The roles of templates consisting of amino acids in the synthesis and application of gold nanoclusters.

Gold nanoclusters (AuNCs) with low toxicity, high photostability, and facile synthesis have attracted great attention. The ligand is of great significance in stabilizing AuNCs and regulating their properties. Ligands consisting of amino acids (proteins and peptides) are an ideal template for synthesizing applicative AuNCs due to their inherent bioactivity, biocompatibility, and accessibility. In this review, we summarize the correlation of the template consisting of amino acids with the properties of AuNCs by analyzing different peptide sequences. The selection of amino acids can regulate the fluorescence excitation/emission and intensity, size, cell uptake, and light absorption. By analyzing the role played by AuNCs stabilized by proteins and peptides in the application, universal rules and detailed performances of sensors, antibacterial agents, therapeutic reagents, and light absorbers are reviewed. This review can guide the template design and application of AuNCs when selecting proteins and peptides as ligands.

The Contribution of Antimicrobial Peptides to Immune Cell Function: A Review of Recent Advances.

The development of novel antimicrobial agents to replace antibiotics has become urgent due to the emergence of multidrug-resistant microorganisms. Antimicrobial peptides (AMPs), widely distributed in all kingdoms of life, present strong antimicrobial activity against a variety of bacteria, fungi, parasites, and viruses. The potential of AMPs as new alternatives to antibiotics has gradually attracted considerable interest. In addition, AMPs exhibit strong anticancer potential as well as anti-inflammatory and immunomodulatory activity. Many studies have provided evidence that AMPs can recruit and activate immune cells, controlling inflammation. This review highlights the scientific literature focusing on evidence for the anti-inflammatory mechanisms of different AMPs in immune cells, including macrophages, monocytes, lymphocytes, mast cells, dendritic cells, neutrophils, and eosinophils. A variety of immunomodulatory characteristics, including the abilities to activate and differentiate immune cells, change the content and expression of inflammatory mediators, and regulate specific cellular functions and inflammation-related signaling pathways, are summarized and discussed in detail. This comprehensive review contributes to a better understanding of the role of AMPs in the regulation of the immune system and provides a reference for the use of AMPs as novel anti-inflammatory drugs for the treatment of various inflammatory diseases.

Sustained release of alginate hydrogel containing antimicrobial peptide Chol-37(F34-R) in vitro and its effect on wound healing in murine model of Pseudomonas aeruginosa infection.

BACKGROUND: Antibiotic resistance is a significant public health concern around the globe. Antimicrobial peptides exhibit broad-spectrum and efficient antibacterial activity with an added advantage of low drug resistance. The higher water content and 3D network structure of the hydrogels are beneficial for maintaining antimicrobial peptide activity and help to prevent degradation. The antimicrobial peptide released from hydrogels also hasten the local wound healing by promoting epithelial tissue regeneration and granulation tissue formation. OBJECTIVE: This study aimed at developing sodium alginate based hydrogel loaded with a novel antimicrobial peptide Chol-37(F34-R) and to investigate the characteristics in vitro and in vivo as an alternative antibacterial wound dressing to treat infectious wounds. METHODS: Hydrogels were developed and optimized by varying the concentrations of crosslinkers and subjected to various characterization tests like cross-sectional morphology, swelling index, percent water contents, water retention ratio, drug release and antibacterial activity in vitro, and Pseudomonas aeruginosa infected wound mice model in vivo. RESULTS: The results indicated that the hydrogel C proved superior in terms of cross-sectional morphology having uniformly sized interconnected pores, a good swelling index, with the capacity to retain a higher quantity of water. Furthermore, the optimized hydrogel has been found to exert a significant antimicrobial activity against bacteria and was also found to prevent bacterial infiltration into the wound site due to forming an impermeable barrier between the wound bed and external environment. The optimized hydrogel was found to significantly hasten skin regeneration in animal models when compared to other treatments in addition to strong inhibitory effect on the release of pro-inflammatory cytokines (interleukin-1ß and tumor necrosis factor-α). CONCLUSIONS: Our results suggest that sodium alginate -based hydrogels loaded with Chol-37(F34-R) hold the potential to be used as an alternative to conventional antibiotics in treating infectious skin wounds.

Erratum: [Corrigendum] Expression of porcine protegrin­1 in Pichia pastoris and its anticancer activity in vitro.

