We investigated the effect of a synthetic complement peptide C3a on the outcome of Brucella abortus 544 infection in a murine macrophage cell line RAW264.7 cell. First, we determined the highest non-cytotoxic concentration of the peptide in the cell line. We also found that the peptide significantly increased the growth of the bacteria at 8 and 24 h. Although the number of bacterial CFU was also elevated at 48 and 72 h, the increases were not significant as compared to controls. We further investigated the effect of C3a peptide on the growth of Brucella by pre-incubating the peptide at various temperatures and found that the effect was reversed at 24 h post-incubation suggesting that incubation of peptide at high temperatures including 65°C or 95°C could inactivate its action. This also could indicate the beneficial effect of high temperature during infection. Although several studies reported the inhibitory effect of different antimicrobial peptides including C3a, the present study preliminarily revealed that it had no positive contribution on the control of B. abortus 544 infection in vitro and indirectly to its receptor, CD88, which belongs to GPCR. Moreover, the encouraged further exploration of the effect of other similar peptides would be performed for the purpose of finding Brucella-host cell interaction for the control of disease progression.

Lysophosphatidic acid (LPA) is a lipid messenger mediated by G protein-coupled receptors (LPAR1-6). It is involved in the pathogenesis of certain chronic inflammatory and autoimmune diseases. In addition, it controls the self-renewal and differentiation of stem cells. Recent research has demonstrated the close relationship between periodontitis and various diseases in the human body. However, the precise role of LPA in the development of periodontitis has not been studied. We identified that LPAR1 was highly expressed in human periodontal ligament stem cells (PDLSCs). In periodontitis-mimicking conditions with Porphyromonas gingivalis -derived lipopolysaccharide (Pg-LPS) treatment, PDLSCs exhibited a considerable reduction in the cellular viability and osteogenic differentiation potential, in addition to an increase in the inflammatory responses including tumor necrosis factor-α and interleukin-1β expression and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Of the various LPAR antagonists, pre-treatment with AM095, an LPAR1 inhibitor, showed a positive effect on the restoration of cellular viability and osteogenic differentiation, accompanied by a decrease in NF-κB signaling, and action against Pg-LPS. These findings suggest that the modulation of LPAR1 activity will assist in checking the progression of periodontitis and in its treatment.

Clear cell odontogenic carcinoma (CCOC), a very rare neoplasm located mostly in the mandible, has been regarded as a benign tumor. However, due to the accumulation of case reports, CCOC has been reclassified as a malignant entity by the World Health Organization. Patients with CCOC present with regional swelling and periodontal indications with variable pain, often remaining misdiagnosed for a long period. CCOC has slow growth but aggressive behavior, requiring radical resection. Histologic analysis revealed the monophasic, biphasic, and ameloblastic types of CCOC with clear cells and a mixed combination of polygonal and palisading cells. At the molecular level, CCOC shows the expression of cytokeratin and epithelial membrane antigen, along with markers that assign CCOC to the sarcoma family. At the genetic level, Ewing sarcoma breakpoint region 1-activating transcription factor 1 fusion is regarded as the key feature for identification. Nevertheless, the scarcity of cases and dependence on histological data delay the development of an efficient therapy. Regarding the high recurrence rate and the potential of distant metastasis, further characterization of CCOC is necessary for an early and accurate diagnosis.

The mesenchymal stem cells (MSCs) that reside in dental tissues hold a great potential for future applications in regenerative dentistry. In this study, we used human dental pulp cells, isolated from the molars (DPCs), in order to establish the organoid culture. DPCs were established after growing pulp cells in an MSC expansion media (MSC-EM). DPCs were subjected to organoid growth media (OGM) in comparison with human dental pulp stem cells (DPSCs). Inside the extracellular matrix in the OGM, the DPCs and DPSCs readily formed vessel-like structures, which were not observed in the MSC-EM. Immunocytochemistry analysis and flow cytometry analysis showed the elevated expression of CD31 in the DPCs and DPSCs cultured in the OGM. These results suggest endothelial cell-prone differentiation of the DPCs and DPSCs in organoid culture condition.

The purpose of this study is to improve practical skill evaluation method of mushroom - trained certified technician's practical skill evaluation which is one of national qualification tests based on national incompetency standards. One of the current National Competency Standards (NCS), mushroom - trained certified technician uses NCS based practical assessment method. In order to improve the current practical evaluation method, we try to improve practical evaluation method based on field customized problem solving ability and improve the practical evaluation method, various evaluation methods should be constructed. On the purpose of identify the diversity and problems of the evaluation method, the experts of the group consultation, the mushroom-related research institute and the related industry collaborated to identify the problems of the actual mushroom - trained certified technician practicum test, This study on the evaluation improvement method was carried out. In this study, the contents of practical test of the current mushroom traits were analyzed and the trends of the latest mushroom industry were widely reflected.

