the OMVs. Results: TEM studies showed the size of the extracted OMVs at 40-200 nm. The presence of pertussis toxin, filamentous hemagglutinin, and pertactin was verified using Western blot and ELISA assay. Conclusion: The presented technique is a simple and effective way to obtain OMVs from Bordetella pertussis. So it can be utilized as an appropriate procedure in the development of an OMV-based vaccine against pertussis.

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.

Cancer remains one of the leading causes of death worldwide. Introduction of natural compounds with anticancer properties can be an effective step in the prevention and treatment of cancer. Antimicrobial peptides (AMPs) are short peptides whose anticancer activities have been proved previously. In the present study, Enterocin-P (EntP) as a bacteriocin of E. faecium was expressed in HEK expression system by pcDNA3.1 + vector. The recombinant peptide was purified from culture medium using Ni2+ affinity chromatography with an average yield of 0.6 mg/ml. The cytotoxic activity of the recombinant peptide was determined toward some cancer cell lines including: SW1353, HUH7, Huh-7.5, C26, B16F0 and NIH3T3 as a normal cell line. Our results showed that EntP peptide had selective cytotoxicity activity only against C26 (IC50: 0.32 mg/ml) and SW1353 (IC50: 1.36 mg/ml) as cancer cell and did not show cytotoxic properties against normal cell. In the second phase of our study, to better understand the mechanisms of EntP peptide, we have tried to predict the possible interaction of this peptide to predominantly negative charge molecules in the cell membrane of cancer cells. Our in-silico analysis revealed that EntP peptide has strong tendency to chondroitin sulfate (− 189 ± 6.24 kJ/mol) and heparan sulfate (− 115 ± 5.12 kJ/mol) as two well-known anionic molecules on surface cancer cells.

Brucella is an important zoonotic pathogen which causes significant commercial loss in industry and considerable disease in livestock and human. Diminishing fertility and also abortion in animal and Malta fever disease in human. In spite of some successful vaccines against animal brucellosis, no satisfactory vaccine has been produced against human brucellosis. Accordingly, OMP31 as a major immunogenic and immunodominant protein in Brucella melitensis has been used to predict substantial epitopes against human and/or sheep MHCII alleles in three different continents (America, Oceania, and Europe) through bioinformatics tools. In order to validate region-based projections, the accuracy of epitopes was assessed by Vaxijen, an online server. The structure of these peptides was predicted through PEP-FOLD server. Predicted epitopes were sifted through docking studies with relevant MHCII receptors and finally, docked complexes were evaluated by molecular dynamic simulations. According to the frequency of human and sheep alleles in different regions, “FGTVRARLG” and “YIGINAGYA” epitopes can be considered for American, Eastern European, and Asian human populations and also sheep flocks of Australia and New Zealand (Corriedale breed), United States (Polypay and Rumbouillet breeds) and France (Rambouillet breed). Besides, “AINNNWTLKSE”, “FLESKVNFH”, “VLGAETDFQG”, and YIGINAGYA can be significant in American and northern Indian human populations and also sheep of France (Suffolk and Columbia breeds), England and Scotland (Cheviot breed) and United States (Polypay breed). Eventually, “AINNNWTLKSE” and “VQAGYNWQL” might be important in American human and Britannia (Scottish Blackface breed), United States (Columbia, Rambouillet, and Polypay breeds) and French (Rambouillet breed) sheep populations.

Three hundred and sixty 1-day-old male broiler chicks were randomly allocated to 4 treatments of 6 replicates to evaluate the effects of cLFchimera, a recombinant antimicrobial peptide (AMP), on gut health attributes of broiler chickens under necrotic enteritis (NE) challenge. Treatments were as follows: (T1) unchallenged group fed with corn-soybean meal (CSM) without NE challenge and additives (NC); (T2) group fed with CSM and challenged with NE without any additives (PC); (T3) PC group supplemented with 20 mg cLFchimera/kg diet (AMP); (T4) PC group supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet (antibiotic). Birds were sampled for villi morphology, ileal microbiota, and jejunal gene expression of cytokines, tight junctions proteins, and mucin. Results showed that AMP ameliorated NE-related intestinal lesions, reduced mortality, and rehabilitated jejunal villi morphology in NE challenged birds. While the antibiotic non-selectively reduced the count of bacteria, AMP restored microflora balance in the ileum of challenged birds. cLFchimera regulated the expression of cytokines, junctional proteins, and mucin transcripts in the jejunum of NE challenged birds. In conclusion, cLFchimera can be a reliable candidate to substitute growth promoter antibiotics, while more research is required to unveil the exact mode of action of this synthetic peptide.