[This corrects the article DOI: 10.3892/etm.2015.2202.].

Immune responses and protective efficacy of a trivalent combination DNA vaccine based on oprL, oprF and flgE genes of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an infectious pathogenic bacteria infecting many different species of animals. Currently, it lacks a commercial vaccine. In this study, three monovalent DNA vaccines (poprL, poprF, and pflgE), three bivalent combination DNA vaccines (poprL+poprF, poprL+pflgE, poprF+pflgE), and a trivalent DNA vaccine (poprL+poprF+pflgE) were constructed. Consequently, we immunised chickens with these DNA vaccines and used inactivated vaccines as the positive controls. Then, the immune efficacy was evaluated through serum antibody detection, a lymphocyte proliferation assay, and cytokine concentration determination. Lastly, we assessed the protection rate through a challenge experiment. Following vaccination, the serum antibody levels induced using these DNA vaccines were different due to the different coating antigens. In the trivalent combination DNA vaccine group, we established that the lymphocyte proliferation (SI values), IFN-γ, IL-2, and IL-4 levels were significantly higher than those of the other six DNA vaccine groups and the inactivated vaccine group. However, the protection provided was slightly lower than that of the inactivated vaccine and higher than those of other DNA vaccines. The protection rate of poprL, poprF, pflgE, poprL+poprF, poprL+pflgE, poprF+pflgE, poprL+poprF+pflgE, and the inactivated vaccine were 50, 45, 60, 75, 80, 80, 90, and 95%, respectively. The results of this study indicated the trivalent DNA vaccine based on oprL, oprF and flgE genes represents a promising approach for the prevention of Pseudomonas aeruginosa infections.

Immune Efficacy of different immunization doses of divalent combination DNA vaccine pOPRL+pOPRF of Pseudomonas aeruginosa.

At present, there is no vaccine available against Pseudomonas aeruginosa, a common zoonotic pathogenic bacterium. In a previous study, the authors prepared a divalent combination DNA vaccine, pOPRL+pOPRF, which exhibited good protective efficacy. To explore the optimal immunization dose of this divalent combination DNA vaccine, in the present study, chickens were vaccinated with 25, 50, 100, and 200 µg doses. The levels of serum antibody, interferon-γ (IFN-γ), and interleukin-2 (IL-2) were determined, and lymphocyte proliferation assays were performed. After challenge with virulent P. aeruginosa, the protective efficacy was evaluated. Following vaccination, the serum antibodies, stimulation index values, and concentrations of IFN-γ and IL-2 were significantly higher in chickens vaccinated with 100 and 200 µg vaccines than in those vaccinated with 25 and 50 µg doses (P<0.05). IFN-γ and IL-2 concentrations in chickens immunized with 100 µg vaccine were slightly higher than those in chickens immunized with 200 µg vaccine, although the difference was not statistically significant. The protective rates were 55%, 65%, 85%, and 85% with 25, 50, 100, and 200 µg of the pOPRL+pOPRF DNA vaccine, respectively. Thus, the immune efficacy of the pOPRL+pOPRF DNA vaccine increased with an increase in immunization dose, but this does not imply that a higher dose necessarily achieves a better outcome. The optimal immunization dose of pOPRL+pOPRF DNA vaccine in chickens was 100 µg.

Cloning and expression patterns of neuromedin U and its receptors in pigs.

Neuromedin U (NMU) is a highly conserved neuropeptide that performs a variety of physiological functions in animals via neuromedin U receptor-1 (NMUR1) and neuromedin U receptor-2 (NMUR2). In this study, we cloned the pig NMU, NMUR1 and NMUR2 genes. Bioinformatics analysis demonstrated that the pig NMU cDNA encoded the amino acids Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2 at the C-terminus and that the NMU receptors, which are G-protein-coupled receptors (GPCRs), contained the seven transmembrane domains typical of GPCRs. Systemic NMU, NMUR1 and NMUR2 mRNA expression was investigated in various pig tissues using real-time RT-PCR. NMU mRNA was expressed both in the central nervous system (CNS) and in peripheral tissues. NMUR1 mRNA was widely expressed in peripheral tissues, whereas NMUR2 mRNA was mainly expressed in the CNS. Immunohistochemistry (IHC) was used to determine the expression patterns of NMU and NMUR1, which were predominantly located in the gastrointestinal tract, genitourinary organs, and immune organs. This study presents molecular and morphological data to aid in additional NMU research in pigs.

