Oocyte is the central factor in the bi-directional communication axis in the ovarian follicles. It controls the cumulus or granulosa cells to perform functions which are beneficial for its own development via secreting paracrine growth factors, including GDF9 and BMP15. The aim of this study was to investigate whether the recombinant GDF9 and BMP15 are able to promote meiotic resumption and cumulus expansion of canine COCs during IVM, as well as to demonstrate the actions of GDF9 and BMP15 in regulating the expression of connexin transcripts in the ovarian granulosa cells. As results, GDF9 and BMP15 significantly improved the meiotic resumption rate and cumulus expansion by activating ERK1/2 signaling. Treatments with GDF9 significantly improved the expression of CyclinB1 but inhibited the expression of Cx43 transcripts. In addition, cumulus expansion genes (MAPK1, Ptgs2, Tnfaip6 and Ptx3) were differentially improved by GDF9 and BMP15. In the ovarian granulosa cells, GDF9 suppressed the expression of Cx43 transcripts by binding ALK4/5/7 receptors and activation Smad2/3 signaling, whereas, BMP15 stimulated the expression of Cx43 transcripts by binding ALK2/3/6 receptors and activating Smad1/5/8 signaling. In conclusion, by regulating functions of granulosa/cumulus cells, oocyte has the potential to enhance the growth and maturation of itself.

Generally, fate of spematogonial stem cells (SSCs) can be determined specifically by microenvironments enclosed with various extracellular matrix (ECM) components and integrins recognizing directly ECM proteins play an pivotal role in transporting ECM-derived signals into cytoplasm, resulting in inducing a variety of biological functions such as cell attachment, self-renewal and differentiation. However, to date, studies on type of integrins expressed on the undifferentiated SSCs remain unclear. Therefore, we tried to investigate systematically what kind of integrin subunits are expressed transcriptionally or translationally in the SSCs derived from testis of hybrid B6CBAF1 mouse. For these, isolation of SSCs from testis were conducted by magnetic activated cell sorting (MACS) using Thy1 antibody. Subsequently, transcriptional and translational level of integrin α and β subunits in the isolated SSCs were measured by real-time PCR and fluorescene immunoassay, respectively. As the results, transcriptional levels of genes encoding total 25 integrin subunits were quantified, and integrin α4, α6, α7, α9, αV, αL and αE and integrin β1, β5 showed higher expression levels than other subunits. By contrast, integrin α3, α5, α 10 and α11 and integrin β2, β3, β4, β7 were weakly transcribed. When translational levels of the integrin α subunits showing high transcription level (α4, α6, α7, α9, αV αL, and αE) were measured, integrin α6, α7, α9, αV and αL were higher than integrin α4 and αE. In case of integrin β subunit, β1 evaluated more expression than β5. From these results, we speculate that the undifferentiated SSCs derived from hybrid B6CBAF1 mouse may express integrin α4β1, α6β1, α7β1, α9β1, αVβ1 and/or αVβ5 on plasma membrane. Moreover, this information will greatly contribute to constructing non-cellular niche supporting self-renewal of SSCs in the future.

Oocyte is the central factor in the bi-directional communication axis in the ovarian follicles. It controls the cumulus or granulosa cells to perform functions which are beneficial for its own development via secreting paracrine growth factors, including GDF9 and BMP15. The aim of this study was to investigate whether the recombinant GDF9 and BMP15 are able to promote meiotic resumption and cumulus expansion of canine COCs during IVM, as well as to demonstrate the actions of GDF9 and BMP15 in regulating the expression of connexin transcripts in the ovarian granulosa cells. As results, GDF9 and BMP15 significantly improved the meiotic resumption rate and cumulus expansion by activating ERK1/2 signaling. Treatments with GDF9 significantly improved the expression of CyclinB1 but inhibited the expression of Cx43 transcripts. In addition, cumulus expansion genes (MAPK1, Ptgs2, Tnfaip6 and Ptx3) were differentially improved by GDF9 and BMP15. In the ovarian granulosa cells, GDF9 suppressed the expression of Cx43 transcripts by binding ALK4/5/7 receptors and activation Smad2/3 signaling, whereas, BMP15 stimulated the expression of Cx43 transcripts by binding ALK2/3/6 receptors and activating Smad1/5/8 signaling. In conclusion, by regulating functions of granulosa/cumulus cells, oocyte has the potential to enhance the growth and maturation of itself.

