Exosomes are Nano-sized lipid vesicles secreted from mammalian cells containing diverse cellular materials such as proteins, lipids, and nucleotides. Multiple lines of evidence indicate that in saliva, exosomes and their contents such as microRNAs (miRNAs) mediate numerous cellular responses upon delivery to recipient cells. The objective of this study was to characterize the different expression profile of exosomal miRNAs in saliva samples, periodically isolated from a single periodontitis patient. Unstimulated saliva was collected from a single patient over time periods for managing periodontitis. MicroRNAs extracted from each phase were investigated for the expression of exosomal miRNAs. Salivary exosomal miRNAs were analyzed using Affymetrix miRNA arrays and prediction of target genes and pathways for its different expression performed using DIANA-mirPath, a web-based, computational tool. Following the delivery of miRNA mimics (hsa-miR-4487, -4532, and -7108-5p) into human gingival fibroblasts, the expression of pro-inflammatory cytokines and activation of the MAPK pathway were evaluated through RT-PCR and western blotting. In each phase, 13 and 43 miRNAs were found to be differently expressed (|FC| ≥ 2). Among these, hsa-miR-4487 (|FC|=9.292005) and hasmiR- 4532 (|FC|=18.322697) were highly up-regulated in the clinically severe phase, whereas hsa-miR-7108-5p (|FC|= 12.20601) was strongly up-regulated in the clinically mild phase. In addition, the overexpression of miRNA mimics in human gingival fibroblasts resulted in a significant induction of IL-6 mRNA expression and p38 phosphorylation. The findings of this study established alterations in salivary exosomal miRNAs which are dependent on the severity of periodontitis and may act as potential candidates for the treatment of oral inflammatory diseases.

The periods of elevated temperature and high humidity has been longer since last ten years and cause problems in program of artificial insemination or at the efficiency of in vitro production of transferable embryos. The aims of this study were to evaluate the relationship between time of heat shock (0, 1, 2 and 4), during in vitro maturation and developmental competence of subsequent embryo after in vitro fertilization. The develpmentat rate and percetage of apoptotic cells were evaluated on matured oocyte and day 8. 41℃ Heat treatment after IVM culture significantly decreased the developmental capacity of IVF embryos. Also the number of apoptotic cell in COCs, morula and blatostcysts was started to increase at 2 hr heat treatment but did not affect on the rate of maturation. These results indicate that heat treatment for 2 to 4 hr at 41℃ have negative effects on maturation rate of COCs and lower the developmental competence of heat shocked oocyte derived embryos.

During the oocyte maturation, antioxidants may be beneficial for futher developmental competence against reactive oxygen species (ROS) because the media for oocytes lack boiomolecules that serve as scavengers. In this study, N-acetyl-L-cysteine (NAC), N-acetyl-L-cysteine amide (NACA), glutathione (GSH) and cysteamime were compared to determine the effects of protection for ROS from GV to MII stage when supplemented during in vitro maturation (IVM) and in vitro culture (IVC) of bovine oocytes. NAC is one of well known ROS scavanger and NACA is modified form of NAC to help permeation into cytosolic area of oocytes. Significant improvement on the development undergoing blastocysts (32%, vs 18%, 22%) were found when cysteamine (0.1mM) was added to the maturation medium than NAC (0.3 mM), NACA (0.2mM) or GSH (0.5 mM) as compared to control medium with antioxydents. However, the addition of NAC(18%) or NACA(21%) to media did not improve the proportion of oocytes undergoing development to morula and blastocysts than control (24%) and GSH (26%). Our study showed that medium supplementation with cysteame during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like NAC, NACA and GSH that caused no improvement.