Expression of porcine protegrin-1 in Pichia pastoris and its anticancer activity in vitro.

Protegrin-1 (PG-1), a ß-hairpin antimicrobial peptide (AMP), is amongst the shortest AMPs in sequence length while remaining active against a variety of microorganisms. The aim of this study was produce recombinant PG-1 and investigate its anticancer activity. A DNA sequence encoding the mature PG-1, fused with a 6His-tag, was cloned into the pPICZα-A vector and transformed into Pichia pastoris. Expression was induced following culture for ~96 h with 1% methanol at 28°C, and ~15.6 mg PG-1 was expressed in 100 ml culture medium. Following purification using a Ni-chelating Sepharose column, ~20 mg pure active PG-1 was obtained from 500 ml culture broth supernatant. The expressed PG-1/6His exhibited strong dose- and time-dependent anticancer activity against HepG2 cells in vitro.

Bursin-like peptide (BLP) enhances H9N2 influenza vaccine induced humoral and cell mediated immune responses.

Vaccination with H9N2 avian influenza whole-inactivated virus (WIV) has been shown to be ineffective at eliciting sufficient humoral and cellular immunity against H9N2 avian influenza virus. This study assessed the effects of a synthetic Bursin-like epitope peptide (BLP) as adjuvant for H9N2 WIV in mice. Titers HI and avian influenza virus neutralizing antibodies, subtypes of HA antibodies, T helper (Th) cytokine levels, cytotoxic T-lymphocyte activities and changes in spleen T-cell subsets and natural killer cells were determined. We found that BLP induced a balance between IgG1 and IgG2a secretion levels. WIV antigen alone induced mainly Th1 cytokines secretion, whereas BLP showed increased secretion of Th1 and Th2 cytokines, including interleukin (IL)-2, interferon-γ (IFN-γ) and IL-4, but not IL-10, and may be resembles a Th0 like response. BLP significantly promoted growth and expansion of natural killer cells and of CD4(+) and CD8(+) T-cell subsets in the spleen. Meanwhile, BLP induced a better cytotoxic T-lymphocyte response to H9N2 virus. Furthermore, virus challenge experiments confirmed that BLP contributed to inhibition replication of the virus from mouse lungs. Taken together, these findings suggest that BLP may be an effective adjuvant for H9N2 avian influenza vaccine.

Imidapril provides a protective effect on pulmonary hypertension induced by low ambient temperature in broiler chickens.

OBJECTIVE: The objective of this article is to explore the role of imidapril on pulmonary hypertension induced by low ambient temperature in broiler chickens. MATERIALS AND METHODS: Ninety chickens were randomly divided into three groups (n = 30): a control group, a low-temperature group and an imidapril group. Chickens in the low-temperature group and imidapril group were exposed to low ambient temperature from 14 days of age until 45 days of age; chickens in the imidapril group were gavaged with imidapril 3 mg/kg once daily for 30 days. The pulmonary arterial pressure, main pulmonary arterial diameter and pulmonary arterial wall thickness were measured, and lung tissue ACE, ACE2 mRNA expression, proliferating cell nuclear antigen (PCNA)-positive cells and Ang II, Ang (1-7) concentration were evaluated. RESULTS: The pulmonary arterial pressure was higher, the main pulmonary arterial diameter was wider and the pulmonary arterial wall was thicker in the low-temperature group than those in the control group and the imidapril group. ACE mRNA and PCNA-positive cells increased significantly in the low-temperature group compared with the control group and imidapril group; lung tissue Ang II concentration in the low-temperature group was higher, but Ang (1-7) content was lower than that in the control group and imidapril group. CONCLUSION: Imidapril provides a protective effect on pulmonary hypertension induced by low ambient temperature in broiler chickens.

Immune responses and protective efficacy of a novel DNA vaccine encoding outer membrane protein of avian Pasteurella multocida.

