Octamer-binding transcription factor 4 (Oct4) is a critical molecule for the self-renewal and pluripotency of embryonic stem cells. Recent reports have shown that Oct4 also controls cell-cycle progression and enhances the proliferation of various types of cells. As the high proliferation of donor fibroblasts is critical to the production of transgenic pigs, using the somatic cell nuclear transfer technique, we analysed the effect of Oct4 overexpression on the proliferation of porcine fibroblasts and embryos. Porcine endogenous Oct4 cDNA was cloned, sequenced and inserted into an expression vector. The vector was transfected into porcine fibroblasts, and a stable Oct4-overexpressed cell line was established by antibiotic selection. Oct4 expression was validated by the immunostaining of Oct4. Cell morphology was changed to sharp, and both proliferation and migration abilities were enhanced in Oct4-overexpressed cells. Real-time RT-PCR results showed that p16, Bcl2 and Myc were upregulated in Oct4-overexpressed cells. Somatic cell nuclear transfer was performed using Oct4-overexpressed cells, and the development of Oct4 embryos was compared with that of wild-type cloned embryos. The cleavage and blastocyst formation rates were improved in the Oct4 embryos. Interestingly, blastocyst formation of the Oct4 embryos was observed as early as day 5 in culture, while blastocysts were observed from day 6 in wild-type cloned embryos. In conclusion, the overexpression of Oct4 enhanced the proliferation of both porcine fibroblasts and embryos.

We investigated the structural complexity and texture of the cytoskeleton and nucleus in human mesenchymal stem cells during early phase differentiation into osteoblasts according to the differentiation–induction method: mechanical and/or chemical stimuli. For this, fractal dimension and a number of parameters utilizing the gray-level co-occurrence matrix (GLCM) were calculated based on single-cell images after confirmation of differentiation by immunofluorescence staining. The F-actin and nuclear fractal dimensions were greater in both stimulus groups compared with the control group. The GLCM values for energy and homogeneity were lower in fibers of the F-actin cytoskeleton, indicating a dispersed F-actin arrangement during differentiation. In the nuclei of both stimulus groups, higher values for energy and homogeneity were calculated, indicating that the chromatin arrangement was chaotic during the early phase of differentiation. It was shown and confirmed that combined stimulation with mechanical and chemical factors accelerated differentiation, even in the early phase. Fractal dimension analysis and GLCM methods have the potential to provide a framework for further investigation of stem cell differentiation.

Sources of variation in nutrient intake have been examined for Western diets, but little is known about the sources of variation and their differences by age and sex among Koreans. We examined sources of variation in nutrient intake and calculated the number of days needed to estimate usual intake using 12 d of dietary records (DR). To this end, four 3 d DR including two weekdays and one weekend day were collected throughout four seasons of 1 year from 178 male and 236 female adults aged 20–65 years residing in Seoul, Korea. The sources of variation were estimated using the random-effects model, and the variation ratio (within-individual:between-individual) was calculated to determine a desirable number of days. Variations attributable to the day of the week, recording sequence and seasonality were generally small, although the degree of variation differed by sex and age (20–45 years and 46–65 years). The correlation coefficient between the true intake and the observed intake (r) increased with additional DR days, reaching 0·7 at 3–4 d and 0·8 at 6–7 d. However, the degree of increase became attenuated with additional days: r increased by 13·0–26·9 % from 2 to 4 d, by 6·5–16·4 % from 4 to 7 d and by 4·0–11·6 % from 7 to 12 d for energy and fifteen nutrients. In conclusion, the present study suggests that the day of the week, recording sequence and seasonality minimally contribute to the variation in nutrient intake. To measure Korean usual dietary intake using open-ended dietary instruments, 3–4 d may be needed to achieve modest precision (r>0·7) and 6–7 d for high precision (r>0·8).

Maslinic acid is found in various natural sources, most notably in pomace olive oil, and exerts pro-apoptotic activities in various cancer cells in vitro. In the present study, DU145 human prostate cancer cells were cultured with 0–25 μm-maslinic acid to examine the effects of maslinic acid on the metastatic capacity of prostate cancer cells. Maslinic acid significantly (P <0·05) inhibited the basal and epidermal growth factor (EGF)-induced migration (27–64 %), invasion (23–60 %) and adhesion (8–40 %) of DU145 cells. Maslinic acid significantly (P <0·05) down-regulated both basal and EGF-stimulated secretion of matrix metalloproteinase (MMP)-9 (25–67 %), MMP-2 (50–86 %), urokinase-type plasminogen activator (uPA, about 100 %), vascular endothelial growth factor (VEGF, 98–100 %) and tissue inhibitors of metalloproteinases (TIMP)-1, as well as expression of uPA receptor (uPAR), intercellular adhesion molecules (22–33 %), vascular cell adhesion molecules (23–46 %) and E-cadherin, whereas it increased TIMP-2 secretion. Maslinic acid dramatically reduced the levels of hypoxia-inducible factor-1α (HIF-1α) protein and mRNA; the reduction was accompanied by reduced stability, nuclear levels and transcriptional activity of HIF-1α. The levels of phospho-Akt and phospho-extracellular signal-related kinase (ERK) were reduced in cells treated with maslinic acid, and the phosphoinositide 3-kinase inhibitor LY294002 and the mitogen-activated protein kinase kinase inhibitor PD98059 reduced HIF-1α levels and VEGF secretion. The results show that maslinic acid markedly inhibited the migration, invasion and adhesion of DU145 prostate cancer cells. Suppressing HIF-1α activation by inhibiting Akt and ERK activation may be part of the mechanism by which maslinic acid inhibited uPAR, E-cadherin, VEGF and MMP expression in DU145 cells.