Purpose: Aims of this study were to investigate the agreement and test-retest repeatability of two methods for measuring magnitude of soft toric lens rotation. The two methods assessed were a newly developed mobile application for iPhone which uses the built-in camera function and the slit lamp biomicroscope. Methods: Agreement of ToriExpertä against known reference source was tested under experimental situation. For clinical measurement, thirty three participants (66 eyes) wore toric lens (prism ballast design) both eyes. Two investigators measured toric lens rotation using the two methods which are slit-lamp measurement(HS-700) and mobile application. First investigator used the same method twice for assessment of test-retest repeatability of each method. Inter- and intra-investigator agreement and repeatability were assessed using Bland-Altman analysis. Results: Against the known reference sources, mean variance was 0.52±0.75 degree and limits of agreement was ±1.47 degree (95% of Cls). The limits of agreement between the silt-lamp biomicroscope and mobile application methods was ±9.1 degree (95% CIs). Measurements using the two different methods showed no statistically significant mean difference (paired t-test, p=0.32). Inter-investigator agreement of lens rotation was ±7.9 degree (95% CIs) using the slit-lamp microscope and ±7.8 degree using mobile application. Intra-investigator repeatability was ±6.6 degree using the slit lamp microscope and ±6.8 using mobile application. Conclusions: The results should be considered in view of the fact that soft toric lenses are not static but move with the blink thus the location of the reference point is unlikely to be at exactly the same location at the different measurement times. Despite this source of variability in the results, the newly developed mobile application provides clinically comparable performance to slit lamp biomicroscope measurement which does not appear to be investigator dependent. This mobile application may provide sufficient precision to those optometric practices have limited access to slit-lamp biomicroscope for measuring soft toric lens rotation.

Insects are among the most diverse groups of animals on the planet, representing more than half of all known living organisms. These insects are found in nearly every environment. Although humans regard certain insects as pests and attempt to control them using insecticides, most insects perform complex ecological functions, and provide either direct or indirect economic benefits to humans. Recently, the importance of insects used as food sources or as pets has increased in many countries, including Korea. In addition, several insects have a strong influence on people's emotion. Insect-mediated mental healthcare program is designed to help people who have disorders with physical, behavior and development. Children who have mental disorder, the experimental group that was provided with an insect-mediated mental healthcare program over a total of 8 sections, one section per week, 60 minutes per section, followed by pre-test and post-test. They responded to therapeutic effect after the completion of the program. Further research on the basis of this study is expected to help children with emotional therapy in other areas.

The baculovirus expression vector system (BEVS) is an effective and widely used method for the production of recombinant proteins in insect cells or larvae. However, the expression efficiency of foreign proteins using the polyhedrin promoter could not obtain the protein yields observed for native polyhedrin. To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of various polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Among the fusion-expressed protein in nucleus and cytoplasm, the most hyper-expression was observed in the fusion of amino acids 19 to 110 and 32 to 59 of polyhedrin. Additionally, the several proteins expressed by the partial polyhedrin-fused expression system was markedly increased. However, we identified that hyper-expression of target protein varied depending on the partial polyhedrin. Therefore, we constructed the virus inducible partial polyhedrin fusion transient expression system. This system amenable for screening of suitable partial polyhedrin to produce the target protein. The present study suggests a new option for higher expression of useful foreign recombinant protein using the partial polyhedrin fusion expression in baculovirus.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of various polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. Among the fusion-expressed protein in nucleus and cytoplasm, the most hyper-expression was observed in the fusion of amino acids 19 to 110 and 32 to 59 of polyhedrin. Also these fragments, some degradation of only the fused polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. The production of luciferase was approximately two folds increased by fusion expression with polyhedrin amino acids 32 to 59, and its activity was measured using Luminometer. This study suggests a new option for higher expression of useful foreign recombinant protein using the partial polyhedrin fusion expression in baculovirus.