Brucellosis is an infectious disease caused by different Brucella species. The outer membrane proteins 25 and 31 play a significant role in stimulation of immunity against Brucella. Herein,...

Nowadays, cancer remains a major cause of death affecting millions of people. Currently, the antimicrobial peptides (AMPs) as potent anticancer therapeutic agents offer specificity and low levels of side effects in cancer therapy. In the present study, a cationic chimeric peptide (cLFchimera), derived from camel lactoferrin, was expressed as a secretory peptide using P170 expression system in L. lactis. Peptide purification was carried out using Ni-NTA agarose column from culture medium with 21 μ/mL concentration. The recombinant peptide was investigated for its activity against four tumor and one normal cell line. The cLFchimera was more active against two tumor cell lines (chondrosarcoma and colorectal cancer cells), but the activity against two other tumor cell lines (hepatoma and breast cancer cell line) and normal cells was low. Finally, to have better insight into the mode of action of the peptide on cytotoxic activity, we examined the interaction of cationic peptide with two glycosaminoglycans (GAGs), heparan sulfate (HS) and chondroitin sulfate (CS), as the two most anionic molecules on the cell surface by molecular dynamic simulation. The results of in silico analysis showed that the cLFchimera interacted with HS and CS with a totally different amino acid profile. Hydrogen bonding screening in GAGs-peptide complexes revealed K21, V23 and I3, R16 are the dominant amino acids involved in peptide-HS and CS interaction, respectively. Overall, the results of this investigation showed the P170 expression system successfully expressed a cationic peptide with potent anticancer activity. Moreover, molecular docking analysis revealed the pattern of peptide interaction with negatively charged membrane molecules.

One major issue in cancer immunotherapy is the large size of mAbs (150 kDa) which usually causes limitation in tumor penetration. Camel nanobodies (

Introduction: In spite of high vaccination coverage, whooping cough (pertussis) is still a worldwide health problem. The main reason for pertussis outbreak is waning immunity of safer acellular vaccines which have replaced the more reactogenic cellular vaccines. A new generation of pertussis vaccines that is potent and safe is desperately needed to control the disease. Previous studies have indicated that outer membrane vesicles (OMVs) obtained from Bordetella pertussis have desirable characteristics which make them a good candidate for application as pertussis vaccine. They contain surface immunogens in a native structure, are self-adjuvant and are easily uptaken by the antigen presenting cells. Methods: B. pertussis Tohama strain was cultured at 35°C in Stainer-Scholte broth. The OMVs were isolated by a new sequential ultracentrifugation method. The extracted OMVs were characterized by electron microscopy, SDS-PAGE and ELISA assays. Results: The existence of pertussis toxin, filamentous haemagglutinin and a 69-kDa antigen in B. pertussis OMVs was verified using an ELISA assay. Electron microscopy showed the size of these OMV’s at 40-200 nm. The ELISA results indicated that the OMVs extracted using this protocol contain major immunogens. Conclusion: We report for the first time a simple protocol for the efficient extraction of B. pertussis OMVs. This protocol can be used in the process of making new generations of B. pertussis vaccines.

