Insect peptides have been extensively studied due to beneficial effects in the treatment of infectious diseases. Melittin, a fundamental component of honeybee venom produced by European honeybee Apis mellifera, has applied to prevent various inflammatory disease and bacterial infections in human. However, the therapeutic application of melittin is limited due to its low stability, hemolytic activity and expensive manufacturing costs. In this study, we aimed to discovery unknown peptides from the Apis mellifera and evaluate its antibacterial activity against Escherichia coli KACC 10005. A total 15,853 peptide sequences were diciphered using Illumina HiSeq 2500 next-generation sequencing (NGS) platform and analyzed based on the Apis mellifera official Gene Set Version 3.2 (amel_OGSv3.2) and the Collection of Anti-Microbial Peptides (CAMPR3) database. All the peptide sequences and annotation data sets were combined and sorted by physicochemical features of antimicrobial peptides (AMPs), such as short peptide length <=50, positive charge, isoelectric point (8.0<=pl<=12), and aggregation propensity (in-vitro: <=500, in-vivo: –40<= Na4vSS <=60). Among the screened peptides, four unknown peptide candidates, named AMP1-4, were chemically synthesized and tested for antimicrobial activity in comparison with a reference peptide, melittin. Inhibition of bacterial growth was observed in the AMP4 treated group from 6 hours to 48 hours post-treatment against E. coli. These results suggest that honeybee-derived peptide sequences can be applied as natural resources to acquire novel AMPs and the peptide sequences derived parameters are enough to recognize antibacterial peptides. In addition, the selected novel peptide candidate, AMP4, has antibacterial activity.

The hypopharyngeal gland (HPG) of the honeybee worker produces royal jelly (RJ) and has a developmental cycle closely related to the division of labor. In this study, we investigated to compare the HPG acini diameter of differently aged worker bees with high royal jelly producing colony (HRC) or less producing colony (LRC). Additionally, we also evaluated whether the fresh weight of the head is a reliable indicator of the developmental status of HPG. The HRC showed a significantly higher RJ production about two-times as compared with those of the LRC. We measured the HG-diameters on days 1, 3, 6, 9, 12, 15. The microscopic analysis revealed that the acini size of the HRC was significantly larger than the LRC. In addition, the acini diameter of HRC was 15% longer than the LRC on the first day after emerging. It was shown that the fastest development during 3 days which is preparing for nurse the brood. The HPG acini diameters increased in both colonies in a similar fashion until day 12 and then decreased. We also compared the fresh head weight of the experimental colonies, differences were similar to the development of HPG. Therefore, high royal jelly production may have a positive correlation between HPG acini size and the fresh head weight.

Regarding therapies for treatment of corneal wounds, ex vivo corneal culture is the most effective for minimizing expensive animal studies. Eighteen porcine enucleated eyes were soaked in 0.2% povidone iodine solution for disinfection prior to cornea excision. Subsequently, corneas were excised from whole eyes and filled with an agar/medium mixture. Corneas were transferred into culture dishes, after which culture medium was added until the limbus was covered. Cultures were then placed onto a plate rocker to mimic blinking action, followed by incubation at 37°C and 5% CO2. Corneas were harvested on Days 0, 3, and 7 after incubation, and optical coherence tomography (OCT) was performed on Day 7. Two eyes from each group were fixed in 2% glutaraldehyde/4% paraformaldehyde for low-vacuum scanning electron microscopy (LV-SEM), and four eyes from each group were fixed in 10% neutral-buffered formalin for histological analysis. OCT results showed that central corneal thickness significantly increased by Day 7 compared to Day 0 (P<0.05). Using LV-SEM, gaps between endothelial cells were detected on Day 7 of ex vivo culture. In the histological evaluation, four to five stratified squamous cell layers, wing cells, and basal cells in the epithelium as well as flat-shaped keratocytes in the stroma were found on Day 0. By Day 7, stratified squamous cells and basal cells had decreased in number, and slightly round-shaped keratocytes were observed; however, the number of keratocytes was similar to that on Day 0. In this short-term ex vivo culture, epithelium and endothelium were sensitive to culture, whereas stroma and keratocytes were well maintained. An additional deswelling method will be needed to obtain more successful results in porcine corneal ex vivo culture.

Antibiotics have been used to prevent disease, promote growth rate, and improve feed efficiency. However, the use of antibiotics in livestock has been restricted worldwide due to problems such as bacterial resistance. Therefore, probiotics among alternatives to antibiotics have gained attention in the livestock feed industry these days. This study was conducted to investigate the effects of dietary supplementation with probiotic 379D on safety, growth rate, and feed efficiency. In this study, bacterial strain 379D was isolated from soil and identified as a Bacillus sp. according to 16S rRNA sequence analysis. In an in vitro test, in-gel activity assay and antimicrobial susceptibility test were conducted to evaluate 379D. In an in vivo study, 379D was administered at concentrations of 0.1% and 1% to broiler chickens for 28 days. The results of in-gel activity assay and antimicrobial susceptibility test showed that strain 379D had broad spectrum antimicrobial activity. Furthermore, no adverse 379D-related effects were observed in 0.1% and 1% groups. Feed efficiency was higher in the 379D-treated groups than in the control group. In conclusion, 379D is expected to be used as a safe alternative to antibiotics in a feed supplement and will improve feed efficiency in broiler chickens.