An organic/inorganic hybrid-type nonvolatile memory TFT was proposed as a core device for the future flexible electronics. The structural feature of this memory TFT was that a ferroelectric copolymer and an oxide semiconductor layers were employed as a gate insulator and an active channel, respectively. The memory TFT with the structure of Au/poly(vinylidene fluoride-trifluoroethylene)/Al2O3/ZnO/Ti/Au/Ti/poly(ethylene naphthalate) could be successfully fabricated at the process temperature of below 150°C. It was confirmed that the TFT well operated as a memory device even under the bending situations.

The miniature pig is regarded as a better organ donor breed for xenotransplantation than other pig breeds because the size of their organs is similar to that of humans. To improve efficiency of cloned miniature pig production, we analysed the effect of breed difference between donor cells and embryo recipients on pregnancy rate and delivery rate. Cloned porcine embryos derived from domestic or miniature pig donor cells were transferred to domestic or miniature recipient pigs. Delivery rate was significantly higher when embryos reconstructed with miniature pig donor cells were transferred to miniature pig recipients as compared with that of embryos transferred to domestic pig recipients. However, pregnancy rates were similar between the two groups. The breed of donor cells, but not of embryo recipients, seems likely to affect litter size. From a 13 610 gene cDNA microarray, 1551 (11.7%) genes showed significantly different levels of expression between the fetuses of the two breeds. Vascular endothelial growth factor and c-kit ligand genes related to implantation and maintenance of pregnancy were significantly down-regulated in miniature pigs. In conclusion, the differential gene expression in fetuses interferes with proper fetal/maternal interactions, and results in late-stage pregnancy loss. Our results indicate that the miniature pig is the preferred embryo recipient breed than domestic pig for producing cloned miniature piglets.

Brain-derived neurotrophic factor (BDNF) signalling via tyrosine kinase B receptors may play an important role in ovarian development and function. It has been reported that metformin elevates the activity of Tyrosine kinase receptors and may amplify BDNF signalling. The objective of this study was to investigate the effect of BDNF during in vitro maturation (IVM) and/or in vitro culture (IVC) (Experiment 1), and to evaluate the collaborative effect of BDNF and metformin treatment on the developmental competence of bovine in vitro fertilized (IVF) embryos (Experiment 2). In Experiment 1, BDNF, which was added to our previously established IVM systems, significantly increased the proportions of MII oocytes at both 10 ng/ml (86.7%) and 100 ng/ml (85.4%) compared with the control (64.0%). However, there was no statistically significant difference in blastocyst development between the control or BDNF-supplemented groups. In Experiment 2, in order to investigate the effect of BDNF (10 ng/ml) and/or metformin (10−5 M) per se, TCM-199 without serum and hormones was used as the control IVM medium. The BDNF (48.3%) and BDNF plus metformin (56.5%) significantly enhanced the proportions of MII oocytes compared with the control (34.4%). Although, BDNF or metformin alone had no effect in embryo development, BDNF plus metformin significantly improved early embryo development to the 8–16-cell stage compared with the control (16.5 vs. 5.5%). In conclusion, the combination of BDNF and metformin may have a collaborative effect during the IVM period. These results could further contribute to the establishment of a more efficient bovine in vitro embryo production system.

Anatomic data are presented of a female abortus with the unusual combination of Ebstein's malformation and atrioventricular septal defect with right isomerism of the atrial appendages, left-hand ventricular topology and discordant ventriculoarterial connection. The common atrioventricular orifice was guarded by an effectively common valve, the right half being guarded by a well-formed three-leaflet valve, but the posterior and left lateral wall of the orifice being deficient of valvar leaflet tissue. The right ventricular inlet was a blind chamber, an imperforate dimple being seen which was surrounded by valvar remnants. The apical trabecular and outlet components of the right ventricle communicated with the left ventricle through an outlet ventricular septal defect in front of the anterosuperior leaflet of the atrioventricular valve, giving the potential clinically for the lesion to be misdiagnosed as double inlet left ventricle.

