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Monocytes and macrophages that originate from common myeloid progenitors perform various crucial roles in the innate immune system. Stimulation with LPS combined with TLR4 drives the production of pro-inflammatory cytokines through MAPKs and NF-κB pathway in different cells. However, the difference in LPS susceptibility between monocytes and macrophages is poorly understood. In this study, we found that pro-inflammatory cytokines—IL-1β, IL-6 and TNFα showed greater induction in phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells than in THP-1 cells. To determine the difference in cytokine expression, the surface proteins such as TLR4-related proteins and intracellular adaptor proteins were more preserved in PMA-differentiated THP-1 cells than in THP-1 cells. MyD88 is a key molecule responsible for the difference in LPS susceptibility. Moreover, MAPKs and NF-κB pathway-related molecules showed higher levels of phosphorylation in PMA-differentiated THP-1 cells than in THP-1 cells. Upon MyD88 depletion, there was no difference in the phosphorylation of MAPK pathway-related molecules. Therefore, these results demonstrate that the difference in LPS susceptibility between THP-1 cells and PMA-differentiated THP-1 cells occur as a result of gap between the activated MAPKs and NF-κB pathways via changes in the expression of LPS-related receptors and MyD88.

The creep behavior of Ni–Cr–W alloy at 950 °C has been investigated by a novel creep testing system which is capable of in-situ measurement of strain. Tubular specimens were pressurized with argon gas for effective stresses up to 32 MPa. Experimental results show that the thermal fatigue reduces the creep life of the tubular specimens and with the introduction of thermal cycling fatigue the primary stage disappears and the creep rate higher than the pure thermal creep (without thermal fatigue). Also the creep behavior of Ni–Cr–W alloy doesn't consist in the secondary stage. A new creep equation has been derived and implemented into finite element method. The results from the finite element analyses are in good agreement with the creep experiment.

Mitochondrial dysfunction is strongly associated with the oocyte quality and aging, wherein the aged oocytes are related to the actin cytoskeleton integrity; however, whether this integrity is associated with mitochondrial dysfunction in oocytes from aged mice remains unclear. In the present study, we investigated the relationship between mitochondrial dysfunction and actin cytoskeleton instability in oocytes from the aged mice. We performed comparable analysis of mitochondrial motility between young, 1.5 μM cytochalasin B (CB)-treated young oocytes, and aged oocytes by confocal live imaging. Moreover, we analyzed the relationships between mitochondrial motility and maturation ratios, including ATP production ratio of the young, CB-treated young, and aged oocytes. Actin cytoskeleton instability in the aged oocytes and CB-treated young oocytes led to a significant decrease in the mitochondrial motility and low ATP productive ratios compared to those in the young group. Our data suggest that the actin cytoskeleton instability is presumably the primary cause for the loss of mitochondrial function in the aged murine oocytes.

Recently, minipig has been considered as an animal model that is appropriate for human disease model to study toxicology, pharmacology, and xenotransplantation. Nevertheless, minipigs are bred in various environment according to their use. Here, we suggest that minipigs used for research should be bred in well-controlled facility, comparing immune status of pigs raised in different breeding environment. DNA microarray was performed with ear skin and placenta of Landrace domestic pigs (DPs) and Minnesota germ-free minipigs (GPs). Their immune transcriptome was analyzed by gene ontology (GO) annotation database, based on criteria of |log2 fold change| ≥1 with P ≤ 0.05. As a result, we found that immune related genes in the ear skin of DPs were highly activated, compared to GPs. On the other hand, no significant s were found in the placenta. Quantitative real-time PCR (qRT-PCR) was performed in five candidate immune genes. Their fold changes were consistent with the results from DNA microarray (P ≤ 0.05). In conclusion, we experimentally proved that porcine immune system was affected by different breeding environment, suggesting the importance of controlling microbes in animal room for the qualified research.

This work presents a methodology combining SEM, EDS, conventional EBSD, and transmission-EBSD to analyse a recrystallised Zircaloy-4 sheet and cold-worked stress-relieved (CWSR) Zircaloy-4 cladding in unprecedented detail. Second-phase precipitates (SPPs) in Zircaloy-4 specimens were revealed after chemical polishing using a solution containing hydrofluoric acid (HF). Pitting corrosion of Zircaloy-4 specimens was revealed after electropolishing using an electrolyte containing HClO4 . A zirconium coupon without SPPs was used to confirm the chemical response of SPPs on surface morphology. Intrinsic features of cold-worked Zircaloy-4 such as relatively small grain sizes, high dislocation density, and complex microstructure make it significantly more difficult to collect excellent EBSD results compared to recrystallised Zircaloy-4. The fine hydride structure of as-hydrided CWSR Zircaloy-4 cladding further increases the level of challenge on EBSD analysis. LAY DESCRIPTION We present a methodology combining multiple microscopic methods to analyse a recrystallised Zircaloy-4 sheet and cold-worked stress-relieved (CWSR) Zircaloy-4 cladding, important alloys of structural materials widely used in nuclear application, and emphasis on the challenge of acquiring a satisfactory electron backscatter diffraction (EBSD) result of CWSR Zircaloy-4 cladding material in great details. EBSD is a powerful technique to characterise the crystallographic distribution and lattice type of conductive crystalline materials, especially for a highly textured material like CWSR Zircaloy-4 alloy. However, zirconium alloys are known to be one of the most difficult materials to prepare for EBSD characterisation. We point out that the configuration of the microstructure of the specimen cause the challenge in the EBSD sample preparations. Moreover, the occurrence of tiny zirconium hydride precipitates in Zircaloy-4 increases the difficulty. We believe that the information of the EBSD sample preparation related results in this paper can provide researchers and scientists in this community a useful reference to speed up the EBSD sample preparation of CWSR Zircaloy-4 cladding material and to expect the corresponding EBSD results.

Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues.