Porphyromonas gingivalis is a gram-negative bacteria of rod shape, and grown in an anerobic condition. It colonizes in subgingival crevice and is known as a major pathogen causing chronic periodontitis. It possesses an invasive property and replicative potential within various cell types, presumably playing an important role in modulating biological behaviors of oral cancer. However, the pathophysiology of P. gingivalis in the malignant transformation of oral cancer has not been fully understood. In this study, we aimed to investigate molecular changes of oral squamous cell carcinoma cells induced by repetitive P. gingivalis infection that clinically resembles chronic periodontitis.

Recently, the importance of inflammation in carcinogenesis has been recognized and studied extensively. As a result, a clear correlation between inflammation and carcinogenesis has been well established in some types of cancers. Despite a high prevalence of chronic periodontitis, one of the most common inflammatory diseases in the general population, there are only a few reports on the role of chronic periodontitis in oral cancer progression. In this study, we aimed to investigate genetic changes in oral cancer cells induced by repetitive Porphryomonas gingivalis infections to mimic chronic periodontitis in a clinical setting. Cells of oral squamous cell carcinoma (OSCC), the most common type of oral cancer, and P. gingivalis 381 were used for the present study. ID1 and ID3 were mRNAs of higher expression in the P. gingivalis-infected group compared to the uninfected control. These mRNAs have been regarded as important modulators participating in cancer progression. Future studies will provide an insight into the roles of the molecules we identified in oral cancer progression. Outcomes from these studies will also shed light on the significance of chronic periodontitis induced by bacterial pathogen, such as P. gingivalis, in progression of oral cancer and relevant molecular mechanisms underlying altered cancer cell behaviors.

Somatic cell nuclear transfer (SCNT) has been considered for preserving genetically valuable or endangered animals. Sapsaree is a Natianal Monument in Korea to maintian a pure pedigree. The aim of this study was to produce azoospermia Sapsaree using SCNT and identify normal reproductive ability of cloned azoospermia Sapsaree. Ear skin biopsy was performed on a thirteen-year-old azoospermia Sapsaree and ear skin fibroblasts were isolated for SCNT as donor cells. The fibroblasts were injected into enucleated in vivo matured oocytes, the couplets were electrical fused by two pulse of direct current (55 V for 15 μs) using titanium and platinum fusion needle and activated by calcium ionophore. Cloned embryos were surgically transferred into oviducts of natuarally estrus cycle synchronized recipient dogs. The fusion rate of platinum needle was 70%, which was higher than those of titanium needle (64.1%). Developmental rate to the 8 cells and 10 cell stages was higher in platinum needle group (24% and 16%, respectively) than those of platinum needle group (14.8% and 3.1%, respectively). Total 35 SCNT embryos were transferred into oviducts of 3 recipient dogs and one recipient finally delivered a puppy by caesarean section. As results, this study demonstrated that platinum fusion needle could be successfully make the reconstructed embryos and improve the efficiency of canine SCNT. Cloning azoospermia Sapsaree may contribute to conserve genetically valuable and unique pedigree. And further study should be confirm whether cloned live dog is azoospermia.

