Insects constitute the largest and most diverse group of animals on Earth. They also serve as the hosts or nutrient sources for an immense assemblage of parasites, pathogens, and predators, ranging from bacteria and fungi to plant and animals. More than 700 known fungal species from 100 genera have adopted an entomopathogenic lifestyle. These fungi are widely distributed, occurring in aquatic, forest, and agricultural habitats, and are often used as active ingredients in microbial insect pest control agents. Their mode of action against insects involves the attachment of conidia to the insect cuticle, followed by germination, cuticle penetration, and internal dissemination throughout the insect. During this process, secreted enzymes, protein toxins, and secondary metabolites can be used by the fungus to overcome the host immune system, modify host behavior, and defend host resources against competing pathogens and saprophytes. In some cases, the host insect relationship has been found to be associated with bioactive fungal metabolites. These metabolites exhibit a wide variety of insecticidal, antibacterial, antifungal, anticancer, antioxidant, and antiviral activities. Using molecular techniques and phylogenetic analyses, both the asexual (anamorphic) stages and sexual (teleomorphic) stages of entomopathogenic fungi have recently been shown as linked together. Therefore, entomopathogenic fungi, especially in complex with the host insect, might be a promising source of bioactive molecules of pharmaceutical and various industrial interests. Here, we evaluated the antimicrobial activity of entomopathogenic fungi metabolites against plant pathogenic bacteria and fungi for the use in agriculture. The radicals scavenging activity and the anticancer activity were also evaluated for pharmaceutical interests.

In this experiment, a highly porous scaffold of biphasic calcium phosphate (BCP) was prepared using the spongereplica method. The BCP scaffold was coated with 58S bioactive glass (BG) and sintered for a second time. The resulting scaffold was coated with gelatin (Gel) and cross-linked with [3-(3-dimethyl aminopropyl) carbodiimide] and N-Hydroxysuccinamide (EDC-NHS). The initial average pore size of the scaffold ranged from 300 to 700μm, with more than 85 % porosity. The coating of BG and Gel had a significant effect on the scaffold-pore size, decreasing scaffold porosity while increasing mechanical strength. The material and surface properties were evaluated by means of several experiments involving scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) and X-ray diffraction (XRD). Cytotoxicity was evaluated using MTT assay and confocal imaging of MC3T3-E1 pre-osteoblast cells cultured in vitro. Three types of scaffold (BCP, BCP-BG and BCP-BG-Gel) were implanted in a rat skull for in vivo evaluation. After 8 weeks of implantation, bone regeneration occurred in all three types of sample. Interestingly, regeneration was found to be greater (geometrically and physiologically) for neat BCP scaffolds than for two other kinds of composite scaffolds. However, the other two types of scaffolds were still better than the control (i.e., defect without treatment).

The Riptortus (stinkbug) has a specialized symbiotic organ, M4 midgut, to harboring symbiont Burkholderia. M4 midgut is located in abdomen and surrounded with insect hemolymph. Recently our group demonstrated that symbiotic Burkholderia showed different physiology after adapting in M4 gut compare with in vitro cultured Burkholderia. And population of symbiotic Burkholderia in the M4 midgut is regulated by special organ. However, the molecular mechanism to prevent spreading and migrating symbiont bacteria to other host tissues from symbiotic organ is not clear. Therefore, we assumed that symbiont Burkholderia are susceptible to host humoral immunity after established infection in M4 midgut to prevent spreading and migrating into the other host tissues through Riptortus hemolymph. To prove this assuming, we tested the susceptibility and survival rate of symbiont Burkholderia in hemolymph of Riptortus in vitro and in vivo. We also examined the susceptibility of symbiont Burkholderia using purified antimicrobial peptides (AMP), pyrrhocoricin-like, thanatin-like and defensin-like AMPs. Finally, we tested inducing ability for AMPs by systemic infection of symbiotic Burkholderia. Gene expression of purified AMPs was not different after systemic infection of both symbiont and in vitro cultured Burkholderia. Surprisingly, in vitro cultured Burkholderia resisted on bacteria injected hemolymph and purified AMPs but symbiont Burkholderia were highly susceptible in bacteria injected hemolymph and purified AMP. These results suggest that symbiont Burkholderia can't survive in the hemolymph after escaping symbiotic organ. Moreover, humoral immunity of host Riptortus is important to prevent spreading and migrating symbiont Burkholderia into the other host tissue or organ from symbiotic organ.