Poly(ADP-ribosyl)ation (PARylation) plays important roles in DNA repair, apoptosis, transcriptional regulation, and cell death, and occurs via the activity of poly(ADP-ribose) polymerases (PARPs). Previous studies have shown that PARylation affects mouse and porcine pre-implantation development and participates in mechanisms of autophagy. However, there have not yet been reported the role of PARylation during in vitro maturation (IVM) of porcine oocytes. Thus, we investigated the effect of PARylation inhibition on this process; cumulus-oocyte complexes (COCs) were cultured with 3-aminobenzamide (3-ABA, PARP inhibitor) during porcine IVM. Full cumulus expansion was significantly reduced (10.34 ± 1.23 [3-ABA] vs. 48.17 ± 2.03% [control]), but nuclear maturation rates were not changed in the 3-ABA treatment group. Especially, we observed that cumulus cells were little expanded after 22 h in 3-ABA treated COCs. The mRNA expression levels of oocyte maturation- and cumulus expansion-related genes were evaluated at 22 and 44 h. GDF9, BMP15, COX-2, and PTX3 expression were upregulated at 44 h, whereas the levels of HAS2 and TNFAIP6 were downregulated in the 3-ABA treated group. Furthermore, 3-ABA treatment significantly decreased the developmental rate (28.24 ± 1.06 vs. 40.24 ± 3.03%) and total cell number (41.12 ± 2.10 vs. 50.38 ± 2.27), but increased the total apoptotic index (6.44 ± 0.81 vs. 3.08 ± 0.51) in parthenogenetically activated embryos. In conclusion, these results showed that PARylation regulates cumulus expansion through the regulation of gene expression and affects developmental competence and quality in parthenogenetic embryos.

• Mitochondrial activation signaling pathways are not clearly identified. • Embryonic mitochondria activity is important for a successful pregnancy and live birth. • Neogenin is a multi-functional receptor that contributes to embryo development. • Neogenin as a receptor is related to mitochondrial activation and replication. • Neogenin could activate mitochondria in pre-implantation embryo development.

In vitro generation of dendritic cells (DCs) is advantageous for overcoming the low frequency of primary DCs and the difficulty of applying isolation techniques for studying DC immunobiology. The culture of bone marrow cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used extensively to generate bone marrow-derived dendritic cells (BMDCs). Studies have reported the heterogeneity of cells grown in murine GM-CSF culture based on the levels of MHCII expression. Although porcine DCs are generated by this classical method, the exact characteristics of the BMDC population have not yet been defined. In this study, we discriminated GM-CSF-grown BMDCs from gnotobiotic miniature pigs according to several criteria including morphology, phenotype, gene expression pattern and function. We showed that porcine BMDCs were heterogeneous cells that differentially expressed MHCII. MHCIIhigh cells displayed more representative of DC-like morphology and phenotype, including costimulatory molecules, as well as they showed a superior T cell priming capacity as compared to MHCIIlow cell. Our data showed that the difference in MHCIIhigh and MHCIIlow cell populations involved distinct maturation states rather than the presence of different cell types. Overall, characterization of porcine BMDC cultures provides important information about this widely used cellular model.

This study was conducted to investigate the effects of dietary oxidized oil and natural polyphenol antioxidants on laying performances and egg quality of laying hens. A total of two hundred, 61-week-old Lohmann Brown laying hens were divided into five groups, consisting in four replicates of 10 birds each. They were fed one of the five experimental diets (fresh oil only, oxidized oil without antioxidants, oxidized oil with vitamin E 200 ppm, oxidized oil with vitamin E 100 ppm + Cabanin CSDⓇ 100 ppm, oxidized oil with Cabanin CSDⓇ 500 ppm)respectively, for 6 weeks. There were no significant differences in laying performances and egg weight among the treatments. However, egg mass in group fed oxidized oil with vitamin E 100 ppm + Cabanin CSDⓇ 100 ppmwas significantly higher than group fed oxidized oil only (p

Transgenic animal producing technology has improved consistently over the last couple of decades. Among the available methods, somatic cell nuclear transfer (SCNT) technology was officially the most popular. However, SCNT has low efficiency and requires a highly skilled individual. Additionally, the allo-SCNT nuclear reprogramming mechanism is poorly understood in the gnotobiotic miniature pig, which is a candidate for xenotransplantation, making sampling in oocytes very difficult compared to commercial hybrid pigs. Therefore, interbreed SCNT (ibSCNT), which is a combination of miniature pig and commercial pig (Landrace based), was analyzed and was found to be similar to SCNT in terms of the rate of blastocyst formation (12.6% ± 2.9% vs. 15.5% ± 2.2%; p > 0.05). However, a significantly lower fusion rate was observed in the ibSCNT compared to normal SCNT with Landrace pig somatic cells (29.6% ± 0.8% vs. 65.0% ± 4.9%). Thus, the optimization of fusion parameters was necessary for efficient SCNT. Our results further revealed that ibSCNT by the whole-cell intracytoplasmic injection (WCICI) method had a significantly higher blastocyst forming efficiency than the electrofusion method (31.1 ± 8.5 vs. 15.5% ± 2.2%). The nuclear remodeling and the pattern of changes in acetylation at H3K9 residue were similar in both SCNT and ibSCNT embryos.

Poly(ADP-ribosyl)ation (PARylation) acts as a modulator of selective autophagic degradation of ubiquitinated aggregates for cellular quality control, functioning in pro-survival role. It was reported previously that the inhibition of PARylation resulted in autophagy defects leading accumulation of ubiquitinated aggregates SQSTM1/p62 and apoptosis in porcine blastocysts. Thus, this study aims to investigate the mechanism between PARylation and autophagy in porcine blastocysts. In vitro produced (IVP) embryos were treated with 3-aminobenzamide (3ABA, poly (ADP-ribose) polymerase inhibitor) and/or rapamycin (RAPA, an mTORC1 inhibitor) during blastocyst formation. Then, these treated blastocysts were analyzed by real-time PCR, immunocytochemistry and TUNEL Assay. We found that the 3ABA treatment increased mTORC1 downstream target, phosphorylation of thr389 p70S6K (p-p70S6K-thr389), suggesting an increase in mTORC1 activity. Co-treatment with rapamycin (RAPA), mTORC1 inhibitor, restored the 3ABA-induced autophagy defects to those of the controls by normalizing mTORC1 activity. Moreover, autophagy induction, with only RAPA treatment, increased the rate of blastocyst development (70.05 ± 0.93 vs. 50.61 ± 3.49%), total cell number (58.48 ± 2.94 vs. 49.58 ± 2.43) and blastomere survival, but decreased the accumulation of SQSTM1/p62 aggregates. In summary, mTORC1 signaling is a key mechanism of PARylation-autophagy and its inhibition improved developmental ability and embryo quality by promoting selective autophagic degradation of ubiquitinated aggregates in porcine blastocysts. Therefore, these findings have significant implications for understanding the importance of autophagy regulation for successful in vitro production of porcine embryos.