Prolonged communication between oocytes and the surrounding somatic cells is one of the unique reproductive physiology in canine. Paracrine Kit ligand (KITL) signaling is a well-known communication between granulosa cells and the oocyte. KITL is a cytokine growth factor secreted by granulosa cells that signals via the c-kit receptor expressed by oocytes. Paracrine factors, including growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), exert their effects by binding with the kinase receptors expressed on the granulosa cells. However, the regulations of GDF9 and BMP15 in the canine KITL expression are currently poorly understood. Therefore, we investigated the effects of GDF9 and BMP15 on the expression of KITL in canine ovarian granulosa cells in vitro. In Annexin V assay recombinant GDF9 and BMP15 did not induce apoptosis in the cultured ovarian granulosa cells. When treated, FSH significantly increased KITL expression, and hCG suppressed its expression. When both FSH and hCG were treated, the expression of KITL was affected by GDF9 and BMP15 in dose and time dependent manner in the luteal granulosa cells. GDF9 (10 ng/mL) significantly decreased KITL expression after12 h. BMP15 (10 ng/mL) significantly also decreased KITL expression after 24 h. Western blot and immunochemistry results indicate that GDF9 activated Smad2/3. After blocking ALK 4/5/7 receptors by SB, GDF9 failed to activate Smad2/3, also BMP15 did not activate Smad1/5/8 after blocking ALK 2/3/6 receptors by DM. So GDF9 exerts its effects via using ALK 4/5/7 receptors to activate SMAD2/3 signaling, and BMP15 binds ALK 2/3/6 receptors to activate SMAD1/5/8 signaling. The expression of KITL was not changed by SB or DM treatment. However, the effect of GDF9 and BMP15, which decreased the expression of KITL, was suppressed by SB or DM treatment. In conclusion, this study provides the first evidence that recombinant GDF9 and BMP15 decrease KITL expression in canine ovarian granulosa cells.

The cancer and Parkinson's disease associated protein DJ-1 is multifunctional protein that involves in diverse cellular process. DJ-1 protein has a cellular protective role and promoted cell survival under an oxidative stress. However, the cellular protective mechanism of DJ-1 is not fully understand, and we needs to be further study their functions in novel organisms. In the present study, we investigated the protective role of DJ-1 against induced oxidative stress in canine cell line. On the basis of these experiments, canine DJ-1 overexpressing and null cell lines were established. The stable overexpression and down regulation of DJ-1 efficiency confirmed by the western blot analysis. Subsequently, the DJ-1 gene transfected cell lines and control cells were subjected to induced the oxidative stress, and then cell viability, cell proliferation assay, cellular apoptosis detection analysis (Annexin V and TUNEL assay), intracellular ROS and mitochondrial activity were measured appropriately. The results showed that DJ-1 overexpressed cells were up-regulated cell viability under oxidative stress conditions induced by the rotenone and hydrogen peroxide (H2O2), whereas loss of DJ-1 cells were down-regulated the cell survival activity. Additionally, overexpression of DJ-1 cells increased cell resistance to oxidative stress and inhibited the elevation of cell death and cellular ROS induced apoptosis. Moreover, DJ-1 overexpressed cells was increased mitochondrial functions by using confocal microscopy with MitoTracker staining. On the contrary to this, DJ-1 null cells show defective cellular protection and mitochondria activity against oxidative stress conditions. Our data indicate that canine DJ-1 protein attenuates cellular apoptosis and ROS generation, enhances the cellular survival activity and promote mitochondrial function under the oxidative stress, likewise other mammalian cells. Importantly, DJ-1 overexpression may be an important part of a protective strategy as a sensor for oxidative stress.

A self-powered time-temperature indicator (TTI) was optimized to enhance the performance of the TTI by modifying a biofuel cell using different immobilization, redox mediators, and status of electrode. The performance of the TTI was measured by output voltage of the TTI. The enzymes and combinations of lacasse mediators (HBT (1-hydroxybenzotriazole), Rupy (Bis-(bipyridine)-(5-aminophenanthroline) ruthenium bis (hexafluorophosphate)), MB (Methylene blue), SDP (4,4- sulfonyldiphenol)) and glucose oxidase mediators (FA (Ferroceneboxaldehyde), DBQ(2,5-dihydroxybenzoquinone), HQS (8-hydroxyquinoline-5sulfonic acid hydrate), FHFP (Ferrocenium hexafluorophosphate)) were immobilized with stabilizers (pyrrole) on a glassy carbon electrode by electrodeposition by applying a square wave, cross-linking and physical immobilization. MWCNTs were used to modify glassy carbon electrodes. MWCNTs coated electrodes produced higher output voltage than uncoated electrodes. The optimum and stable performance of the self-powered TTI was that the output voltage of 64 mV and duration time was 3hr at 25°C, when the combination of Rupy, MB for laccase mediators and HQS, FHFP for glucose oxidase mediators were immobilized on MWCNTs coated electrodes by applying a square wave method. In the application, the concentrations of enzyme and glucose were adjusted to prolong the shelf-life of TTI at much lower output voltage.