Insect constitute the largest and most diverse group of animals on world and also serve as the hosts or nutrient sources. In addition, several insects have a strong influence on people's emotion. To utilize the preference and interest of insects in the field of mental healthcare, a survey study was conducted with individual living in Korea. As results, the most people had a high preference and interest of insect, but some were disagreeable to the insect itself. The preference and interest of insect were high on male, adult and practician experienced insect-related events than female, student and non-practician, respectively. The most favored insects were familiar or pet insects such as Papilio xuthus, Lucanus maculifemoratus, Allomyrina dichotoma and Lampyridae. These results may be useful to develop a healing program for mental healthcare using insects. Further research is needed to determine the effects of these insect in the mental therapy for this purpose.

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.

Flexible transparent conducting films (TCFs) were fabricated by dip-coating single-wall carbon nanotubes (SWCNTs) onto a flexible polyethylene terephthalate (PET) film. The amount of coated SWCNTs was controlled simply by dipping number. Because the performance of SWCNT-based TCFs is influenced by both electrical conductance and optical transmittance, we evaluated the film performance by introducing a film property factor using both the number of interconnected SWCNT bundles at intersection points, and the coverage of SWCNTs on the PET substrate, in field emission scanning electron microscopic images. The microscopic film property factor was in an excellent agreement with the macroscopic one determined from electrical conductance and optical transmittance measurements, especially for a small number of dippings. Therefore, the most crucial factor governing the performance of the SWCNT-based TCFs is a SWCNT-network structure with a large number of intersection points for a minimum amount of deposited SWCNTs.

Entomopathogenic fungi are natural pathogens of insects and contribute to the regulation of host insect populations in the environment. Several these fungi produce a wide range of secreted enzymes, secreted protein toxins and secondary metabolites to overcome host defenses and ultimately kill the host, and to defend host resources against competing pathogens and saprophytes. Therefore, this study was performed to select the antimicrobial activity of entomopathogenic fungi form Korea soils against plant pathogenic bacteria Ralstonia solanacearum and plant pathogenic fungi Botrytis cinerea using dual culture technique on SDYA. In addition, we also performed to screening of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging activity compounds from liquid culture filtrates of entomopathogenic fungi and investigate to it’s anticancer activity. As results, 12 isolates, 6 isolates and 25 isolates showing of these fungal metabolites produced antibacterial, antifungal and radicals scavenging activity compounds, respectively. The preferential antimicrobial and radical scavenging activities give evidence that these entomopathogenic fungal metabolites might be useful as a source for plant pathogen control and pharmaceutical interests.

Government encourages small size learning circle activities as a part of ‘Learning Organizing Scheme’ to enhance the performances of the company through making SME employees develop themselves by giving them vision and hope who have poor working and learning conditions. Especially as the importance of learning circle (learning shift) activities is getting bigger, diversified studies on learning circles have been made. Accordingly, this study tried to verify the effects of activation of learning circle activities in SMEs on the quality performance of the organization and propose factors related to quality performance of the company.

The bulb mite (Rhizoglyphus echinopus) damages garlic, shallot and onion in the bulbs, corms and tubers. It has recently become a serious problem because of the continuous use of acaricides resulting in resistance among bulb mite population. Thus, there is need to find alternative control measures to suppress bulb mite population. Here, we report the screening result of pathogenic fungi for the control of R. echinopus. Initial screenings were performed using 352 isolates of entomopathogenic fungi from Korea soils. As results, 15 isolates of acaropathogenic fungi showed the pathogenicity to bulb mite supporting fungal conidiation. These isolates were identified as 3 isolates of Metarhizium flavoviride var. pemphigi and 12 isolates of Metarhizium pingshaense by microscopic examination and genetic sequencing of the ITS region and elongation factor-1 alpha. Selected 15 isolates were tested for their virulence against adult R. echinopus and the thermotolerance and the activity to UV-B irradiation of conidia. Additionally, the activities of chitinases and proteases produced by M. pingshaense were compared according to the medium. These acaropathogenic fungi would be considered promising for biological control of bulb mite.