Over the last decade, global increase in antibiotic consumption is a major concern in the word. Antimicrobial peptides (AMPs) known as potential alternative and were considered as a safe antimicrobial agent. However, current approaches for production and purification of AMPs are costly and time-consuming. Here we show that heterologous expression of a chimeric peptide was successfully developed in Lactococcus lactis as a safe and cost-effective recombinant protein expression platform. Minimum inhibitory concentrations (MICs) of His-tag purified peptide was determined against a broad spectrum of human pathogenic bacteria consistence of Gram-positive, Gram-negative and resistance strains in deferent range from 7.24 ± 0.4 to 156.24 ± 3.0 μg/mL. Furthermore, our results showed that the peptide was not toxic to HEK and HeLa cells and even at concentrations as high as 250 μg/mL exhibited minimal hemolysis against RBCs. Additional characteristics such as thermal, protease and 50% human plasma stability were determined for cLFchimera. Molecular modeling analysis demonstrated that fusion of His-tag to the C-terminal of chimeric peptide increased peptide stability during 10 ns simulation in water. Overall, the chimeric peptide has a considerable antibacterial activity with low hemolysis, low or none in toxicity and good temperature resistance and also high stability in serum. We anticipate the established expression system could be developed and used more effectively in probiotic strains in future studies.

Over the last decades, poultry industry faced to the rapid emergence of multidrug-resistant bacteria as a global concern. Antimicrobial peptide (AMPs) known as potential antibiotic alternative and were considered as a new antimicrobial agent. Current methods of production and purification of AMPs have several limitations such as: costly, time-consuming and killing the producing host cells in recombinant form. In the present study, a chimeric peptide derived from camel lactoferrin was produced in Escherichia coli periplasmic space using a pET-based expression system and its antibacterial activity was determined on some avian pathogens in vitro. A carboxy-terminal polyhistidine tag was used for purification by Ni2+ affinity chromatography with an average yield of 0.42 g/L. The His-tagged chimeric peptide showed different range of antimicrobial activity against clinically isolated avian pathogens with low chicken blood hemolysis activity and high serum stability. Overall, the results of this investigation showed the recombinant chimeric peptide was successfully expressed in pET-based expression system and could be considered as a proper alternative for some currently used antibiotics in poultry industry and drugs veterinary medicine.

Probiotics are beneficial microorganisms and have long been used in food production as well as health promotion products. Bioengineered probiotics are used to express and transfer native or recombinant molecules to the mucosal surface of the digestive tract to improve feed efficiency and promote health. Lactococcus lactis is a potential probiotic candidate to produce useful biological proteins. The aim of this investigation was to develop a recombinant Lactococcus lactis with the potential of producing phytase. To enhance the efficiency of expression and secretion of recombinant phytase, usp45 signal peptide was added to the expression vector containing phytase gene (appA2) derived from Escherichia coli. Sequencing of recombinant plasmid containing appA2 showed the correct construction of plasmid. Total length of the phytase insert was 1.25 kbp. A Blast search of the cloned fragment showed 99% similarity to the reported E. coli phytase sequence in the GenBank (accession number: AM946981.2). A plasmid containing usp45 and appA2 electrotransferred into Lactococcus lactis. Zymogram with polyacrylamide gel revealed that the protein extract from the supernatant and the cell pellet of recombinant bacteria had phytase activity. Enzyme activity of 4 U/ml was obtained in cell extracts, and supernatant maximal phytase activity was 19 U/ml. The recombinant L. lactis was supplemented in broiler chicken feed and showed the increase of apparent digestibility on phytate phosphorus in the digestive tract and it was same as performance of E. coli commercial phytase.

Two specific characteristics of stem cells, self-renewal and their differentiation capacity have made them important for vast scientific areas, particularly in the molecular and cell biology of ste...