The pasteurization is employed for extending shelf-life and keeping the quality of products constant. However the pasteurization undesirably accelerates the oxidation of beer which renders volatile compounds concerning off-flavor. The pasteurization conditions was optimized to avoid off-flavor of beer during pasteurization. Under the isothermal condition, thermal destruction kinetics such as D and Z value of Lactobacillus brevis which is reported the most common beer spoilage, was determined. The binomial data (detected or non-detected of off-flavor) was treated with logistic regression to estimate off-flavor development (OFD) times. The temperature dependence of OFD times was established in terms of Arrhenius relationships. Optimized pasteurization temperature and time were found at which OFD times was not detected. The constraint for optimization was that the pasteurization degree should be larger than 5 decimal reduction time. The optimization was conducted through mathematical simulation using kinetics and temperature-dependent models for microbial death and OFD times. The optimized results were validated by the corresponding experimentations, which met the requirements that the concentration of Lactobacillus brevis was 5-Log reduction and the OFD times not detected.

A novel nanocomposite LDPE film with UV protective properties was developed for active packaging applications. Initially, undoped and Mn-doped TiO2 nanoparticles (NPs) were synthesized by the sol-gel method and the resulting particles were characterized. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed an agglomerated nature and spherical morphology. X-ray diffraction (XRD) studies indicated that all products were crystalline and in the form of rutile. The reflectance spectrum of undoped TiO2 NPs demonstrated a characteristic sharp edge at 410 nm. Subsequently, nanocomposite (NC) LDPE samples were prepared with the NPs by solvent precipitation followed by film casting. The optical and thermal properties of the NC samples were investigated. Incremental increases in Mn concentration from 0.25 mol % to 1.00 mol % were associated with progressive decreases in light transmission in the UV region. The melting and maximum decomposition temperatures of all NCs were 107 and 442-449 °C, respectively. The UV protective LDPE-based NC films exhibited superior photostability. Absorption in the FTIR spectra at 1716 and 1734 cm-1 changed after 4-wk exposure to UV for all film samples as a consequence of photodegradation. Finally, the photooxidation of perilla oil was assessed as an example of a UV protective packaging application. After 12 days, protection with 1.00 mol% Mn-doped TiO2-LDPE was associated with a gradual increase in PV, while protection with TiO2-LDPE was associated with a significant increase and protection with the control treatment was associated with a dramatic increase in PV. Hence, a 1.00 mol% Mn-doped TiO2-LDPE NC showed promise for UV shielding packaging applications.

Makgeolli is rich in nutrients such as beneficial microbial species, essential amino acids, oligosaccharides, and organic acids. The lactic acid bacteria contained in Makgeolli provides a healthy function as probiotics for customers. The purpose of this study was to enhance the nutritional properties, lactic acid bacteria propagation and alcohol contents of makgeolli by controlling redox potential balance. An automatic control system was created using ORP sensor, Labview control kit, and air supplier, in which air concentration was controlled by on & off mode. Makgeolli was fermented at three different redox potentials -50 mV, -100 mV, -150 mV. Regulating of aeration according to the redox potentials could give prominence to nutritional benefits of Makgeolli. The profiles of redox potential appeared bathtub curves, and related to lactic acid bacteria growth curve and byproducts. We could find the optimum redox potential balance that affects the factors such as LAB’s, alcohol contents and byproducts. In conclusion it was essential to control redox potential balance in order to produce nutrients rich Makgeolli.