Entomopathogenic fungi are natural pathogens of insects and contribute to the regulation of host insect populations in the environment. Several these fungi produce a wide range of secreted enzymes, secreted protein toxins and secondary metabolites to overcome host defenses and ultimately kill the host, and to defend host resources against competing pathogens and saprophytes. This study was performed to evaluate the antimicrobial activity of 207 entomopathogenic fungi form Korea soils against plant pathogenic bacteria Ralstonia solanacearum and plant pathogenic fungi Botrytis cinerea using dual culture technique on SDYA. As results, twelve isolates (5.7%) and six isolates (2.8%) showing the greatest inhibition against R. solanacearum and B. cinerea, respectively. The culture supernatant of these selected isolates completely suppressed the growth of the pathogen, indicating that suppression was due to the presence of antimicrobial compound in the culture filtrate. The stability test of the culture filtrate showed that the antimicrobial component was heat stable and not protein. These entomopathogenic fungal metabolites may be a good feature to be used in the development of a new biocontrol method of R. solanacearum and B. cinerea.

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.

Hyphantria cunea is a fall webworm is considered an agricultural pest. It is a major pest of many board-leaved trees. H. cunea nucleopolyhedrovirus (HcNPV) and H. cunea granulovirus (HcGV) were isolated from the fall webworm cadavers in Korea. To better understand HcNPV and HcGV, their genomic sequences were determined, analyzed and compared to two viruses together. The entire nucleotide sequence of the HcNPV genome was fully sequenced using 454 pyrosequencing. The genome of the HcNPV was 131,302 bp with a 45 % G+C content. Computer assisted analysis predicted 146 open reading frames (ORFs) of 50 or more amino acids that showed minimal overlap. Further more, when the phylogenetic relationship was analyzed, HcNPV was closely related to Orgyia pseudotsugata MNPV (OpMNPV) which belong to Group I NPV. The HcGV genome was 114,557 bp with a 39% G+C content and contained 130 putative ORFs of 50 or more amino acids. When phylogenetic relationships were analyzed, HcGV was closely related to Xestia c-nigrum granulovirus, which belong to the Type-II GV. HcNPV shares 48 ORFs with HcGV. The most significant difference between HcNPV and HcGV is fgf gene. HcNPV contains one fgf gene, whereas HcGV contains three fgf genes. The presence of fgf reduces the time and efficient systemic infection it takes the virus to kill its host. The difference of fgf number from HcNPV and HcGV suggested that different affect for the speed of systemic infection.

Artificial insemination (AI) has been performed widely in swine industry using fresh liquid sperm instead of frozen type of sperm. However fresh sperm are not able to preserve more than three days with optimal motility and other sperm parameters for the successful fertilization, since in vitro stored sperm has an oxidative stress that resulted increase of abnormality and acrosome reation. To overcome these major problems, novel preservative formulation is needed to neutralize the oxidative stress and to provide suitable physiological environment for sperm in in vitro. In this study, naturally derived substances such as Poncirus trifoliate (Trifoliate orange), Garcinia mangostana (Mangosteen), pig placenta and testis extracts were tested as sperm preservative agents. Placenta extracts (PE), trifoliate orange extracts (TOE), testes extracts (TE) and mangosteen extracts (ME) were applied to analyze specific parameters for sperm motion characteristics individually and combinatorial. Each individual extract treatment can accelerate the sperm motility but noticeably TOE, TE and ME treatments exhibited the considerable and significant preservation of sperm motility. PE, TE and ME showed a significant (p<0.05) increase in ALH after one week. Further we evaluated the five different combinations of these extracts on sperm motility and its motion characteristics. Surprisingly even after one week ME, TOE and TE combination significantly preserved the sperm motility about 75%. It is noteworthy that unlike individual extract treatment, combination of ME, TOE and TE simultaneously protect the sperm motility and its motion characteristics. Taken together these data conclude that addition of ME, TOE and TE can be effective for preservation of pig sperm.