The use of anticancer peptides (ACPs) as an alternative/complementary strategy to conventional chemotherapy treatments has been shown to decrease drug resistance and/or severe side effects. However, the efficacy of the positively-charged ACP is inhibited by elevated levels of negatively-charged cell-surface components which trap the peptides and prevent their contact with the cell membrane. Consequently, this decreases ACP-mediated membrane pore formation and cell lysis. Negatively-charged heparan sulphate (HS) and chondroitin sulphate (CS) have been shown to inhibit the cytotoxic effect of ACPs. In this study, we propose a strategy to promote the broad utilization of ACPs. In this context, we developed a drug repositioning pipeline to analyse transcriptomics data generated for four different cancer cell lines (A549, HEPG2, HT29, and MCF7) treated with hundreds of drugs in the LINCS L1000 project. Based on previous studies identifying genes modulating levels of the glycosaminoglycans (GAGs) HS and CS at the cell surface, our analysis aimed at identifying drugs inhibiting genes correlated with high HS and CS levels. As a result, we identified six chemicals as likely repositionable drugs with the potential to enhance the performance of ACPs. The codes in R and Python programming languages are publicly available in https://github.com/ElyasMo/ACPs_HS_HSPGs_CS. As a conclusion, these six drugs are highlighted as excellent targets for synergistic studies with ACPs aimed at lowering the costs associated with ACP-treatment. This article is protected by copyright. All rights reserved.

The current study was conducted to investigate the effects of cLFchimera, a recombinant antimicrobial peptide (AMP), on various productive performance and gut health attributes of broilers experimentally challenged withClostridium perfringens(Cp). Three hundred and sixty 1-day-old chickens were randomly allocated to 4 treatments of 6 replicates as follows: T1) unchallenged group fed with corn-soybean meal (CSM) without Cp challenge and additives; T2) challenge group fed with CSM and challenged with Cp without any additives; T3) peptide group challenged with NE supplemented with 20 mg cLF36/kg diet (AMP); T4) antibiotic group challenged with NE and supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet (antibiotic). Birds had free access to feed and water, sampling for villi morphology and ileal microbiota were performed on days 10 and 22, while jejunal section was sampled for gene expression of cytokines, tight junctions proteins, and mucin only on day 22. Results showed that AMP ameliorated NE-related lesion in the jejunum and ileum and reduced mortality in challenged birds compared to challenge group with Cp without any additives. Also, supplementing challenged birds with AMP improved growth performance and reconstructed villi morphology. While antibiotic non-selectively reduced the count of bacteria, AMP positively restored ileal microflora in favor of good bacteria (i.e.Bifidobacteria spp.andLactobacillus spp.). AMP beneficially regulated the expression of cytokines, junctional proteins, and mucin in the jejunum of challenged birds with Cp. Since cLFchimera ameliorated NE lesion score, reduced mortality, improved productive performance and gut health attributes in chickens compared to challenged group and also were mostly similar with those of antibiotics and therefore, it could be concluded that this chimeric peptide can be a worthy candidate to substitute growth promoter antibiotics, while more research is required to unveil the exact mode of action of this synthetic peptide.Author summaryNecrotic enteritis (NE) is a detrimental enteric disease in the poultry industry worldwide. The etiological factor of this disease isClostridium perfringens, which is gram-positive anaerobic bacterium. This bacterium is common inhabitant of the intestine in lower counts (105), but it becomes pathogenic in higher counts and can secrete NetB toxin, which is the main cause of inducing NE in broilers. Due to the emergence of antibiotic-resistant bacteria, new generation of antimicrobial additives such as antimicrobial peptides (AMPs) has been introduced to the poultry industry. AMPs are small molecules with 12-50 amino acids having antibacterial activity. Recently, we extracted new AMP from camel milk, expressed inE. coli, refined and lyophilized to produce purified peptides. The current study investigated the effects of this peptide on prevention of NE in broilers. Results showed that AMP ameliorated lesion scores in the intestine and reduced mortality in challenged birds. AMP improved growth performance and reconstructed villi morphology in NE-challenged broilers. While antibiotic non-selectively reduced the count of bacteria, AMP positively restored ileal microflora. AMP beneficially regulated the expression of cytokines, junctional proteins, and mucin in the jejunum of NE-challenged birds.