Somatic cells could be directly converted into induced neural stem cells (iNSCs) by ectopic expression of defined transcription factors. However, the underlying mechanism of direct lineage transition into iNSCs is largely unknown. In this study, we examined the effect of genetic background on the direct conversion process into an iNSC state. The iNSCs from two different mouse strains exhibited the distinct efficiency of lineage conversion as well as clonal expansion. Furthermore, the expression levels of endogenous NSC markers, silencing of transgenes, and in vitro differentiation potential were also different between iNSC lines from different strains. Therefore, our data suggest that the genetic background of starting cells influences the conversion efficiency as well as reprogramming status of directly converted iNSCs.

Xenotransplantation has been considered an alternative to the moderate shortage of donor organs for transplantation. To achieve successful xenotransplatation, there is the need to overcome immune rejection. Although, hyperacute rejection has been overcome by α1,3-galactosyltransferase knockout pig, cellular immune rejection remains as a subsequent barrier. Interleukin-10 (IL-10) is known as an anti-inflammatory and immunomodulatory cytokine which has been shown to limit inflammatory responses by inhibiting macrophage activation in several animal experiments. To study the effect of human IL-10 (hIL-10) on pig-to-human xenotransplantation, porcine kidney epithelial cell line (PK(15)) expressing hIL-10 was established. The cytotoxicity of macrophages decreased by hIL-10 from transgenic cells. Furthermore, there is a decreased production of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-23, and increased anti-inflammatory cytokines like IL-10, but not transforming growth factor beta, in the presence of hIL-10. Also, macrophage polarization toward M2-like phenotype were induced by hIL-10 from transgenic PK(15) cells. Finally, we suggest that the cytotoxicity of human macrophages was reduced by hIL-10 from transgenic cells, inducing M2-like macrophage polarization. Therefore, these results show that hIL-10 transgenic pig can be used as a model to overcome acute immune rejection in pig-to-human xenotransplantation., Highlights • The effect of human IL-10 (hIL-10) on pig-to-human xenotransplantation was studied. • Cytotoxicity of macrophages decreased by hIL-10 from transgenic cells. • hIL-10 induced macrophage polarization toward M2-like phenotype.

A new in-situ biaxial creep-fatigue testing system was built to study the creep-fatigue interaction of Alloy 617, a nickel base alloy with potential structural applications in the Very High Temperature Reactor, up to 70 MPa at 950 °C. The results show that creep-fatigue life of Alloy 617 is reduced with increasing hold time and the cracking mode seems to be intergranular in all creep-fatigue samples. At the microstructural level, both cellular network dislocations and slip bands were observed in the creep-fatigue tested specimens. The portion of slip bands seems to increase with increasing fatigue damage fraction. Moreover, the dislocation density increased with increasing effective stress.

Recently, diabetes mellitus (DM) has shown rapid global increases with about five million deaths annually. Animal models are imperative to understand disease mechanisms and develop diagnostic, preventive, and therapeutic interventions in translational research. Rodent and mini-pig models have been established and widely used for DM research. However, domestic pig models are limited in spite of advantages such as pharmacokinetic and physiopathological availability. This study examines the potential use of domestic pigs expressing recombinant human erythropoietin (rhEPO) as disease and therapeutic response models for DM. We previously generated transgenic pigs (n = 16, EPO Tg) in which rhEPO was expressed and circulated in all organs. Thirty-two pigs, including 16 controls, were fed high fat (HF) diets for 42 weeks. Subsequently, blood samples for chemical and metabolic analysis were collected after fasting for 24 h and glucose loading for oral glucose tolerance tests (OGTTs). We found increased activation of the PI3 K/Akt signaling pathway under hypoxic conditions after rhEPO treatment, and HF diet-inducible-obesity in the EPO Tg and control pigs. OGTTs showed lower fasting glucose levels in the EPO Tg pigs than in controls before and after the HF diet, suggesting that rhEPO may affect glucose concentrations. Insulin and C-peptide concentrations responded slowly to glucose administration and returned to initial levels after 2 h. The blood test results suggest that EPO might affect metabolic and chemical components such as glucose, high-density lipoprotein, glucagon, triglyceride, and free fatty acid. Our findings support the use of rhEPO transgenic domestic pigs as model animals for translational DM research.

Poly(ADP-ribosyl)ation (PARylation) prevents apoptosis through its involvement in pro-survival autophagy in cultured cells; whether or not the same is true for pre-implantation embryos has not yet been documented. In this study, we investigated the participation of PARylation and autophagy in in vitro porcine pre-implantation embryo development. The transcript levels of autophagy-related genes and poly(ADP-ribose) polymerase 1 (PARP1), an enzyme required for PARylation, were transiently up-regulated by fertilization, decreased at the late 1-cell stage, and maintained until the blastocyst stage. LC3, a marker of autophagosomes, and poly(ADP-ribose) (PAR) polymer were present in all stages of pre-implantation development. Exposure of embryos to 3-methyladenine, an autophagy inhibitor, or 3-aminobenzamide, a PARP inhibitor, suppressed the development of blastocysts. Pharmacological inhibition of PARylation further suppressed pro-survival autophagy by decreasing the expression of autophagy-related genes (ATG5, BECLIN1, and LC3) and decreasing LC3 protein abundance while increasing the rate of apoptosis in blastocysts. Deficiency in autophagy also induced abnormal accumulation of SQSTM1/p62 aggregates in porcine blastocysts. Collectively, these data suggest that PARylation is involved in selective autophagic degradation of ubiquitinated proteins, functioning in a pro-survival role, in porcine in vitro-produced embryos. These pro-survival regulatory mechanisms may be important for the control of embryo quality.

Recently, p16INK4a/Ki-67 dual immunostaining has been introduced as a new biomarker protocol for early detection of uterine cervical dysplasia and cancer in liquid-based cytology (LBC). We performed the p16INK4a/Ki-67 dual immunostaining using a CINtec® PLUS kit in a total of 109 LBC cases of cervicovaginal smear and compared its results with those from LBC, HPV hybrid capture II (HC II) test and histological diagnosis. Expression of p16INK4a and Ki-67 was significantly associated with cases of LSIL or higher in cytological diagnosis and cases of cervical intraepithelial neoplasia (CIN) 1 or higher in histological diagnosis (p

The discovery of activating mutations in EGFR in a subset of lung adenocarcinomas was a major advance in our understanding of lung adenocarcinoma biology, and has led to groundbreaking studies that have demonstrated the efficacy of tyrosine kinase inhibitor therapy. Cytologic specimen procedures have become increasingly popular for obtaining diagnostic material in lung carcinomas. However, frequently the small amount of material or sparseness of tumor cells obtained from cytologic preparations limit the number of specialized studies, such as mutation analysis, that can be performed. In this study we used microdissection to isolate small numbers of tumor cells to assess for EGFR mutations from 76 cytological smear slides of patients with lung adenocarcinomas. We compared our results with previous molecular assays that had been performed on either surgical or cytology specimens as part of the patient’s initial clinical work-up. Not only were we able to detect the identical EGFR mutation through the pyrosequencing, but we were also able to consistently detect the mutation from as few as 25 microdissected tumor cells. Furthermore, isolating a purer population of tumor cells resulted in increased sensitivity of mutation detection as we were able to detect mutations from microdissection-enriched cases. Therefore, microdissection can not only significantly increase the number of lung adenocarcinoma patients that can be screened for EGFR mutations, but can also facilitate the use of cytologic samples in the newly emerging field of molecular-based personalized therapies.