The current study was conducted to evaluate the biocompatibility of α-1,3 galactosyltransferase knockout pig bone graft in a rat calvarial defect model. Porcine cancellous bones were harvested from general and alpha-gal KO pigs and washed with 70% ethanol solution and normal saline. Bone pieces of the alpha-gal KO pig underwent a chemical treatment process to delipidize and deproteinize the bone. Bone graft particles were freeze-dried and stored at −70°C until use. Each bone graft was implanted into the rat calvarial defect in a fresh general pig, fresh transgenic pig, and chemical-treated pig bone group. There was no systemic adverse effect on hematology or necropsy findings in all groups at 1 week and 4 weeks. In the microcomputed tomography analysis, bone volume increased significantly in the chemical-treated transgenic pig bone group, whereas bone mineral density decreased significantly in the fresh general pig bone group compared with other groups. Histological evaluation showed cellular infiltration located at the margin of the bone graft particles, especially in the fresh general pig bone group. These results indicate that fresh general pig bone can elicit a greater local inflammatory response than fresh transgenic pig bone. Further, chemical-treated transgenic pig bone graft was less immunogenic than fresh bone graft. In conclusion, transgenic pig bone is a more biocompatible graft material. In addition, chemical treatment can reduce bone graft immunogenicity by delipidizing and deproteinizing bone.

This study evaluated the possibility of clinical application using matrigel-based bioceramic/polymer scaffolds treated with bone morphogenetic protein, angiogenic factor, and mesenchymal stem cells (MSCs) for new bone formation. In the in vitro study, bone morphogenetic protein (BMP-2) and vascular endothelial growth factor (VEGF) containing matrigel, which is a basement membrane gel, was injected into HA/PCL scaffolds to estimate the release rates of growth factors. In the in vivo study, BMP-2, VEGF, and MSCs with matrigel-based scaffolds were implanted into rat femoral segmental defects, and new bone formation was evaluated at 4 and 8 weeks. In the results, the release rates of BMP-2 and VEGF explosively increased by day 5. For the in vivo study results, radiological evaluation revealed that the matrigel-based HA/PCL scaffolds with BMP-2 and VEGF grafted (M+B+V) and matrigel-based HA/PCL scaffolds with BMP-2, VEGF, and MSC grafted (MSC) groups showed increased bone volume and bone mineral density. Moreover, in the histological evaluation, large new bone formation was observed in the M+B+V group, and high cellularity in the scaffold was observed in the MSC group. In conclusion, grafted matrigel-based HA/PCL scaffolds with BMP-2, angiogenic factor, and MSCs increased new bone formation, and in clinical cases, it may be effective and useful to enhance healing of delayed fractures.

In the livestock feed industry, antibiotics are used to prevent disease, promote growth rate, and improve feed efficiency. However, antibiotic supplementation to animal feed results in increased bacterial resistance to antibiotics as well as antibiotic residues in animal products, which can negatively affect human health. Therefore, alternative sources of antibiotics are need- ed. Probiotics as an alternative to antibiotics in animal feed have been shown to increase feed efficiency and growth rate by improving microbial balance. Further, Bacillus sp. produces a wide spectrum of antibacte- rial peptides. The present study was conducted to investigate the effects of dietary supplementation with CS-32 on safety, growth rate, and feed efficiency. Antibacterial substance (5697.9 molecular weights) produced by CS-32 was isolated and purified from culture broth. Moreover, the results of minimal inhibi- tory concentration (MIC) test confirmed the excellent antibacterial effect of CS-32. In vivo, 0.1% and 1% CS-32 were fed to broiler chickens for 28 days. Feed efficiency was slightly higher in groups of chickens supplemented with 0.1% and 1% CS-32 than those of the control group. CS-32 had no significant effect on necropsy findings, hematology, or serum biochemistry, and there was no mortality. These results suggest that CS-32 among various biologically active substances may be safe and effective as a feed additive to improve growth rate and feed efficiency.

The current study was conducted in order to investigate bone formation using matrigel and angiogenic factors with HA and poly ε-caprolactone (HA/PCL) in a rat calvarial defect model. Calvarial defect formation was surgically created in Sprague Dawley rats (n=36). Rats in the control group (CD group, n=6) did not receive a graft. The HA/ PCL scaffold was grafted with matrigel (M-HA/PCL group, n=6) or without matrigel (HA/PCL group, n=6); and 100 ng of vascular endothelial growth factor with HA/ PCL scaffold containing matrigel (VEGF100 group, n=6), 100 ng (PDGF100 group, n=6) and 300 ng (PDGF300 group, n=6) of PDGF with HA/PCL scaffold containing matrigel were grafted in calvarial defects, respectively. Four weeks after surgery, bone formation was evaluated with micro computed tomography (micro CT) scanning, and histologically. According to the results, bone mineral density was significantly increased in the VEGF100, PDGF100, and PDGF300 groups compared to the HA/PCL group, in which angiogenic factors were not applied. In histological evaluation, more new bone formation around scaffolds was observed in the PDGF100 and the PDGF300 groups, compared with the VEGF100 group. Thus, the results indicate that HA/PCL containing matrigel with VEGF and PDGF is an effective grafting material for enhancement of bone formation in critical-sized bone defects. Especially, due to its price and capacity for bone formation, PDGF may be more effective than VEGF.