This study investigated the effects of an antimicrobial peptide (AMP), cLF36, on growth performance and the histophysiological changes of the intestine in E. coli-challenged broiler chickens. A total number of 360 day old male chicks were randomly assigned to 4 groups of 6 replicates as follows: T1) negative control diet based on corn-soybean meal without E. coli challenge and additives; T2) positive control diet based on corn-soybean meal and challenged with E. coli without any additives; T3) positive control diet challenged with E. coli and supplemented with 20 mg AMP (cLF36)/kg diet; T4) positive control diet challenged with E. coli and supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet. Results showed that T3 improved growth performance and the jejunal morphology of E. coli-challenged chickens similar to those of T4. While antibiotic non-selectively decreased the population of ileal bacteria, AMP increased the population of Lactobacillus spp. and decreased harmful bacteria in the ileum of E. coli-challenged chickens. Supplementing E. coli-challenged chickens with AMP improved the gene expression of immune cells and upregulated the expression of tight junction proteins compared to other challenged groups. In conclusion, although cLF36 beneficially affected growth performance and the intestinal morphology of E. coli-challenged chickens similar to those of the antibiotic group, this AMP drastically improved the intestinal microbiome, immune cells, and junctional proteins compared to other E. coli-challenged birds, and can be nominated as an alternative for growth promoter antibiotics.

BackgroundFoodborne pathogens and their biofilms are considered as one of the most serious problems in human health and food industry. Moreover, safety of foods is a main global concern because of the increasing use of chemical food additives. Ensuring food safety enhances interest in discovery of new alternative compounds such as antimicrobial peptides (AMPs), which can be used as bio-preservatives in the food industry. In this study, the most important antimicrobial peptides of camel milk lactoferrin (lactoferrampin and lactoferricin) were recombinantly expressed in the form of chimeric peptide (cLFchimera) in a food-gradeL. lactisstrain. P170 expression system was used to express secreted cLFchimera using pAMJ1653 expression vector which harbors a safe (non-antibiotic) selectable marker.ResultsPeptide purification was carried out using Ni-NTA agarose column from culture medium with concentration of 0.13 mg/mL. The results of disk diffusion test revealed that cLFchimera had considerable antimicrobial activity against a number of major foodborne bacteria. Furthermore, this chimeric peptide showed strong and weak inhibitory effect on biofilm formation againstP. aeruginosa,S. aureus E. faecalis,andE. coli,respectively. Antioxidant activity and thermal stability of the chimeric peptide was determined. The results showed that cLFchimera had antioxidant activity (IC50: 310 μ/mL) and its activity was not affected after 40 min of boiling. Finally, we evaluated the interaction of the peptide with LPS and DNA in bacteria using molecular dynamic simulation as two main intra and extra cellular targets for AMPs, respectively. Our in silico analysis showed that cLFchimera had strong affinity to both of these targets by positive charged residues after 50 ns molecular dynamic simulation.ConclusionsOverall, the engineered food-gradeL. lactisgenerated in the present study successfully expressed a secreted chimeric peptide with antimicrobial properties and could be considered as a promising bio-preservative in the food industry.

Brucellosis is a zoonetic disease that is caused by Brucella melitensis, which affects human health and animal husbandry industry. Omp31 is the most exposed outer membrane proteins (OMPs) and is a dominant antigen in smooth strains of B. melitensis, thus it is considered a desirable protein in subunit vaccine against brucellosis disease. Besides, IL-2 acts as a growth and differentiation stimulator of T-cells in cellular immune responses, therefore, it can serve as an adjuvant in subunit vaccines against intracellular pathogens. It seems that chimera scaffold, consisting of some copies of OMP31 experimental epitope region and IL-2, can provide a good immunization. In this regard, these tow fragments are linked together through using rigid linker and splicing overlap extension (SOE) PCR technique. Successful PCR products were TA cloned and then sub-cloned into pET-22b (+) vector as an expression vector. Recombinant protein was expressed in Escherichia coli BL21 (DE3) as an expressing lineage bacterium and then confirmed by SDS-PAGE and Western-blotting analysis. Secondary and tertiary structure prediction of recombinant protein and its immunity characterization were evaluated using in silico process. Further studies on the immunogenicity properties of this protein are expected to be carried out in the future to confirm the results in vivo.