Next-generation sequencing (NGS) technologies make it possible to obtain the entire genomic content of microorganisms in metagenome samples. Thus, many studies have developed methods for the processing and analysis of metagenomic NGS reads, including analyses for predicting functions and their enrichments in environmental metagenome samples. Especially, comparative functional studies by using multi-metagenome samples are essential for identifying and comparing different characteristics of multiple environmental samples. In this paper, we introduce a pipeline for functional characterization of multiple metagenome samples to infer major functions as well as their quantitative scores in a comparative metagenomics manner. The pipeline performs the annotation of functions related to expected proteins in the metagenome samples, calculates their enrichment scores based on the reads per kilobase per million reads (RPKM) measure, and predicts the relative abundance of associated functions by a statistical test. The results from single sample analysis are then used to find common and sample-specific major functions. By applying the pipeline to six different environmental metagenome samples, including two ocean (Antarctica aquatic and Baltic Sea) and four terrestrial (Acid mine drainage, human gut microbiome, Amazon River, and Wasca soil) samples, we were able to predict common functions as well as environment-specific functions. Our pipeline is available at http://bioinfo.konkuk.ac.kr/FCMM/ .

BACKGROUND The microdissection method has greatly facilitated the isolation of pure cell populations for accurate analysis of mutations. However, the absence of coverslips in these preparations leads to poor resolution of cellular morphological features. In the current study, the authors developed the MultiTech DNA extraction solution to improve the visualization of cell morphology for microdissection and tested it for the preservation of morphological properties of cells, quality of DNA, and ability to detect mutations. METHODS A total of 121 cytological samples, including fine-needle aspirates, sputum, pleural fluid, and bronchial washings, were selected from hospital archives. DNA extracted from microdissected cells was evaluated by epidermal growth factor receptor (EGFR) mutation analysis using pyrosequencing, Sanger sequencing, and peptide nucleic acid (PNA)-mediated real-time polymerase chain reaction clamping. Morphological features of cells as well as DNA quality and quantity were analyzed in several cytological samples to assess the performance of the MultiTech DNA extraction solution. The results were compared with previous EGFR mutation tests. RESULTS The MultiTech DNA extraction solution improved the morphology of archived stained cells before microdissection and provided a higher DNA yield than the commercial QIAamp DNA Mini Kit in samples containing a minimal number of cells (25-50 cells). The authors were able to detect identical EGFR mutations by using different analysis platforms and consistently identified these mutations in samples comprising as few as 25 microdissected cells. CONCLUSIONS The MultiTech DNA extraction solution is a reliable medium that improves the resolution of cell morphology during microdissection. It was particularly useful in EGFR mutations of samples containing a small number of cells. Cancer (Cancer Cytopathol) 2015;123:401-12. © 2015 American Cancer Society.

The aim of this study was to define the critical warning sign of real-time brainstem auditory evoked potential (BAEP) for predicting hearing loss (HL) after microvascular decompression (MVD) for hemifacial spasm (HFS).Nine hundred and thirty-two patients with HFS who underwent MVD with intraoperative monitoring (IOM) of BAEP were analyzed. We used a 43.9 Hz/s stimulation rate and 400 averaging trials to obtain BAEP. To evaluate HL, pure-tone audiometry and speech discrimination scoring were performed before and one week after surgery. We analyzed the incidence for postoperative HL according to BAEP changes and calculated the diagnostic accuracy of significant warning criteria.Only 11 (1.2%) patients experienced postoperative HL. The group showing permanent loss of wave V showed the largest percentage of postoperative HL (p 0.001). No patient who experienced only latency prolongation (≥1 ms) had postoperative HL. Loss of wave V and latency prolongation (≥1 ms) with amplitude decrement (≥50%) were highly associated with postoperative HL.Loss of wave V and latency prolongation of 1 ms with amplitude decrement ≥50% were the critical warning signs of BAEP for predicting postoperative HL.These findings elucidate the critical warning sign of real-time BAEP.

To overcome shortages of human donor organs for organ failure patients, we made a commitment to develop gnotobiotic miniature swine as an alternative organ donor source for xenotransplantation. For this, we have constructed an absolute barrier-sustained gnotobiotic facility. Pregnant sows of gnotobiotic miniature swine, were procured and germfree piglets were obtained by hysterectomy. These were maintained in germfree isolators for about 4 weeks, deprived of colostrum and were fed sterilized soybean milk. They were associated with di-flora, anaerobic Lactobacillus sp. and Streptococcus sp. After confirmation of successful associations, gnotobiotic piglets were transferred into the facility aseptically. The piglets are maintained on high-efficiency particle air-filtered air in and out; maintaining constant room air pressure of 33 ± 3 mmAq, and sterile water and diet. In 10 sessions of hysterectomy, 18 male and 32 female piglets were obtained of which piglets (M six, F eight) died within 5 days. Among live piglets, piglets (M eight, F 12) were confirmed to be germfree by microbiological monitoring. For research of xenotransplantation, one consistent experimental result was essential. Therefore, major histocompatibility complex class II which related innate immunity, homozygotic gnotobiotic miniature swine was developed. As a result, genotyping revealed 14 individuals to be homozygous for major histocompatibility complex class II (DRB, DQB) as 0301, three individuals were homozygous as 0201 and each of two were homozygous for DQB as 0701 and DRB as 0404, respectively. Genetic modifications and immunological research for ideal alternative organ sources are in progress.