This study compared the developmental competence of somatic cell nuclear transfer (SCNT) embryos reconstructed with different donor cells and analysed gene expression in the resulting embryos. Bovine fetal/adult ear fibroblasts and cumulus cells were used as donor cells and the developmental competence of the reconstructed embryos was monitored. The cell number and allocation in blastocysts were determined by differential staining. The Bax, E-cad, IF-tau, Hsp (heat shock protein) 70, Igf2r (insulin-like growth factor 2 receptor), DNMT (DNA methyltransferase) 1 and Mash (mammalian achaete-scute homologue) 2 genes were selected for gene expression analysis. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by semiquantitative reverse transcription-polymerase chain reaction. In experiment 1, development of SCNT preimplantation embryos and the cell numbers of inner cell masses and trophoblasts were not different among SCNT embryos derived from different cell types. In experiment 2, the relative expression of GAPDH and Hsp 70 transcripts was similar in all embryos. The expression of Bax, Igf2r and Mash2 transcripts was significantly increased in SCNT embryos reconstructed with adult fibroblasts. The E-cad transcript levels were reduced in SCNT embryos reconstructed with fetal fibroblasts. Relative abundance of DNMT1 in SCNT embryos derived from fetal fibroblasts was increased, and IF-tau expression in SCNT embryos derived from cumulus cells was increased. In conclusion, depending on the type of donor cells, preimplantation SCNT embryos displayed marked differences in gene expression. This may affect the developmental competence of SCNT embryos reconstructed with different cell types after implantation or during fetal growth in vivo.

High plasma level of cholesterol is a well-known risk factor for atherosclerotic diseases. Oxidized LDL induces cellular and nuclear damage that leads to apoptotic cell death. We tested the hypothesis that flavonoids may function as antioxidants with regard to LDL incubated with 5 μm-Cu2+ alone or in combination with human umbilical vein endothelial cells (HUVEC). Cytotoxicity and formation of thiobarbituric acid-reactive substances induced by Cu2+-oxidized LDL were examined in the presence of various subtypes of flavonoid. Flavanols, flavonols and flavanones at a non-toxic dose of 50 μm markedly inhibited LDL oxidation by inhibiting the formation of peroxidative products. In contrast, the flavones luteolin and apigenin had no such effect, with >30 % of cells killed after exposure to 0.1 mg LDL/ml. Protective flavonoids, especially (−)-epigallocatechin gallate, quercetin, rutin and hesperetin, inhibited HUVEC nuclear condensation and fragmentation induced by Cu2+-oxidized LDL. In addition, immunochemical staining and Western blot analysis revealed that anti-apoptotic Bcl-2 expression was enhanced following treatment with these protective flavonoids. However, Bax expression and caspase-3 cleavage stimulated by 18 h incubation with oxidized LDL were reduced following treatment with these protective flavonoids. The down-regulation of Bcl-2 and up-regulation of caspase-3 activation were reversed by the cytoprotective flavonoids, (−)-epigallocatechin gallate, quercetin and hesperetin, at ≥10 μm. These results suggest that flavonoids may differentially prevent Cu2+-oxidized LDL-induced apoptosis and promote cell survival as potent antioxidants. Survival potentials of certain flavonoids against cytotoxic oxidized LDL appeared to stem from their disparate chemical structure. Furthermore, dietary flavonoids may have therapeutic potential for protecting the endothelium from oxidative stress and oxidized LDL-triggered atherogenesis.

Porcelain (veneer layer)/alumina (core layer) is a typical dental crown structure. Due to its high incidence of failure, a new porcelain/mullite (buffer layer)/alumina trilayer structure is designed, fabricated, and evaluated. Alumina green bodies were prepared by gel-casting process, and then calcined at 900 and 1100°C to infiltrate mullite precursor slurry of silica-rich (Al2O3·2SiO2) composition into the bodies. Porosity in the bodies is not dependent on calcination temperature, resulting in a similar infiltration depth. Porcelain was coated on the alumina sintered at 1600°C with and without mullite buffer layer. There are no delamination or cracks observed after firing the layered materials. Rod type microstructure and continuous composition are indicated at the interface in the case of the layered structure with mullite buffer layer. To investigate the cracking resistance behavior for this new structure, Vickers indentation and Hertzian contact fatigue tests were conducted. Cracks do not penetrate the interface with mullite buffer layer into the porcelain, showing a reversal case for the layered structure without mullite buffer layer. The layered structure with mullite buffer layer shows higher critical load for fracture than that without mullite buffer layer. Fracture mode of the layered structures in cyclic fatigue shows a top layer (porcelain) fracture at relatively low load (P = 250 N) and higher cycles (n = 106), and a bottom layer (alumina) fracture at higher load (P = 300 N) and relatively low cycles (n = 105).