This study is investigated the growth characteristics(number of available stipe, pileus diameter, pileus thickness, stipe length, stipe thickness, object weight, comparison yield ability of 1 cycle) and storage characteristics of ‘Sanjo 701’ (S7) cultivars according to relative humidity. The S7 growth characteristics were investigated by quantifying the growth and the characteristics according to the relative humidity, The storage stability was investigated every 5 days and freshness was measured by ‘Minamide Method’. S7 pileus diameter is The higher the relative humidity was confirmed becomes larger and the more increased relative humidity also increase the comparison yield ability of 1 cycle. However, pileus diameter or stipe length This could not see the big difference in the three treatment groups, the plieus thickness was no significant difference in the treatment of 80% and 95%. The fresh weight of S7 decreased significantly at 80% and 95% relative humidity after 10 days of storage, but decreased continuity in 65% humiditiry. The elongation percentage of S7 pileus was observe in 95% relative humidity, values of L, a and b (SCI), showed the highest L value in 65% and the a value in 95%. b values were similar in 65%, 80% and 95% treatments. On the ‘Minamide Method’ measure freshness was changed from the 10th day of storage at relative humidity of 95%, but humidity of 65% and 80% treatments, it changed after 15 days of storage. In this study growth characteristics and yield were increased at higher relative humidity, but storage stability was decreased. Therefore, it is deemed necessary to change the relative humidity to produce high quality mushrooms.

This study was conducted to analyze the muscle activity in the right forearm and shoulderwhile doing the sowing activity by muscle type and work section, and to find the possibility of the utilization of sowing as a muscle exercise and rehabilitative activity based on the results of the analysis. The results showed that the activity of 7 muscles including the upper trapezius near the neck, the upper trapezius near the shoulder, the middle deltoid, the biceps brachii, the triceps brachii, the brachioradialis, and the flexor carpi ulnaris were significantly higher than the stable state. In addition, the muscle activity in the sections of mixing the soil, filling the tray with the soil, sowing, writing the label, and watering was significantly higher than the stable state. In particular, the muscle activity of the biceps brachii and the triceps brachii was statistically significantly high in the section of filling the tray with the soil, which indicates that the task of filling the tray with the soil can be utilized as an exercise to activate the biceps brachii and the triceps brachii. In addition, the muscle activity of the brachioradialis was significantly high in all the work sections, which indicates that the sowing process can be utilized to exercise the brachioradialis in particular. These results can be used as the basic data about the activated muscle types and degrees throughout the sowing process. These results can be also used to induce the activation of specific muscles, and the overall exercise and rehabilitation of the forearms.

Hepatocytes and hepatic progenitors derived from human ES cells may be a useful source for clinical application. Therefore, identification and purification of these cell types would be following important issues. There are very few candidate surface markers that can be used to identify and purify hepatic progenitor cells. In addition, indocyanine-green can be uptaken by mature hepatocytes, but cannot be applied for fluorescence activated cell sorting (FACS) due to its long emission wavelength. In the present study, we tested EpCAM as a potential marker for magnetic-activated cell sorting (MACS) of hepatic progenitors and also modified indocyanine-green into fluorescent indomonocarbocyanine for FACS-mediated sorting of mature hepatocytes after differentiation of human ES cells. Hepatic progenitor cells were sorted by MACS after incubation with anti-human EpCAM antibodies. After the final differentiation, the differentiated cells and mouse primary hepatocytes (control group) were incubated with indomonocarbocyanine and were sorted by FACS. MACS and immunocytochemistry data showed that approximately 45% of differentiated cells were EpCAM-positive cells. EpCAM-positive cells expressed α-fetoprotein, FOXa2, HnF4a, and CK18. Differentiation efficiency into albumin-positive cells was significantly higher in EpCAM-positive cells, compared to EpCAM-negative cells. Importantly, indomonocarbocyanine successfully stained cells that expressed ALB. Furthermore, FACS analysis data showed that the purity of hepatocytes that expressed albumin was significantly increased after purification of indomonocarbocyanine-positive cells. Our data demonstrated that human ES cell-derived hepatic progenitors can be efficiently isolated by MACS using EpCAM antibody. In addition, we also showed that indomonocarbocyanine can be successfully used to identify and purify mature hepatocytes using FACS.