The spinal cord does not spontaneously regenerate, and treatment that ensures functional recovery after spinal cord injury (SCI) is still not available. Recently, fibroblasts have been directly converted into induced neural stem cells (iNSCs) by the forced expression defined transcription factors. Although directly converted iNSCs have been considered to be a cell source for clinical applications, their therapeutic potential has not yet been investigated. Here we show that iNSCs directly converted from mouse fibroblasts enhance the functional recovery of SCI animals. Engrafted iNSCs could differentiate into all neuronal lineages, including different subtypes of mature neurons. Furthermore, iNSC-derived neurons could form synapses with host neurons, thus enhancing the locomotor function recovery. A time course analysis of iNSC-treated SCI animals revealed that engrafted iNSCs effectively reduced the inflammatory response and apoptosis in the injured area. iNSC transplantation also promoted the active regeneration of the endogenous recipient environment in the absence of tumor formation. Therefore, our data suggest that directly converted iNSCs hold therapeutic potential for treatment of SCI and may thus represent a promising cell source for transplantation therapy in patients with SCI.

Isolating total DNA from small samples using traditional methods is difficult and inefficient mainly due to loss of DNA during filtration and precipitation. With advances in molecular pathology, DNA extraction from micro-dissected cells has become essential in handling clinical samples. Genomic DNA extraction using small numbers of cells can be very important to successfully PCR amplify DNA from small biopsy specimens. We compared our experimental genomic DNA extraction method (A) with two other commercially available methods: using spin columns (B), and conventional resins (C), and determined the efficacy of DNA extraction from small numbers of cells smeared on a glass slide. Approximately 50, 100, 200, 500 and 1000 cells were isolated from fine needle aspiration biopsy (FNAB) slides aspirated from histologically proven papillary thyroid carcinoma masses. DNA was extracted using the three techniques. After measuring DNA quantity, PCR amplification was performed to detect the b-globin and BRAF V600E gene mutations. DNA extracted by method (A) showed better yield than the other methods in all cell groups. With our method, a suitable amount of genomic DNA to produce amplification was extracted from as few as 50 cells, while more than 100 to 200 cells were required when methods (B) or (C) were applied. Our genomic DNA extraction method provides high quality and improved yields for molecular analysis. It will be especially useful for paucicellular clinical samples which molecular pathologists often confront when handling fine needle aspiration cytology, exfoliative cytology and small biopsy specimens.

Adipose-derived mesenchymal stem cells (AD-MSCs) are abundant in adipose tissue from animals of all ages, are easily isolated, can differentiate into multi-lineage cells, and have a clinical application. This promising potential may only be achieved if the cells are expanding in a large number while maintaining their stemness in sequential passages. In this study, canine AD-MSCs (cAD-MSCs) were individually isolated from five dogs and subjected to proliferative culture with seven sub-passages. The cells at each sub-passage were characterized for properties associated with multipotent MSCs such as proliferation kinetics, expression of MSCs-specific surface markers, expression of molecules associated with self-renewal and differentiation capabilities into mesodermal lineage cells. Proliferation of the cells plateaued at passage 5 by cumulative population doubling level, while cell doubling time gradually increased with passage. MSCs surface markers (CD44, CD90, and CD105) and molecules (Oct 3/4, Sox-2, Nanog and HMGA2) associated with self-renewal were all expressed in the cells between passages 1 to 6 by RT-PCR. In addition, the cells at passage 1, 3 or 6 underwent adipogenic and chondrogenic differentiation under specific induction conditions. However, the level of adipogenic and chondrogenic differentiation was negatively correlated with the number of sub-passage. The present study suggests that sequential sub-passages affect multipotent properties of cAD-MSCs, which should be considered in their therapeutic application in regenerative medicine.

Terminally differentiated cells can be directly converted into different types of somatic cells by using defined factors, thus circumventing the pluripotent state. However, low reprogramming efficiency, along with the absence of proliferation of some somatic cell types, makes it difficult to generate large numbers of cells with this method. Here we describe a protocol to directly convert mouse fibroblasts into self-renewing induced neural stem cells (iNSCs) that can be expanded in vitro, thereby overcoming the limitations associated with low reprogramming efficiency. The four transcription factors required for direct conversion into iNSCs (Sox2, Klf4, Myc (also known as c-Myc) and Pou3f4 (also known as Brn4)) do not generate a pluripotent cell state, and thus the risk for tumor formation after transplantation is reduced. By following the current protocol, iNSCs are observed 4-5 weeks after transduction. Two additional months are required to establish clonal iNSC cell lines that exhibit retroviral transgene silencing and that differentiate into neurons, astrocytes and oligodendrocytes.

Treatment of donor cells and/or cloned embryos with cytidine analogues, having an Aza group at its 5th carbon (5-Aza), such as 5-Azacytidine (5-Aza-C) or 5-Aza-2'-deoxycytidine (5-Aza-dC) improves the in vitro development of cloned embryos produced by somatic cell nuclear transfer (SCNT). In vitro maturation (IVM) of immature pig oocytes treated with 5-Aza-C not only results in greater (P 0.05) meiotic maturation to the MII stage but also enhances the capacity of 5-Aza-C treated oocytes for early embryonic development after parthenogenetic activation (PA), in vitro fertilization (IVF) or SCNT in a dose-dependent manner (0-10 μM). Cloned embryos generated from 5-Aza-C (0.01 μM) treated oocytes had an increased capacity to develop to the blastocyst stage (14.1 ± 1.5% compared with 9.6 ± 1.8%), greater probability of hatching (61.8 ± 1.5% compared with 45.0 ± 3.9%) and contained a greater number of cells per blastocyst (38.5 ± 4.4 compared with 30.5 ± 3.4) than those produced from non-treated control oocytes (P 0.05). Data from the present study indicate that treatment of oocytes with 5-Aza-C may be an important approach to enhance the meiotic maturation and subsequent in vitro development of pig embryos. Future studies should be conducted to determine the underlying mechanism of improved early embryonic development of 5-Aza-C treated oocytes.

