Search

BACKGROUND: Intestinal organoids have evolved as potential molecular tools that could be used to study host-microbiome interactions, nutrient uptake, and drug screening. Gut epithelial barrier functions play a crucial role in health and diseases, especially in autoimmune diseases, such as inflammatory bowel diseases (IBDs), because they disrupt the epithelial mucosa and impair barrier function. METHODS: In this study, we generated an in vitro IBD model based on dextran sodium sulfate (DSS) and intestinal organoids that could potentially be used to assess barrier integrity. Intestinal organoids were long-term cultivated and characterized with several specific markers, and the key functionality of paracellular permeability was determined using FITC-dextran 4 kDa. Intestinal organoids that had been treated with 2 µM DSS for 3 h were developed and the intestinal epithelial barrier function was sequentially evaluated. RESULTS: The results indicated that the paracellular permeability represented epithelial characteristics and their barrier function had declined when they were exposed to FITC-dextran 4 kDa after DSS treatment. In addition, we analyzed the endogenous mRNA expression of pro-inflammatory cytokines and their downstream effector genes. The results demonstrated that the inflammatory cytokines genes significantly increased in inflamed organoids compared to the control, leading to epithelial barrier damage and dysfunction. CONCLUSION: The collective results showed that in vitro 3D organoids mimic in vivo tissue topology and functionality with minor limitations, and hence are helpful for testing disease models. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13770-020-00278-0) contains supplementary material, which is available to authorized users.

Background The liver is one of the vital organs involved in detoxification and metabolism. The sex-based differences between the functionality of male and female liver have been previously reported, i.e., male’s liver are good in alcohol clearance and lipid metabolism, while female’s liver are better in cholesterol metabolism. To date, studies on novel drug toxicity have not considered the sex-specific dimorphic nature of the liver. However, the use of hepatocyte-like cells to treat liver diseases has increased recently. Methods Mouse embryos were isolated from a pregnant female C57BL/6J mouse where mouse embryonic fibroblasts (MEFs) were isolated from back skin tissue of each embryo. MEFs were transduced with human transcription factors hHnf1α, hHnf4α, and hFoxa3 using the lentiviral system. The transduced MEFs were further treated with hepatocyte-conditioned media followed by its analysis through RT-qPCR, immunofluorescence, functional assays, and finally whole-transcriptome RNA sequencing analysis. For in vivo investigation, the mouse hepatocyte-like cells (miHep) were transplanted into CCl4-induced acute liver mouse model. Results In this study, we evaluated the sex-specific effect of miHep induced from male- and female-specific mouse embryonic fibroblasts (MEFs). We observed miHeps with a polygonal cytoplasm and bipolar nucleus and found that male miHeps showed higher mHnf4a, albumin secretion, and polyploidization than female miHeps. Transcriptomes from miHeps were similar to those from the liver, especially for Hnf4a of male miHeps. Male Cyps were normalized to those from females, which revealed Cyp expression differences between liver and miHeps. In both liver and miHeps, Cyp 4a12a and Cyp 4b13a/2b9 predominated in males and females, respectively. After grafting of miHeps, AST/ALT decreased, regardless of mouse sex. Conclusion In conclusion, activation of endogenic Hnf4a is important for generation of successful sex-specific miHeps; furthermore, the male-derived miHep exhibits comparatively enhanced hepatic features than those of female miHep.

Probiotics have been defined as live microorganisms that are administered in an appropriate amount to provide health benefits to the host animal. In this study, we investigated the effect of L. salivarius DJ-sa-01 secreting the 3D8 single-chain variable fragment (3D8 scFv) on the growth performance, cytokine secretion, and intestinal microbial flora of chickens. The experiment was divided into the control group and L. salivarius expressing 3D8 scFv experimental group. Chicken was fed 109 colony-forming units (CFUs) of wild-type (WT) L. salivarius or 3D8 scFv-secreting L. salivarius daily for 35 days. The administration of L. salivarius expressing 3D8 scFv significantly improved the body weight of chickens compared with the administration of WT L. salivarius. A 16S ribosomal RNA metagenomic analysis showed that Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the dominant phyla in both experimental groups. At the genus level, Lactobacillus was more abundant (22.82%) in the L. salivarius/3D8 group compared with the WT L. salivarius group. The serum levels of cytokines, such as IL-8, TNF-α, IL-1β, IFN-γ, IL-4, and IGF1, were significantly reduced in the L. salivarius/3D8-treated chickens. In summary, our results suggest that L. salivarius expressing 3D8 scFv could be considered a feed additive for improving the growth performance, immune function, and disease resistance of poultry.