Time-temperature indicators or integrators (TTIs) indicate food quality changes based on time-temperature history. Whilst many types of TTIs have been developed and commercialized, educated consumers often refuse to purchase food products with attached TTI labels showing even a slight color change. In this study, a novel on-off diffusion-based TTI coupled with polydiacetylene/silica nanocomposites has been proposed. The prototype TTI tag has a multilayer structure comprised of a self-adhesive base layer, a middle microporous sheet, and an upper opaque white layer coupled with a square reservoir of Tween 20 attached to an activation stripe. At the end of the diffusion path, polydiacetylene/silica nanocomposites were injected into a loading site as a fine blue stripe. After activation, Tween 20 diffused and reached the loading site, where it rapidly changed from blue-to-red via solvatochromism. This alternative and innovative TTI continuously showed a blue color until reaching the end point, at which stage a red color rapidly appeared, indicating product rejection. Thus, this novel TTI it is of great benefit to the brand owner. The developed prototype was characterized and evaluated for its ability to monitor microbial quality based on published, isothermal, microbial growth data of modified-atmosphere packaged minced beef, Mediterranean fish, and ground pork. The diffusion of Tween 20 in the TTI system was measured under various isothermal conditions and a kinetic model, based on the association between diffusion and time-temperature, was investigated. The Gaussian-estimated activation energy value was 51.082kJ mol-1. Tween 20 diffusion of 6.10, 5.15 and 6.15mm along the TTI systems were considered to be end points and the 95% confidence interval between the times taken for TTI to display OFF and for the foods to reach their deterioration thresholds were 23.30-23.70, 23.00-23.50 and 23.44-24.05h for total aerobic bacteria, Shewanella putrefaciens, and Pseudomonas spp. respectively. The TTI performance test for reproducibility and accuracy revealed a normal frequency distribution with 35004.90, 1200.254.82 and 549.811.09min at 0, 11 and 25C, respectively in accordance with the investigation of diffusion in the TTI.

Microbe have been considered as potential control agents for pest, as alternative to chemical control methods. Among entomopathogens, fungi cause the mortality by penetrating the cuticle of pest and/or by metabolites such as toxin. Not only this direct control effect of fungi, but repellency of fungi also may be used to prevent the pest. Repellence effect of fungi is considered as inhibitory factor to control termite. A study was reported in Japan that termite was able to detect and remove the conidia of fungi on their surface. The termite can escape from fungal infection and protect their colony. There is few study that insect pest such as moth can detect and avoid the virulence fungi. Therefore, we has been conducting the detection and avoidance of beet armyworm to high pathogenic fungi, Paecilomyces fumosoroseus. Adult of the beet armyworm avoided oviposition at Chinese cabbage treated with P. fumosoroseus compare to control. This result may be used to prevent the infestation of moth in crop production.

This study attempts to manufacture a Ni-Cr-Al-Y coating layer using a kinetic spray process and investigates the microstructure and physical properties of the manufactured layer. The Ni-22Cr-10Al-1Y (wt.%) composition powder is used, and it has a spherical shape with an average diameter of 23.7 μm. Cu plate is used as the substrate. Optical microscope, X-ray diffraction, scanning electron microscope and Vickers hardness test are carried out to characterize the macroscopic properties of the coating layer. Furthermore, the coating layer underwent vacuum heat treatment at temperatures of 400˚C and 600˚C for 1 hour to check the effect of heat treatment temperature on the properties. The manufactured coating layer is 1.5 mm thick, and featured identical phases to those found in the powder. The porosity of the coating layer is measured at 2.99%, and the hardness is obtained at 490.57 Hv. The layer shows reduced porosity as heat treatment temperature increased, and hardness is reduced at 400˚C but shows a slight increase at 600˚C. Based on the findings described above, this study also discusses possible manufacturing methods for a Ni-Cr-Al-Y coating layer using the kinetic spray process.