Egg consumption has been limited to avoid cardiovascular diseases such as atherosclerosis and hyperlipidemia, because the yolk contains high levels of cholesterol. This study was conducted to evaluate the effect of the water-soluble component of egg-yolk on the growth efficiency, immune modulation, and changes in serum lipid levels in BALB/c mice. A total 5 wk old 120 BALB/c male mice were divided into 4 groups and were fed 0, 2, 10, and 20 mg/d water-soluble egg yolk extract (WSEYE) for 5 wk. Water-soluble egg yolk extract (WSEYE) uptake resulted in a significant reduction in daily weight gain and feed efficiency rate (FER). The mouse groups treated with 2 and 20 mg/d WSEYE showed a significant increase in populations of monocytes at the third wk and B-lymphocyte activity at the fifth wk. In addition, WSEYE uptake did not influence serum immunoglobulin E levels. In serum lipid-profile studies, treatment of WSEYE did not alter total cholesterol and low-density lipoprotein levels; however, blood triglyceride levels were significantly diminished in mice treated with 2 mg/d at the third wk (p

Urokinase-type plasminogen activator is a serine protease that is clinically used in humans for the treatment of thrombolytic disorders and vascular diseases such as acute ischemic stroke and acute peripheral arterial occlusion. This study explored the feasibility of using chickens as a bioreactor for producing human urokinase-type plasminogen activator (huPA). Recombinant huPA gene, under the control of a ubiquitous Rous sarcoma virus promoter, was injected into the subgerminal cavity of freshly laid chicken eggs at stage X using the replication-defective Moloney murine leukemia virus (MoMLV)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein. A total of 38 chicks, out of 573 virus-injected eggs, hatched and contained the huPA gene in their various body parts. The mRNA transcript of the huPA gene was present in various organs, including blood and egg, and was germ-line transmitted to the next generation. The level of active huPA protein was 16-fold higher in the blood of the transgenic chicken than in the nontransgenic chicken (P < 0.05). The expression of huPA protein in eggs increased from 7.82 IU/egg in the G0 generation to 17.02 IU/egg in the G1 generation. However, huPA-expressing embryos had reduced survival and hatchability at d 18 and 21 of incubation, respectively, and the blood clotting time was significantly higher in transgenic chickens than their nontransgenic counterparts (P < 0.05). Furthermore, adult transgenic rooster showed reduced (P < 0.05) fertility, as revealed by reduced volume of semen ejaculate, sperm concentration, and sperm viability. Taken together, our data suggest that huPA transgenic chickens could be successfully produced by the retroviral vector system. Transgenic chickens, expressing the huPA under the control of a ubiquitous promoter, may not only be used as a bioreactor for pharming of the huPA drug but also be useful for studying huPA-induced bleeding and other disorders.

This study explored the possibility of producing transgenic cloned embryos by interspecies somatic cell nuclear transfer (iSCNT) of cattle, mice, and chicken donor cells into enucleated pig oocytes. Enhanced green florescent protein (EGFP)-expressing donor cells were used for the nuclear transfer. Results showed that the occurrence of first cleavage did not differ significantly when pig, cattle, mice, or chicken cells were used as donor nuclei (p>0.05). However, the rate of blastocyst formation was significantly higher in pig (14.9±2.1%; p0.05). Moreover, NCSU23 medium, which was designed for in vitro culture of pig embryos, was able to support the in vitro development of cattle, mice, and chicken iSCNT embryos up to the blastocyst stage. Taken together, these data suggest that enucleated pig oocytes may be used as a universal cytoplast for production of transgenic cattle, mice, and chicken embryos by iSCNT. Furthermore, xenogenic transfer of mitochondria to the recipient cytoplast may not be the cause for poor embryonic development of cattle–pig iSCNT embryos.

SummaryInsulin, transferrin and selenium (ITS) supplementation to oocyte maturation medium improves the post-fertilization embryonic development in pigs. ITS is also commonly used as a supplement for the in vitro culture (IVC) of embryos and stem cells in several mammalian species. However, its use during IVC of pig embryos has not been explored. This study investigated the effect of ITS supplementation to IVC medium on the in vitro development ability of pig embryos produced by parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). We observed that ITS had no significant effect on the rate of first cleavage (P > 0.05). However, the rate of blastocyst formation in ITS-treated PA (45.3 ± 1.9 versus 27.1 ± 2.3%), IVF (31.6 ± 0.6 versus 23.5 ± 0.6%) and SCNT (17.6 ± 2.3 versus 10.7 ± 1.4%) embryos was significantly higher (P < 0.05) than those of non-treated controls. Culture of PA embryos in the presence of ITS also enhanced the expansion and hatching ability (29.1 ± 3.0 versus 18.2 ± 3.8%; P < 0.05) of blastocysts and increased the total number of cells per blastocyst (53 ± 2.5 versus 40.9 ± 2.6; P < 0.05). Furthermore, the beneficial effect of ITS on PA embryos was associated with significantly reduced level of intracellular reactive oxygen species (ROS) (20.0 ± 2.6 versus 46.9 ± 3.0). However, in contrast to PA embryos, ITS had no significant effect on the blastocyst quality of IVF and SCNT embryos (P > 0.05). Taken together, these data suggest that supplementation of ITS to the IVC medium exerts a beneficial but differential effect on pig embryos that varies with the method of embryo production in vitro.

Background In pig-to-human xenotransplantation, hyperacute rejection of pig organs could be overcome by the production of α1,3-galactosyltransferase knockout pigs. However, macrophage-mediated acute rejection is another obstacle that needs to be overcome. Among the various candidate genes involved in acute rejection, CD47 inhibits monocyte/macrophage-mediated phagocytosis by identifying the CD47 signal regulatory protein alpha (SIRP-α) as self/non-self. Tissue factor pathway inhibitor (TFPI) is involved in the regulation of the coagulation pathway and is able to bind to another ligand of CD47, thrombospondin-1 (TSP-1). When TSP-1 binds to CD47, phagocytosis in macrophages is increased. Methods The 2A peptide system was used to establish pig kidney cells (PK15) simultaneously expressing human CD47 and human TFPI, and they were cultured with activated THP-1 cells. After staining with 7-aminoactinomycin D, flow cytometry analysis was carried out. TFPI siRNA analysis and recombinant human TFPI (rhTFPI) treatment were performed to determine the potentiating effect of TFPI on pig cells for activated THP-1 cells in the presence of CD47. Related inflammatory cytokines produced by activated THP-1 cells were analyzed using qPCR and Western blot technique. In addition, the tyrosine phosphorylation level of SIRP-α in activated THP-1 cells was analyzed using immunoprecipitation and Western blot. Results hCD47/hTFPI-PK15 cells survived better than hCD47-PK15, hTFPI-PK15, or normal PK15 cells on cytotoxicity tests using activated THP-1 cells. TSP-1, derived from these activated THP-1 cells, served as a mediator for this enhancing effect, and it also played a role in activated adherent peripheral blood mononuclear cells (PBMCs). The tyrosine phosphorylation level of SIRP-α in activated THP-1 cells was further increased in the case of co-expression of CD47/TFPI than in individual non-expression or expression of CD47 or TFPI alone. Conclusions When hCD47 was expressed, the expression of hTFPI leaded to tyrosine phosphorylation of SIRP-α in activated THP-1 cells via hTSP-1 inhibition, and consequently, it might improve the effect of hCD47-SIRP-a signaling.