Pluripotent stem cells can be derived from both pre- and post-implantation embryos. Embryonic stem cells (ES cells), derived from inner cell mass (ICM) of blastocyst are naïve pluripotent and epiblast stem cells (EpiSCs) derived from post-implantation epiblast are primed pluripotent. The phenotypes and gene expression patterns of the two pluripotent stem cells are different each other and EpiSCs thought to be in a more advanced pluripotent (primed pluripotent state) than mouse ES cells (naïve pluripotent state). Therefore, we questioned whether EpiSCs are less potential to be differentiated into specialized cell types in vitro. EpiSCs were isolated from 5.5～6.5 day post coitum mouse embryos of the post-implantation epiblast. The EpiSCs could differentiate into all tree germ layers in vivo, and expressed pluripotency markers (Oct4, Nanog). Interestingly, EpiSCs also were able to efficiently differentiate into neural stem cells (NSCs). The NSCs differentiated from EpiSCs (EpiSC-NSCs) expressed NSC markers (Nestin, Sox2, and Musasi), self-renewed over passage 20, and could differentiate into two neural subtypes, neurons, astrocytes and oligodendrocytes. Next, we compared global gene expression patterns of EpiSC-NSCs with that of NSCs differentiated from ES cells and brain tissue. Gene expression pattern of brain tissue derived NSCs were closer to ES cell-derived NSCs than EpiSC-NSCs, indicating that the pluripotent stem cell-derived somatic cells could have different characteristics depending on the origin of pluripotent stem cell types. * This work was supported by the Next Generation Bio-Green 21 Program funded by the Rural Development Administration (Grant PJ 008009).

True hermaphrodites are animals of equivocal sex in which both male and female gonads develop simultaneously. The frequency of true hermaphroditism is higher in pigs than in other domestic animals. Two Korean pigs were diagnosed with true hermaphroditism showing ovotestes, epididymes, penes, and uteri. Histomorphologically, the testicular tissues consisted of Sertoli cells that were devoid of spermatogenic germ cells and showed proliferation of interstitial cells. However, the uteri were of normal architecture and had well-developed uterine endometrial glands. The samples were 38, XX female karyotype without the sex-determining region Y (SRY) gene. The findings of this study could contribute to the understanding of true hermaphroditism in the Korean pig industry. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.

Interferon induced transmembrane protein-1 (IFITM1) is one of transmembrane protein which is differentially expressed in uterus during estrus cycle and pregnancy, that IFITM1 gene is highly expressed in estrus stage by the effect of estrogen, and in parturition by the effect of PGF2 alpha. This genes are also up-regulated in cells with hyperactivation of the WNT/β-catenin signaling pathway. In this study, to identify a function of IFITM1, the binding partner of IFITM1 were determined using immunoprecipitation and LC- MASSMASS methods. 1, 3 and 5 ug of polyclonal anti-IFITM1 antisera were used for immunoprecipitation, and the 75 kDa of specific band was detected in silver stained polyacylamide gel. This band were chracterized using LC-MASS-MASS, and revealed this band is glucose regulated protein 75 (GRP75) which binds to p53 and inhibits the p53 action in nucleus. To identify the localization of GRP75 in cells, immunocytochemical approach has been applied, and GRP75 is expressed in mitochondria of L929 murine connective tissue cells. Co-localization study between IFITM1 and GRP75 in L929 cell identified that these two proteins were closely expressed in mitochondria. Although the role of the interaction of these two protein need to be clarified in various biological phenomena, this data suggest that close interaction of IFITM1 and GRP75 may regulate cellular functions in uterus on sets of estrus cycle and pregnancy.