Highly homogeneous and functional stem cell-derived hepatocyte-like cells (HLCs) are considered a promising option in the treatment of liver disease and the development of effective in vitro toxicity screening tool. However, the purity of cells and expression and/or activity of drug metabolizing enzymes in stem cell-derived HLCs are usually too low to be useful for clinical or in vitro applications. Here, we describe a highly optimized differentiation protocol, which produces more than 90% albumin-positive HLCs with no purification process. In addition, we show that hepatic enzyme gene expressions and activities were significantly improved by generating three-dimensional (3D) spheroidal aggregate of HLCs. The 3D differentiation method increased expressions of nuclear receptors that regulate the proper expression of key hepatic enzymes. Furthermore, a significantly increased hepatic functions such as albumin and urea secretion were observed in 3D hepatic spheroids and HLCs in the spheroid exhibited morphological and ultrastructural features of normal hepatocytes. Importantly, we show that repeated exposures to xenobiotics facilitated the functional maturation of HLC, as confirmed by increased expression of genes for drug metabolizing enzymes and transcription factors. In conclusion, the 3D culture system with repeated exposures to xenobiotics may be a new strategy for enhancing hepatic maturation of stem cell-derived HLCs as a cell source for in vitro high-throughput hepatotoxicity models.

Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells have received extensive attention in the development of drug screening and toxicity testing. However, it has been reported that stem cell-derived HLCs showed hepatic functions that were too limited to be of use in drug screening and toxicity testing, possibly due to the lack of sufficient intercellular communication under conventional two-dimensional (2D) culture conditions. Therefore, a 3D differentiation system may overcome the in vitro limitation of 2D culture to produce stem cell-derived hepatocytes with mature metabolic functions. In this study, the feasibility of using a silicone-based spherofilm, specifically designed to produce spherical cell clusters, to generate uniformly sized 3D hepatic spheroids from hESCs was investigated. Hepatic spheroids generated on the spherofilm showed more homogenous size and shape than those generated in conventional low-attachment suspension culture dishes. Results of immunohistochemical analysis showed that expression of the mature hepatic marker albumin (ALB) increased over time during the hepatic maturation process. Furthermore, the 3D culture system mimicked the in vivo 3D microenvironment. Laminin, which is an important component of hepatic ECM, was expressed in hepatic spheroids. The results of immunohistochemical analysis indicated that the 3D culture environment is capable of generating an in vivo-like microenvironment. In addition, quantitative PCR analysis showed that the mature hepatic marker ALB and cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A7 were expressed at higher levels in 3D culture than in 2D culture. This indicates that the 3D culture system is suitable for hepatic maturation and that our size-controlled 3D culture conditions might accelerate hepatic function. These results suggest that 3D hepatic spheroids significantly enhance metabolic maturation of hepatocytes derived from hESCs

MFG-E8 (Milk fat globule-epidermal growth factor VIII), also called lactadherin or BA46, SED1 is a glycoprotein found in milk and mammary epithelial cells, it is a major protein component associated with milk fat globule membrane. Previously, our study showed that expression of MFG-E8 is gradually increased with hepatic differentiation of human embryonic stem cells (hESCs). Therefore, we hypothesized that MFG-E8 would be an early cancer stem cell marker, which may predict cancer progression. Our results showed that MFG-E8 was expressed in various human cancer cell lines such as HepG2, Hep3B, and Huh7. Production and secretion of the MFG-E8 were also confirmed in the conditioned media of those three cell lines using enzyme-linked immunosorbent assay. Next, we analyzed the MFG-E8 expression in 11 clinical cases of cholangiocellular carcinoma (CC) and 33 cases of hepatocellular carcinoma (HCC) by immunohistochemistry and examined the potential correlation with β-catenin and AFP, which are known cancer markers. According to hitological criteria, the progression of HCC and CC was evaluated and classified into high, low, metastatic, and well-, moderate-, poor-differentiated, respectively. Statistical analysis indicated that incidence of both HCC and CC is significantly associated with male compared to female (P<0.05). Tumor size also has positive correlation with age (r2=08948). Our immunohistochemistry data showed that MFG-E8 was expressed both HCC and CC tissue. Interestingly, the MFG-E8 expression was significantly increased with cancer progression (P<0.05) in both cases. Additionally, b-cateninexpression was increased and its localization was changed from membrane to cytoplasm and nucleus with the degree of HCC. Likely b-catenin, AFP was also increased with the degree of HCC but it was not correlated with severalty of CC. Importantly, both AFP and b-catenin were highly co-localized with MFG-E8 in HCC. These results suggest that MFG-E8 may have important physiological roles and its expression in HCC and CC would be considered as an important prognostic factor.