To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of polyhedrin fragments were investigated by fusion expression them with the enhanced green fluorescence protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the minimal region for self-assembly and the KRKK nuclear localization signal (NLS). The increase of EGFP production by fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production of EGFP was shown by the mutation of the NLS within the fused polyhedrin fragment. Among the fusion expressed protein in cytoplasm, the most hyper-expression was observed in the fusion of amino acids 32 to 59 of polyhedrin. Polyhedrin fragment fusion expression with classical swine fever virus E2 protein also resulted hyper-enhanced expression of E2 protein. However, the fusion expression of porcine circovirus ORF2 with polyhedrin fragment did not show significant enhance of ORF2 production. These results suggested that the enhancement of foreign protein production when fused with polyhedrin is caused by the enhanced stability of expressed protein.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). To enhance the expression level of baculovirus vector system, we constructed several fusion vectors using various fragments of the polyhedrin. The polyhedrin fragments were genetically fused to the enhanced green fluorescent protein (eGFP) under the control of polyhedrin promoter, and their expressions were analyzed in Sf21 insect cells. Expression of the fusion protein was identified by SDS-PAGE and Western blot analysis using anti-GFP and anti-Polyhedrin. The expression level of eGFP was markedly increased by the fusion of partial polyhedrin. Also, the fluorescence intensity of fusion proteins was higher than that of non-fusion protein. Confocal laser scanning microscopy demonstrated that fusion proteins were localized to the cytosol or nucleus of insect cells. In additional, the glycoprotein E2 (gE2) of classical swine fever virus (CSFV) expressed by the these vectors was dramatically increased and its immunogenicity was proofed using experimental animal guinea pigs that were immunized with the partial polyhedrin containing gE2. This study provides a new option for the higher expression of useful foreign recombinant protein by using the partial polyhedrin in BEVS.

Microsatellite loci are increasingly used as markers in the human, animal and plant genomes. Being highly mutable, microsatellite regions are able to differentiate between related taxa, even at the level of individual isolates in a single species. Studies on mushroom population structure, gene flow and dispersal between natural and cultivated species have become central in breeding programmes and the knowledge of new polymorphic, codominant markers will be a promising avenue to exploit wild genetic resources. The molecular phylogeny in 50 different commercial cultivated strains of Pleurotus eryngii using PCR amplification with URP primers and mitochondrial microsatellite primer was studed. The sizes of the polymorphic fragments obtained were in the range of 200 to 2000 bp. RAPD analysis techniques were able to detect genetic variation among the tested strains. With these isolated PCR amplification with URP primers we intend to analyse the population structure of the P. eryngii species complex and investigate the structure of the basidiomycete genome which deserves. A few single-locus microsatellite markers have been isolated in Pleurotus eryngii and Pleurotus ferulae. This technique is useful in those species where microsatellite loci are rare in the mitochondria.

The mating-type genes control formation of the dikaryon from two haploid strains. These genes are now used in mating-type-assisted breeding programs for economically important mushrooms, especially the oyster mushroom, Pleurotus ostreatus, aiming at high-yield and high-quality standard mushroom production. However, it improves the breeding program when the breeder is able to quickly identify compatible strains in a given set of progeny. The two mating factors with their mating-type loci are used as markers for breeding and have been incorporated in a chromosome mapping investigation. The linkage maps include not only genetic markers such as the mating types that can be cored, but also molecular markers such as PCR-assisted approaches, e.g. RAPD analyses, or RFLP markers. Once mating-type genes within progeny may be more easily identified by the use of PCR-directed cloning of partial mating-type genes. We analyzed homeodomain (HD1 and HD2) and pheromone receptor(rcb1, 2 and 3) genes as molecular markers for breeding using mating type A and B of Pleurotus eryngii and Pleurotus ferulae by direct PCR.

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. The objective of this study was to enhance production of E2 protein by fusion with partial polyhedrin of nucleopolyhedrovirus in insect cells. We generated various E2 form by fusion with different combinations of the partial polyhedrin and deletion of the C-terminal transmembrane region (TMR). Expression of the E2 protein was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. The fusion expression of an E2 protein with the partial polyhedrin markedly increased expression levels. Also, expression of E2 proteinlacking TMR region was higher than that of intact E2 protein. As a result, the fusion expression of E2 protein lacking the C-terminal TMR with partial polyhedrin was significantly increased in insect cells. These suggest that the fusion of target foreign protein with partial polyhedrin could enhance significantly the production of target protein.

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The genome of CSFV is a positive single-stranded RNA molecule 12.3 kb and contains a single large open reading frame (ORF). The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. To determine the characteristics of the CSFV, LOM strain, we investigated the nucleotide sequence of the glycoprotein E0, E1 and E2. Comparison of the LOM with the other strains revealed nucleotide sequence identity ranging from 97 to 98%. Expression of the glycoprotein E2 was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies in Sf21 cells. The expression levels of glycoprotein E2 were observed from day 3 and 5 days maximum. In addition, its expression efficiency by media and cell line was investigated. The result showed that High-Five cells and Grace’s insect media for Sf21 were the best conditions for the expression of the glycoprotein E2.