The viral vector-mediated overexpression of the defined transcription factors, Brn4/Pou3f4, Sox2, Klf4, and c-Myc (BSKM), could induce the direct conversion of somatic fibroblasts into induced neural stem cells (iNSCs). However, viral vectors may be randomly integrated into the host genome thereby increasing the risk for undesired genotoxicity, mutagenesis, and tumor formation. Here we describe the generation of integration-free iNSCs from mouse fibroblasts by non-viral episomal vectors containing BSKM. The episomal vector-derived iNSCs (e-iNSCs) closely resemble control NSCs, and iNSCs generated by retrovirus (r-iNSCs) in morphology, gene expression profile, epigenetic status, and self-renewal capacity. The e-iNSCs are functionally mature, as they could differentiate into all the neuronal cell types both in vitro and in vivo. Our study provides a novel concept for generating functional iNSCs using a non-viral, non-integrating, plasmid-based system that could facilitate their biomedical applicability.

Cryopreservation of bovine embryos can be performed by a variety of methods with variable degree of success. Here, we report a new, easy to perform, simple, inexpensive, and successful method for vitrification of bovine blastocysts. In vitro produced bovine blastocysts were exposed to vitrification solution (5.5 m ethylene glycol, 10% serum and 1% sucrose) in one single step for 20 s, loaded on a paper container prepared from commonly available non-slippery, absorbent writing paper, and then were directly plunged into liquid nitrogen for storage. Vitrified blastocysts were warmed by serial rinsing in 0.5, 0.25 and 0.125 m sucrose solution for 1 min each. Results showed that one step exposure of bovine blastocysts to cryoprotective agents was sufficient to achieve successful cryopreservation. Under these conditions, more than 95% of blastocysts survived the vitrification-warming on paper containers which was significantly higher than those obtained from other containers, such as electron microscope (EM) grid (78.1%), open pulled straw (OPS; 80.2%), cryoloop (76.2%) or plastic straw (73.9%). Embryo transfer of blastocysts vitrified-warmed on paper container resulted in successful conception (19.3%) and full-term live birth of offspring (12.3%) which were lower (P0.05) than those obtained from non-vitrified blastocysts (38.0 and 32.7%) but were comparable (P0.05) to those obtained from blastocysts vitrified-warmed on EM grid (23.3 and 14.2%). Our results, therefore, suggest that paper may be an inexpensive and useful container for the cryopreservation of animal embryos.

To investigate reproductive disorder in human erythropoietin (EPO)-expressing pig, we performed comparative proteomic analyses of testicular tissues from human erythropoietin (hEPO) gene-harboring transgenic pigs and wild type pigs born from natural conception. In hEPO TG pigs, we found relatively low sperm motility and higher death rate indicating impaired sperm development. Consistently, plasma concentration of testosterone was significantly lower in the transgenic post-pubertal boars compared with wild type boars. Normalized protein spots showing higher than 2-fold differential expression intensity in two-dimensional polyacrylamide gel electrophoresis were selected for matrix associated laser desorption/ionization time-to-flight mass spectrometry analysis. Specific proteins were identified by searching the NCBI protein sequence databases. Among 55 proteins selected, 12 proteins were identified as those differentially expressed between transgenic and wild type pigs. Three downregulated proteins (β-globin, carbonyl reductase 1, and peroxiredoxin 6) and nine upregulated proteins (cytoskeletal β-actin, α 2,3-sialyltransferase, apolipoprotein A-I, tubulin α-1A chain, tropomodulin 3, thioredoxin, heat shock Protein 70.2, ch4/domains of swine IgM, and albumin), all of which are closely related to apoptosis and cytoskeletal development, were found in the transgenic boar testes. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay confirmed the increased occurrence of apoptosis in the transgenic boar testes compared with the wild type boar testes. Reproductive defects of the hEPO-expressing transgenic pigs may be caused by the abnormal expression of the genes identified in this study.

UV radiation-mediated photodamage to the skin has been implicated in premature aging and photoaging-related skin cancer and melanoma. Little is known about the cellular events that underlie premature senescence, or how to impede these events. In the present study we demonstrate that PPARδ (peroxisome-proliferator-activated receptor δ) regulates UVB-induced premature senescence of normal keratinocytes. Activation of PPARδ by GW501516, a specific ligand of PPARδ, significantly attenuated UVB-mediated generation of ROS (reactive oxygen species) and suppressed senescence of human keratinocytes. Ligand-activated PPARδ up-regulated the expression of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and suppressed the PI3K (phosphatidylinositol 3-kinase)/Akt pathway. Concomitantly, translocation of Rac1 to the plasma membrane, which leads to the activation of NADPH oxidases and generation of ROS, was significantly attenuated. siRNA (small interfering RNA)-mediated knockdown of PTEN abrogated the effects of PPARδ on cellular senescence, on PI3K/Akt/Rac1 signalling and on generation of ROS in keratinocytes exposed to UVB. Finally, when HR-1 hairless mice were treated with GW501516 before exposure to UVB, the number of senescent cells in the skin was significantly reduced. Thus ligand-activated PPARδ confers resistance to UVB-induced cellular senescence by up-regulating PTEN and thereby modulating PI3K/Akt/Rac1 signalling to reduce ROS generation in keratinocytes.

The active metabolite of vitamin D3, 1α,25(OH)2D3, displays anticancer effects by regulating cell cycle and apoptosis in many cancer cells. However, it has not been determined whether 1α,25(OH)2D3 increases the susceptibility of cancer cells to NK cells. Here, we investigated the anticancer effect of 1α,25(OH)2D3 in human melanoma cell lines by investigating enhancement of NK susceptibility and elucidating the mediator of NK cytotoxicity. 1α,25(OH)2D3-resistant melanoma cells (G-361 and SK-MEL-5) treated with 1α,25(OH)2D3 showed higher susceptibility to NK cells with up-regulation of Fas expression. Furthermore, G-361 cells treated with 1α,25(OH)2D3 showed significantly increased caspase activity. In addition to Fas up-regulation, expression of heat shock protein 60 (Hsp60) was elevated by 1α,25(OH)2D3. Increased expression of Hsp60 by 1α,25(OH)2D3 was related to not only up-regulation of Fas expression but also to NK susceptibility of G-361 cells. Taken together, our data suggest that 1α,25(OH)2D3 acts as an anticancer agent by increasing expression of Fas on the surface of melanoma cells through Hsp60 induction and strengthens caspase sensitivity to Fas-mediated apoptotic pathway by NK cells. 1α,25(OH)2D3 treatment may therefore have a preventive role in melanoma occurrence or potentiate the anticancer effects of NK-cell immune therapy.