Hepatocytes derived from human embryonic stem cells (hESCs) may be a useful source for the treatment of diseased or injured liver. However, a low survival rate of grafted hepatocytes and immune rejection are still major obstacles to be overcome. We previously showed that secreted proteins (secretome) from hESC-derived hepatocytes had a potential therapeutic power in the tissue repair of injured liver without cell transplantation. The purpose of the present study was to discover key protein(s) in the secretome of hESC-derived hepatocytes using proteomic analysis and to study the tissue repair mechanism which may be operated by the secretomes. Purified indocyanine green+ hepatocytes derived from hESCs displayed multiple hepatic features, including expression of hepatic genes, production of albumin, and glycogen accumulation. The nano-LC/ESI-QTOF-MS analysis identified 365 proteins in the secretome of hESC-derived hepatocytes and the protein functional network analysis was conducted using the MetaCore TM from GeneGO. In addition, 20 tissue regeneration-related transcription factors (TFs) were extrapolated through further proteomic analysis. After intraperitoneal injection, the secretome significantly promoted the liver regeneration in a mouse model of acute liver injury. Protein functional network analysis on the secretome-induced regenerating liver confirmed 20 transcription factors (TFs) which were identified in the ICGhigh cells. The upreguation of these tissue repair-related TFs were validated by qPCR and western blotting on the regenerating liver tissues. These results demonstrate that application of the secretome analysis in combination with the protein functional network mapping would provide a reliable tool to discover new tissue-regenerating proteins as well as to expand our knowledge of the mechanisms of tissue regeneration.

Estrogens are ubiquitous signaling molecules that influence nearly every cell type, and exert profound effects on embryonic development, and differentiation. Wnt pathway, which recruits β-catenin into nuclei, and activates The Wnt-dependent transcription factors, also plays an important role in embryonic development and stem cell maintenance, and differentiation. Accumulating evidences indicate that potential convergence between these two pathways in carcinoma cells. However, physiological roles of estrogens in development and differentiation of human embryonic stem cells (hESCs) are relatively unknown. Here, we demonstrated that estrogenic compounds 17α-ethinylestradiol (EE2) and genistein (GEN) significantly increased β-catenin expression in undifferentiated hESCs cultured in feeder-free media. Interestingly, GEN treatement induced an increased trend of mesendodermal gene expressions, and significantly inhibited ectodermal gene expressions (Nestin and Pax6) in embrioid body (EB). Expectantly, GEN increased epithelial-mesenchymal transition (EMT) related gene expression (Snail2, and Twist), whereas decreased E-cadherin on day 6 of EB development. Taken together, these suggest that estrogens may in part the powerful effects on normal hESC differentiation. Mechanistic studies of estrogen signaling continue to suggest novel drug targets for stem cells and will also improve screening methods of developmental toxicity.

We previously reported that purified hepatocyte-like cells derived from human embryonic stem cell (hESC) promoted the liver tissue recovery not only by cell replacement, but also by delivering proteins (secretome) that enhance endogenous host liver regeneration. In this study, we investigated possible therapeutic effects of secretomes obtained from undifferentiated hESC and mesenchymal stem cell (hMSC), and explored the underlying mechanism in a mouse model of chronic liver injury. Mice pre-intoxicated with dimethylnitrosamine (DMN) were treated with single intraperitoneal injection of secretome or medium used to support the growth of hESCs or hMSCs. Both hESC- and MSC-secretomes induced robust host liver regeneration, as determined by biochemical and histological analyses. The expression of MMP2 was significantly increased in the liver that received hESC- or hMSC-secretome, compared to control groups. In contrast, expression of α-SMA, a hallmark of activated hepatic stellate cells, was profoundly decreased after administration of both secretomes. These results suggest that hESCs and MSCs may release soluble factors that support the host tissue regeneration of chronically injured liver.

Previously, the wheat non-specific lipid transfer proteins (TaLTP), members of a small multigene family, appear to show a complex pattern of expression regulation. For further assessment of expression diversity of the TaLTP genes, we have attempted to evaluate their expression profiles of responses to abiotic stresses via the semi-quantitative RT-PCR method. The expression profiles revealed that the TaLTP genes in group A evidenced highly similar (but not identical) responses against abiotic stresses, whereas much differential expression pattern among genes in each group. The four promoters of TaLTP1, TaLTP7, and TaLTP10 of group A and TaLTP3 of group B were fused to a GUS reporter gene and the recombinant genes were introduced into Arabidopsis. The promoters of TaLTP1, TaLTP7 and TaLTP10 of group A, drove strong but various GUS expression in cotyledons, hypocotyls, epidemic and sub-epidemic cells of young shoots and leaves, floral organs as well as siliques. By contrast, the promoter of TaLTP3 just directed trace expression in cotyledons, young emerged leaves and epidemic cells of flower ovaries. The promoter of TaLTP1 directed the expression in root system whereas the promoters of TaLTP1 and TaLTP10 showed some degree of expression during seed development. The expression diversity of TaLTP genes suggests their multiple physiological functions, evidencing subfunctionalization over evolutionary time.