Lactoferrin is the most dominant protein in milk after casein. This protein plays a crucial role in many biological processes including the regulation of iron metabolism, induction and modulation of the immune system, the primary defense against microorganisms, inhibiting lipid peroxidation and presenting antimicrobial activity against various pathogens such as parasites, fungi, bacteria, and viruses. The major antimicrobial effect of lactoferrin is related to its N-terminal tail where different peptides for instance lactoferricin and lactoferrampin which are important for their antimicrobial abilities are present. The growth rate of bacterial cells in camel milk is lower than that of the cow milk due to having more antimicrobial compounds. In this study, we have fused a codon-optimized partial camel lactoferrcin and lactoferrampin DNA sequences in order to construct a fused peptide via a lysine. This chimeric 42-mer peptide consists of complete and partial amino acid sequence of camel lactoferrampin and lactoferricin, respectively. Human embryonic kidney 293 (HEK-293) cells were used for synthesizing this recombinant peptide. Finally, the antibacterial activities of this constructed peptide were investigated under in vitro condition. The result showed that, all construction, cloning and expression processes were successfully performed in HEK-293. One His-tag tail was added to the chimera in order to optimize the isolation and purification processes and also reduce the cost of production. Additionally, His-tag retained the antimicrobial activity of the chimera. The antimicrobial tests showed that the growth rate in the majority of bacterial plant pathogens, including gram negative and positive bacteria, was inhibited by recombinant chimera as the level of MIC values were evaluated between 0.39 and 25.07 μg/ml for different bacterial isolates.

Brucellosis is one the serious infectious diseases caused deleterious health and economic losses. Vaccination with subunit vaccines is the efficient alternative way than live attenuated vaccines against infectious diseases. Herein a new chimeric OMP25-BLS antigen emulsified in Chitosan Nanoparticles was designed and its immune responses were compared with control groups. Also, the role of heat shock protein 60 kDa in combination with OMP25-BLS antigen was assessed. Structural and antigenic features of chimeric antigen were predicted using bioinformatics tools. Moreover, the humoral and cellular immune responses were measured by ELISA in seven different groups. Observations showed rOMP25-BLS structure was highly stable and antigenic. Cytokines analysis showed rOMP25 and rOMP25-BLS + rHSP60 induced higher titer of INF-γ than rHSP60 and rOMP25-BLS. There was no statistically significant difference between positive control group and rOMP25-BLS + rHSP60 in inducing TNF-α (p .05). Additionally, the highest titer of IL-4 was dedicated to rOMP25 among other immunized treatments, while there were no significant differences between positive control group and other immunized groups with recombinant proteins (p .05). In addition, rOMP25-BLS and rHSP60 induced higher titer of total antibody compared to other groups. Also, rHSP60 could improve IgG2a to IgG1 ratio when it used in combination with chimeric antigen. Moreover, the lymphocyte proliferation index was higher in chimeric rOMP25-BLS + HSP60 antigen. In conclusion, while rOMP25-BLS chimeric antigen unable to induce efficient cellular response than individual injection of rOMP25, its injection in combination with rHSP60 could improve cellular immunity.

Brucellosis caused by the bacterium Brucella affects various domestic and wild species. The outer membrane proteins 25 and 31 play key roles on stimulation of cell-mediated immune response against Brucella. GroEL as one of the major Brucella antigens stimulates the immune system and increases intracellular survival of bacteria. In the present study, we assumed injection of GroEL in combination with OMP25 and OMP31 would offer higher immunity levels. So, the impact of GroEL with different concentrations of recombinant outer membrane proteins emulsified in Chitosan Nanoparticles on immune responses was evaluated in mice model. Results showed both univalent (except rGroEL) and divalent immunized groups induced higher IFN-γ, TNF-α, and IL-4 titers in comparison to negative control groups. While GroEL showed negative effect on TNF-α titer, there were positive increase trends in IFN-γ in some treatments. Analysis of humoral antibody response revealed both univalent and divalent immunized groups induced higher IgG2a titer than IgG1 titer, indicating strong bent of Th1 immune response. Also, results showed GroEL can have positive impact on lymphocyte proliferation response. Overall, mice immunization using individual OMP25 or OMP31 demonstrated more effective cell-mediated immunity, although some combinations of rGroEL and rOMP31 vaccines were more efficient than other divalent ones.