We investigated the adsorption of Na on graphene and graphene oxide, which are used as anode materials in sodium ion batteries, using density functional theory. The adsorption energy for Na on graphene was -0.507 eV at the hollow sites, implying that adsorption was favorable. In the case of graphene oxide, Na atoms were separately adsorbed on the epoxide and hydroxyl functional groups. The adsorption of Na on graphene oxide-epoxide (adsorption energy of -1.024 eV) was found to be stronger than the adsorption of Na on pristine graphene. However, the adsorption of Na on graphene oxide-hydroxyl resulted in the generation of NaOH as a by-product. Using density of states (DOS) calculations, we found that the DOS of the Na-adsorbed graphene was shifted down more than that of the Na-adsorbed graphene oxide-epoxide. In addition, the intensity of the DOS around the Fermi level for the Na-adsorbed graphene was higher than that for the Na-adsorbed graphene oxide-epoxide.

Chronic inflammation has long been considered as an important contributing factor to the development of malignant tumors in various tissues. In this study, we aimed to investigate a potential association between chronic periodontitis, a representative inflammatory disease in the oral cavity, and oral squamous cell carcinoma (OSCC), the most common form of malignant tumors in the oral cavity. A retrospective study was designed to include the cases and controls, each of which consisted of patients first diagnosed with OSCC and temporomandibular disorders, respectively. The existence or a history of periodontal disease was quantitatively estimated based upon the level of alveolar bone loss (ABL) from panoramic radiographs in these groups. Unlike other covariates, including LDH, WBC count and hemoglobin, the levels of ABL measured at three independent regions (second premolar and first/second molar) were significantly higher in the OSCC group, regardless of the patients’age in most cases. Our results thus support the hypothesis that chronic periodontitis, represented by significant ABL, is an important and clinically relevant factor potentially associated with the development of OSCC.

Chronic inflammation is widely considered to predispose individuals to cancer. Microorganisms facilitate recruitment and activation of inflammatory cells and thus allow release of inflammatory mediators. These molecules can then promote accumulation of mutations, leading to tumor development in the host. Porphyromonas gingivalis, a pathogen causing chronic periodontitis, is detected in oral squamous cell carcinoma (OSCC) tissues. Considering a strong link between chronic inflammation and tumor development, functional consequences of P. gingivalis infection may include malignant transformation of the host cells. In this study, we monitored transcriptional changes induced by invasion of P. gingivalis in OSCC cells using microarrays. Our preliminary results suggest changes in a wide range of genes involved in inflammation, apoptosis and autophagy, tumor progression, and carcinogenesis. Further studies on molecular mechanisms underlying these changes will lay a useful foundation to elucidate the role of microorganism-related inflammation and for the development of preventive and therapeutic agents for oral cancer.

Spermatogonial stem cells (SSCs) developed into sperms through spermatogenesis have been utilized as a useful tool in the field of regenerative medicine and infertility. However, a small number of highly qualified SSCs are resided in the seminiferous tubule of testis, resulted in developing effective in-vitro culture system of SSCs for solving simultaneously quantitative and qualitative problems. Presently, SSCs can be enriched on testicular stromal cells (TSCs), but there are no systematic researches about TSC culture. Therefore, we tried to optimize culture condition of TSCs derived from mouse with different strains. For these, proliferation and viability were measured and compared by culturing ICR outbred or DBA/2 inbred mouse-derived TSCs at 35 or 37℃. In case of ICR strain, primary TSCs cultured at 37℃ showed significantly higher proliferation and viability than those at 35℃ and significant increase of proliferation and viability in sub-passaged TSCs was detected in the 35℃ culture condition. Moreover, sub-passage of primary TSCs at 35℃ induced no significant effects on proliferation and viability. In contrast, in case of DBA/2 strain, significantly improved proliferation were detected in the primary TSCs cultured at 35℃, which showed no significant difference in the viability, compared to those at 37℃. Furthermore, sub-passaged TSCs cultured at 37℃ showed no significant differences in proliferation and viability, compared to those at 35℃. However, with significant decrease of proliferation induced by sub-passage of primary TSCs at 35℃, no significant effects on proliferation and viability were resulted from sub-passage of primary TSCs at 37℃. From these results, culture temperature of primary TSCs derived from outbred and inbred strain of mouse could be separately optimized in primary culture and subculture.