The transplantation of adult stem cells into recipients is a method used widely in mammals to determine the fate of transferred cells, and for the production of progenies. This study is the first report, to our knowledge, to demonstrate the successful production of chickens using cells transdifferentiated from adult chicken bone marrow cells (BMCs) transplanted into the testes. BMCs from the enhanced green fluorescent protein (eGFP) transgenic (Tg) chickens were induced via in vitro transdifferentiation to male germ cells and injected into the testes of normal recipients. The multipotency of BMC was found with RT-PCR, immunocytochemistry, and FACS using specific markers, such as OCT4 and SSEA-1, -3, and -4. Localization and in vivo transdifferentiation of injected cells in the seminiferous tubules of recipients were traced for up to 40 days' post-injection by GFP expression and immunocytochemical analyses. The integration of the eGFP and the neo(R) genes in sperm gDNAs of recipient was confirmed via PCR analysis. A subsequent testcross of the recipient roosters with non-Tg hens resulted in the production of eGFP Tg progenies, demonstrating the successful transdifferentiation of the adult BMC to the germ cells in the testis. Therefore, we suggest that the use of adult BMCs is a new and promising approach to the production of Tg poultry, and may prove helpful in the study of avian developmental biology.

This study evaluated the effect of 3-hydroxyflavone (a flavonoid having hydroxyl group at 3 carbon position) on embryos using parthenogenetic activation (PA) and cloned pig embryos as model system. There was no evidence for embryo toxicity of 3-hydroxyflavone in a wide concentration range of 1–100 μM. On the contrary, 3-hydroxyflavone significantly improved the in vitro development and quality of PA embryos that was associated with the activation of ERK signaling molecules and reduction in Caspase 3 expression. Furthermore, 3-hydroxyflavone rescued the in vitro development and embryo quality of in vitro aged oocytes by inhibiting ROS activity and activating ERK signaling. The beneficial effects of 3-hydroxyflavone on PA embryos were consistent both in PVA- and BSA-containing embryo culture medium and in cloned embryos. These results suggest that, contrary to those of other structurally related flavonoid molecules, 3-hydroxyflavone may be useful as a therapeutic drug for improving the developmental potential of aged oocytes in assisted reproductive technologies.

The effects of two antioxidants, superoxide dismutase (SOD) and the flavonoid 3,4-dihydroxyflavone (DHF), on bovine embryo development in vitro were examined. Blastocyst development, total cell and inner cell mass (ICM) numbers, intracellular levels of reactive oxygen species (ROS), apoptotic indices and gene expression levels were examined before and after treatment of day 2 bovine embryos (≥2-4 cells) with various concentrations of 3,4-DHF or SOD for 6 days. Statistical analysis was performed using analysis of variance, with significance defined at the P

Fatty liver hemorrhagic syndrome (FLHS) is characterized by increased hepatic triacylglycerol content associated with liver hemorrhages and results in a sudden decline in egg production. Genetic, environmental, nutritional, and hormonal factors have all been implicated in the etiology of FLHS, but the exact cause of FLHS is still unknown. Estrogens have been implicated in the development of excess fat content of the liver and in the etiology of FLHS. This study investigated estradiol (E(2)) administration in hens and its effect on lipid metabolism. Hy-Line Brown laying hens were intramuscularly injected with E(2) on a daily basis for 3 wk. The dosages were 0, 0.5, and 1.0 mg/kg of BW, with corn oil injections used as a control. Egg production and quality were measured among the groups, with no significant difference seen in egg production. Liver weights of hens treated with E(2) were greater than those of control hens, but the increase was not statistically significant. Serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase activities and E(2) plasma concentrations increased in a dose-dependent manner, with plasma concentration of E(2) increasing from 6,900 to 19,000 pg/mL. No significant differences in free cholesterol or phospholipids were observed, but there was a significant increase in hepatic triacylglycerol levels. Injection with E(2) showed an increased expression of mRNA for peroxisome proliferator-activated receptor γ (23-fold), but not for peroxisome proliferator-activated receptor α. A statistically significant increase was seen for fatty acid synthase, apolipoprotein B, and adenosine triphosphate citrate lyase, but not for acetyl coenzyme A carboxylase, apolipoprotein VLDL-II, microsomal triglyceride transport protein, or malic enzyme. For proteins involved in the oxidation of E(2), only cytochrome P450 3A37 showed a statistically significant increase. The present results suggest that E(2) upregulates the synthesis of fatty acids and triacylglycerols and the accumulation of hepatic lipids by increasing mRNA expression related to lipid metabolism, and that excess E(2) in the blood leads to activation of E(2) catabolic metabolism (cytochrome P450 3A37)-related mRNA expression.

Adult tissues contain stem cells that can transdifferentiate into other cell lineages besides forming differentiated cells of their own tissue of origin. However, human adult skin-derived stem cells have a very low efficiency. Here we established a novel culture system involving bone morphogenetic protein-4 and a floating culture system with sphere-producing medium that can enrich adult stem-cell populations in vitro. Adult stem cells were isolated from useless human scar tissue. Like mesenchymal stem cells, cultured human scar tissue-derived stem cells (hSTSCs) altered their morphology and significantly increased the number of Nestin-positive cells in proportion to the alkaline phosphatase-positive cell ratio. Moreover, the expression of the pluripotency regulator Oct-4 and its target transcripts, Sox-2, c-kit, and Rex-1, was also stimulated by this culture system. Differentiation of neurogenic progenitor cells using basic fibroblast growth factor and Neurogen 2 was successfully performed in vitro more rapidly than previous reports. Neuronal differentiation results showed that our hSTSCs expressed marker of neurogenic genes, such as glial fibrillary acid protein, neural cell adhesion molecules, neuron filament-M, and microtubule-associated protein 2. These results suggest that bone morphogenetic protein-4 and the floating culture system with sphere-producing medium induced significant proliferation of hSTSCs and mediated reprogramming of the cells from adult somatic tissue into precursor state to some degree. It is thought that this new culture system might be a simple, effective, and easily manageable process for regenerative tissue repair and autotransplantation.