Background: PhiC31 integrase system provides a new platform in various felid of research, mainly in gene therapy and creation of transgenic animals. This system enables integration of exogenous DNA into preferred locations in mammalian genomes, which results in robust, long-term expression of the integrated transgene. Objectives: Identification of a novel pseudo attP site. Materials and Methods: Genomic DNA was extracted from primary bovine fetal fibroblast cells, which were stably transfected with EGFP and phiC31 integrase cDNAs carrying vectors. An inverse PCR was carried out for production of mini-circle DNAs and followed by sequencing. Results: A new specific pseudo attP site termed BF5 was identified in bovine genome. This site is located in an intergenic AT rich region on chromosome 5 with similar features of other mammalian attP pseudo sites. Furthermore, direct sequencing of generated attL site confirmed that site-specific transgene recombination was occurred at this site. Conclusions: This finding confirmed that phiC31 integrase could be feasible for production of transgenic animals for biotechnological applications.

Silver nitrate is an inorganic compound with chemical formula AgNO3. Silver or silver ions have long been used in many areas due to their strong antimicrobial activity against pathogenic microbes such as bacteria, yeast, fungi and algae. The protein Pal (peptidoglycan-associated lipoprotein) is anchored in the outer membrane (OM) of Gram-negative bacteria and interacts with Tol proteins. We used Molegro virtual docker (MVD). The results obtained from docking showed us that the best pose which is derived from MolDock score for Chitinase was -23.4702 with reranking score equal to -33.6883. Bioinformatic studies show that silver nitrate has interaction with protein Pal.

Background: PhiC31 integrase is a DNA site-specific recombinase integrates DNA into the chromosomes between the two sites of attB and attP. Several pseudo attPs have been identified in mammalian genomes with critical features for long-term expression of transgene. In this manuscript, we report a novel intrinsic pseudo attP site named CHOL1 in the Chinese hamster genome implementing an inverse Polymerase Chain Reaction (IPCR). Objectives:Identification of pseudo attP site(s) of Tenecteplase cDNA integration in the genome of stable transformed CHO cell line. Materials and Methods: First, genome was extracted from a stable transformed CHO cell line expressing Tenecteplase. By creating of minicircle DNA in the last step, sequenc‌ing was performed. Results: We obtained one band. BLAST analysis of the respective sequence of inverse PCR band identified a pseudo attP site. Conclusions: Data demonstrated that the phiC31 integrase provides a suitable insertion site in the genome to express the gene of interest.

Despite widespread application of intracytoplasmic sperm injection (ICSI) in human-assisted reproductive techniques (ART), the efficiency of this method is still far from satisfactory in livestock, particularly in the bovine species with its unique sperm condensation. On the basis of the natural chemical structure of chromatin in condensed sperm, we developed a novel combined heparin-reduced glutathione (GSH) sperm pretreatment that improves the efficiency of bovine ICSI via selection of the most appropriate sperm at the time of ICSI. Assessment of sperm DNA integrity revealed that this pretreatment can be considered as a safe and efficient approach for in vitro sperm decondensation when compared to conventional sperm pretreatments with dithiothreitol (DTT). Injection of completely decondensed bull sperm derived from this pretreatment significantly improved fertilization and blastocyst formation rates compared to untreated or intact sperm injection (34.8 ± 2.7 and 29.1 ± 1.5 vs. 12.0 ± 3.2 and 15.9 ± 1.2%, respectively; p0.05). Real-time PCR analysis revealed that expression of pluripotent and anti-apoptosis markers in blastocysts derived by injection of completely decondensed sperm from heparin-GSH pretreatment were comparable to IVF when compared to the DTT pretreatment and control ICSI groups (p0.05). The results of this study suggested that the degree of sperm decondensation derived from heparin-GSH pretreatment may affect ICSI efficiency in bovine.