Avian Pasteurella multocida is a causative agent of fowl cholera. Two proteins OmpH and OmpA are the major immunogenic antigens of avian P. multocida, which play an important role in inducing immune responses that confer resistance against infections. In the present study, we used pcDNA3.1(+) as a vector and constructed DNA vaccines with the genes encoding the two antigens mentioned above. These DNA vaccines include monovalent (pcDNA-OMPH, pOMPH and pcDNA-OMPA, pOMPA), divalent combination (pcDNA-OMPH+pcDNA-OMPA, pOMPH+pOMPA) and fusion of two gene vaccines (pcDNA-OMPH/OMPA, pOMPHA). The immune responses to these DNA vaccines were evaluated by serum antibody titers, lymphocyte proliferation assay and titers of a cytokines, IFN-γ. The protective efficacy after challenging with a virulent avian P. multocida strain, CVCC474, was evaluated by survival rate. A significant increase in serum antibody levels was observed in chickens vaccinated with divalent combination and fusion DNA vaccines. Additionally, the lymphocyte proliferation (SI value) and the levels of IFN-γ were both higher in chickens immunized with divalent combination and fusion DNA vaccines than in those vaccinated with monovalent DNA vaccines (P<0.05). Furthermore, the protection provided by divalent combination and fusion DNA vaccines was superior to that provided by monovalent DNA vaccines after challenging with the avian P. multocida strain CVCC474. And the protective efficacy in chickens immunized three times with the fusion DNA vaccine was equivalent to the protective efficacy in chickens vaccinated once with the attenuated live vaccine. This suggests that divalent combination and fusion DNA vaccines represent a promising approach for the prevention of fowl cholera.

Expression of 4kD scorpion defensin and its in vitro synergistic activity with conventional antibiotics.

The 4kD scorpion defensin (SD) is a potent disulfide-linked peptide. In this study, we expressed it in methylotrophic yeast Pichia pastoris and purified it using Ni-NTA His Bind Resin. We investigated its in vitro antibacterial activity and effect in combination with several conventional antibiotics. We first examined its antibacterial activity towards several Gram-positive and Gram-negative bacteria. Then we used the broth microdilution method to test drugs alone and in combination and used the fractional inhibitory concentration (FIC index) to classify the drug interactions. Our study showed the expressed SD peptide has antibacterial activity against Salmonella typhimurium, E. coli and S. aureus etc. Synergy or additive interaction was observed between SD and Norfloxacin, Polymyxin B and Ampicillin. Cell growth tests showed that combination of SD and Norfloxacin can improve their activity against bacteria. This result maybe permit lower using of the conventional antibiotic agents more effectively and safely.

[Expression of bursin receptor on membranes of hybridoma cell and identification of binding peptide of bursin from 12-merrandom phage display peptide library].

OBJECTIVE: Bursin (BS) could greatly promote the production of monoclonal antibody in hybridoma cell. We studied the interaction between BS and hybridoma cell. METHODS: Fluorescence microscopy, Laser Scanning Confocal Microscope (LSCM) and fluorescence activated cell sorter (FACS) were used. RESULTS: Fluorescence microscopy revealed specific binding of FITC-BS to hybridoma cell. FACS analysis demonstrated that the binding was specific, saturable and reversibility. BS was used as target protein to screen its binding peptides from 12-mer random phage display peptide library. After four rounds of biopanning, 20 phage clones were randomly selected and identified. ELISA and competitive inhibition test results indicated that 2 phage clones were identified as positive clones. The amino acid sequences analysis shown that the sequences were ACTKHLCLLQPL or MSCNDTLCLLPN, which sharing a conservative sequence LCLL. In vitro experiments suggested that the two binding peptides can inhibit BS specific binding to hybridoma cell. CONCLUSION: These results confirmed that existence of BS receptor in the membrane of hybridoma cell, which involved in hybridoma cell secreting monoclonal antibody signal pathway.

Molecular cloning and sequence analysis of the duck enteritis virus UL5 gene.

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious, and fatal disease. In the present article, the DEV UL5 gene was cloned and sequenced from a vaccine virus. According to the consensus sequence of herpesvirus UL5 and UL3 gene degenerate oligonucleotide primers were designed and were used in the polymerase chain reaction (PCR) to amplify DNA products with 4577 bp in size. DNA sequence analysis revealed a 2568 bp open reading frame (ORF) encoding a 855 amino acid polypeptide homologous to herpesvirus UL5 proteins. The DEV UL5 gene has a base composition of 769 adenine (29.95%), 556 cytosine (21.65%), 533 guanine (20.76%) and 710 thymine (27.65%). Sequence comparison revealed that the nucleotide sequence of the DEV UL5 gene was highly similar to other alphaherpesviruses. Phylogenetic tree analysis showed that the fifteen herpesviruses viruses analyzed fell into four large groups, and the duck enteritis virus itself branched and was most closely related to meleagrid herpesvirus 1, gallid herpesvirus 2 and gallid herpesvirus 3 subtrees.