Peptidyl prolyl cis/trans isomerases (PPIases) catalyze the slow cis/trans isomeraization of proline peptide (Xaa-Pro) bonds in oligopeptides and accelerates slow, rate-limiting steps in the folding of several proteins. We studied the characterization of Cyclophilin A (bCyp A) isolated from Bombyx mori . The cDNA of bCyp A is 947 bp. There is a 5´-untranslated region of 91 nucleotides followed by an initiating ATG codon. The TAA termination codon occurs at nucleotide 588. Thus translation of the sequence from nucleotides 91 to 588 would produce a protein of 166 amino acids with a calculated molecular mass of 18.2kDa. The 'AATAAA' consensus polyadenylation signal and poly A tail are present in the 3´-untranslated region. To analysis of PPIase activity, we expressed the bCyp A protein in Sf9 cell by using baculovirus expression vector system (BEVS). SDS-PAGE and Western blot analysis showed that the molecular weights of intracelluar expressed protein was approximately 28.2 kDa. The PPIase activity assay was monitored by proteolytic cleavage of the chromophore p-nitroanilide byα-chymotrypsin. As substrate the synthetic tetrapeptide succinyl-Ala-Ala- Pro-Phe-p-nitroanilide was used.

In the field of reproductive medicine, assessment of sperm motility is a key factor for achieving successful artificial insemination, in vitro fertilization, or intracellular sperm injection. In this study, the motility of boar sperms was estimated using real-time imaging via confocal microscopy. To confirm this confocal imaging method, flagellar beats and whiplash- like movement angles were compared between fresh and low-temperature-preserved (17℃ for 24 h) porcine sperms. Low-temperature preservation reduced the number of flagellar beats from 11.0±2.3 beats/s (fresh sperm) to 5.7±1.8 beats/s and increased the flagellar bending angle from 19.8°±13.8° (fresh) to 30.6°±15.6°. These data suggest that sperm activity can be assessed using confocal microscopy. The observed motility patterns could be used to develop a sperm evaluation index and automated confocal microscopic sperm motility analysis techniques.

Humankind has been searching for medicinal materials from various plant sources in an attempt to treat disease. Mistletoe is one indubitable plant source for these materials due to its effectiveness in treating various diseases, but it has almost disappeared from the mountainous areas of Korea due to excessive harvesting. In this study, in order to select host tree species for Korean mistletoe [Viscum album var. coloratum (Kom.) Ohwi] by seed inoculation and to clarify the effect of host specificity among various tree species were conducted for the purpose of gaining basic information for the artificial cultivation of Korean mistletoe. Almost all the seeds of Korean mistletoe germinated in vitro at the temperature of 15℃. Among host trees used in this study, Prunus mume showed the highest parasitic affinity with inoculated Korean mistletoe, compared with any other host plants. However, treatment of hormones could not increase the low survival rate of Korean mistletoe on the host trees.

광나무(L. lucidum)는 ursolic acid와 oleanolic acid를 다량 포함하고 있다. 본 연구에서는 광나무 열매, 줄기, 잎 세 부위 추출물의 항주름 효능을 평가하였다. 광나무 추출물은 human skin fibroblasts에서 독성이 없을 뿐만 아니라 MMP-1과 MMP-2의 발현을 감소시키고 COL1A1의 발현을 증가시켰다. 이들 추출물은 모두 농도 의존적으로 COL1A1의 발현을 증가시켰으며 MMP-1과 MMP-2의 발현을 감소시켰다. 광나무 세 부위 추출물 가운데, 열매 부위에 가장 많은 양의 ursolic acid 와 oleanolic acid가 함유되어 있었으며 가장 강한 COL1A1 upregulating 효과와 MMP-1과 MMP-2 downregulating 효과를 나타냈다. 이처럼 항주름 효능을 보이는 광나무 열매 추출물은 기능성 화장품 소재로 개발될 수 있는 가능성이 있다.