The objective of the current study was to describe in vitro embryo production in Hanwoo, analyzing oocytes yield and embryo production. The effects of oocytes production and the number of OPU procedures per animal on embryo production were also evaluated. OPU was done every 3～4 days during experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, we compared the recovery rate of oocytes based on OPU session (Experiment 1). The average of collected oocytes was calculated from every 10 session. The average number of total oocytes recovered per animalonsessionwas 5.16 (mean). Second, we compared the recovery rate base on collection period of OPU (Experiment 2). The following results show the difference of the number of recovered oocytes in every month during the procedure between the months of session. Every animal shows the constant number of recovered oocytes for the first 5 months. However, the recovery rate of oocytes was decreased from month 6 to 8. Third, we compared the developmental rate to blastocyst in two groups (Experiment 3). Oocytes by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24～48 h after in vitro fertilization (IVF) was 75.8% and blastocyst development rate was 18.8% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (61.1%), blastocyst development rate was higher compared with needle puncture group (28.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio- technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being.

The objective of this study was to evaluate in vitro production of bovine embryos in Hanwoo. Oocytes were collected by ovum pick up (OPU) from ovaries of genetically high-value Hanwoo or by needle puncture from ovaries of slaughtered cattle. OPU was done every 3 4 days duing experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, We compared the in vitro maturation rate in two groups (Experiment 1). 545 oocytes were recoverd from 4 females by 32 trials of OPU and then 433 oocytes were shown MⅡ stage after in vitro maturation (79.4%). In case of needle puncture group, 1905 oocytes were collected and then 1420 oocytes were matured to MⅡ stage during in vitro culture(74.5%). Second, we compared the developmental rate to blastocyst in two groups (Experiment 2). 1420 oocyte by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24 48 h after in vitro fertilization (IVF) was 88.6% and blastocyst development rate was 20.5% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (84.8%), blastocyst development rate was higher compared with needle puncture group (26.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio-technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being. This research was suppoted by Imsil-gun agricultural technology service center.

The green peach aphid, Myzus persicae Sulzer, is one of the most important pests affecting protected and open-grown crops, because they cause direct damage by feeding on crops and indirect damage as virus vectors. It has recently become a serious problem because of the continuous use of insecticide resulting in resistance among green peach aphid population. Thus, the development of entomopathogenic fungi as aphid biocontrol agents has received increasing interest as part of integrated control strategies. In this study, we report the screening result of pathogenic fungi for the control of green peach aphid. Initial screenings were performed using 347 isolates of putative pathogenic fungi from Korea soils. As results, 20 isolates of entomopathogenic fungi were isolated from cadavers of green peach aphid supporting fungal conidiation. These isolates were identified as three strains of Lecanicillium attenuatum, nine strains of Beauveria bassiana, one strain of Metarhizium anisopliae, one strain of Metarhizium flavoviride, five strains of Paecilomyces lilacinus, one strain of Aspergillus sp. by microscopic examination, genetic sequencing of the ITS region and Universally Primed PCR (UP-PCR). Based on the screening results, twenty isolates were tested for their pathogenicity against adult green peach aphid. All fungal isolates were pathogenic to green peach aphid but mortality varied with isolates. These entomopathogenic fungi may be useful to develop eco-friendly insecticide to control green peach aphid.

The polyhedrin is responsible to form polyhedra of nucleopolyhedrovirus(NPV) and highly conserved in most completely sequenced in lepidopteran NPVs. Previously, we have reported that the substitution of polyhedrin of Autographa californica NPV(AcNPV) with that of Spodoptera exigua NPV(SeNPV) or Bombyx mori NPV(BmNPV) result the change of polyhedra morphology. In this study, we investigated the influence of changed polyhedra morphology to the virulence of AcNPV. The recombinant AcNPVs were propagated in Spodoptera frugiperda clone 9, 21 cells and S. exigua larvae. Each collected recombinant polyhedra were used in bioassays using S. exigua larvae. The recombinant AcNPVs show that difference virulence according to the polyhedra morphologies. Internal and external morphological features of each recombinant AcNPV were also compared on the electron microscope. Our results suggest that the morphology of polyhedra influence the virulence of NPV and is well worth considering for the development viral insecticide.