The molecular design of a recombinant antimicrobial peptide CP and its in vitro activity.

Antibacterial peptides from various sources express different antibacterial activity. In order to obtain a high activity antibacterial peptide, the sequences of four antimicrobial peptides--Protegrin-1, 4 kDa Scorpion Defensin, Metalnikowin-2A and Sheep Myeloid Antibacterial Peptide SMAP-29--were exploited to generate a synthetic antimicrobial peptide cp gene, which was then cloned into the expression vector pPICZalpha-A. The constructed recombinant expression vector pPICZalpha-cp was transformed into Pichia pastoris X-33, in which the synthetic antimicrobial peptide (CP) could be expressed under the control of the inducible AOX1 promoter and secreted via the alpha mating factor leader of Saccharomyces cerevisiae. Results showed that recombinant plasmid is highly stable, and In vitro experiments showed that the recombinant antimicrobial peptide CP is heat and acid-stable, and it has high antibacterial activity against several Gram-positive and -negative bacteria. Only 1 microg of the recombinant antimicrobial peptide CP has an antibacterial activity equivalent to 64 U ampicillin. Thus, this recombinant antimicrobial peptide could serve as an attractive candidate for the development of therapeutic antimicrobial drugs.

[Secreted expression of the combinant antimicrobial peptide PL in Pichia pastoris and its antibacterial activity in vitro].

In order to obtain a high activity antibacterial peptide, An expression vector pPICZalphaA-pl is constructed with a tandem of four antimicrobial peptides in the same direction,which includes Protegrin-1 (PG-1), Scorpion Defensin (SD), Metalnikowin-2A and Sheep Myeloid Antibacterial Peptide (SMAP-29) (serial number in GenBank are AAB27599, AAAB27538, P80409 and P49928 respectively). At the same time the expression vector pPICZalphaA-sd which express Scorpion Defensin was contructed. The expression vectors of pPICZalphaA-pl and pPICZalphaA-sd were linearized and transformed into the yeast host strain X-33 respectively. Under the control of the promoter AOX1 (alcohol oxidase1), the peptides PL and SD were secreted expressed. Their heat-stable property, acid-stable property and MIC were detected in vitro. The results suggest the peptides PL and SD have good heat-stable and acid-stable properties, and the combinant PL peptide showes higher antibacterial activity against several Gram-positive bacteria (G+) and Gram-negative bacteria (G-) than the peptide SD, especially against Escherichia coli. The antibacterial activity of combinant antimicrobial peptide PL shows its far exploiting perspective.

[Influence of epitope A modification and N-linked glycosylated site mutation of PRRSV NJ-a strain ORF5 gene on the ability to induce neutralizing antibodies and T cell proliferation response].

To enhance the DNA immunogencity of PRRSV ORF5 gene, CpG sequence and the universal helper T cell antigen epitope (PADRE) sequence were inserted between the decoy epitope and the neutralizing epitope. At the same time, site-mutations were introduced at N33 and N51 to diminish the coverage effect to epitope B from the polysaccharides. Subsequently, the modified ORF5 gene (MORF5) and PRRSV ORF6 gene were cloned into the eukaryotic expression vector pcDNA3.0 under the control of two CMV promoters, respectively. With indirect immunofluorescence assay and Western-blot the expression in vitro of the two genes was confirmed, then six-week-old Balb/C mouse were immunized with the modified expression plasmid pcDNA-M5A-6A. The non-modified expression plasmid pcDNA-5A-6A, the blank eukaryotic expression plasmid pcDNA3.0, living attenuated vaccine and inactivated vaccine were used as controls. The PRRSV specific neutralizing antibodies and the T cell proliferation response were elevated with virus neutralization assay and MTf method. Results indicate that the modified plasmid pcDNA-M5A-6A can elicit not only higher titer of neutralizing antibodies in a rapid time, but also more vigorous T cell proliferation response compared with the non-modified plasmid pcDNA-5A-6A and commercial vaccines, indicating that DNA vaccine pcDNA-M5A-6A maybe a promising candidate for PRRS prevention.