The aim of this study was performed to evaluate the effects of ice-binding protein from the arctic yeast Leucosporidium (LeIBP) supplementation on cryopreservation of boar sperm. The collected semen was diluted (1.5×108/ml) in lactose egg yolk (LEY) and cooled at 5°C for 3 h. The cooled semen was then diluted (1×108/ml) in LeIBP containing LEY with 9% glycerol and maintained at 5°C for 30 min. The semen was divided into six experimental groups (control, 0.001, 0.005, 0.01, 0.05 and 0.1 mg/ml of LeIBP). The straws were kept on above the liquid nitrogen (LN2) vapors for 20 minutes and then plunged into LN2. After thawing, computer-assisted sperm analysis was used for sperm motility and flow cytometry was performed to assess the viability, acrosome integrity (FITC-PSA/PI), ROS (DCF/PI), lipid peroxidation (BODIPY C11/PI) and apoptosis (Annexin V/PI), respectively. No significant responses were observed for sperm motility. However, sperm viability was significantly increased on 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). In addition, acrosome integrity was significantly increases LeIBP groups (P < 0.05) and both ROS and lipid peroxidation level were lower in all LeIBP groups than those of control (P < 0.05). On the other hand, a significant higher apoptosis rate was observed in 0.05 and 0.1 mg/ml of LeIBP groups compared to control (P < 0.05). It can be assumed that a supplementation of LeIBP in boar sperm freezing extender is an effective method to increase the sperm qualities after cryopreservation.

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig (GalT-MCP/-MCP). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain GalT-MCP/-MCP/CD39/CD73 pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from GalT-MCP/-MCP pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate GalT-MCP/-MCP/CD39/CD73 pig expressing CD39 and CD73 at endothelial cells.

The identification of biomarkers of a living tissues is essentially required to understand specific functions of the cells. In previous study, we reported IGFBP 3 as one of the putative biomarkers, by showing specific expression at porcine spermatogonial stem cells (SSCs) of early stage of porcine testis. In this study, we analyzed the expression of seven members of IGFBP family (IGFBPs) in SSCs and histological expression pattern of pregnancy-associated plasma protein-A (PAPP-A), which plays a role on the growth promoting enzyme by cleavage of IGFBPs in testis of 5 days old pig. RT-PCR analysis showed that IGFBP 1, 2, 3, 4, and 6 were expressed at high level specifically in porcine SSCs compared with whole testis. We performed immunohisotochemical staining of testis sections with PAPP-A and protein gene product 9.5 (PGP9.5) which are the known biomarkers for SSCs. We were not able to find co-expression of PAPP-A and PGP9.5; PAPP-A was expressed only in Sertoli cells and PGP9.5 expression was confirmed in spermatogonium. Additionally, we were able to confirm the GATA4 expression in Sertoli and Leydig cells as a regulator of Sertoli cell function was not detected PGP9.5 expressing cells, indicating indirect evidence of that cytolocalization of PAPP-A expression is limited in Sertoli cells. These results suggested that the PAPP-A expressed in Sertoli cells may play role on regulation of development and differentiation of testicular cells through the IGF axis in neonatal porcine testis.

Previously, Escherichia coli harboring the codon-optimized 3D8scFv gene (E. coli 3D8scFv) was developed as a feed additive for use in preventing norovirus infection. Here, we evaluated whether the 3D8scFv gene affects the colonization of E coli when E. coli 3D8scFv passes through the mouse gastrointestinal tract. To determine the colonization ability of E. coli 3D8scFv, E. coli cells with or without the 3D8scFv gene were fed to mice. Total DNA was extracted from the animals’ stools, stomach, small intestine and colon. All samples were amplified using 3D8scFv gene-specific primer sets. E. coli 3D8scFv begins to be excreted 1 h after feeding and that all E. coli 3D8scFv cells were excreted between 12 and 24 h after the last feeding of the cells. The previously measured gastrointestinal transit time of the mice was between 8 h and 22 h. The results of this study therefore show that E. coli 3D8scFv cannot colonize the gastrointestinal tracts of mice. In addition, if the purified 3D8 scFv protein is used as a feed additive, any associated E. coli 3D8scFv bacteria will not colonize the gastrointestinal tracts of the livestock. Thus, this feed additive meets the safety assessment criteria for the commercial use of bacteria., Highlights • It is evaluated whether E. coli 3D8scFv colonizes in the gastrointestinal tracts of mice. • Orally ingested E. coli 3D8scFv is excreted from mice without colonization of the gastrointestinal tract. • Purified 3D8 scFv is suitable for a feed additive according to the concept of substantial equivalence.

Transplantation is considered to be a very useful approach to improve human welfare and to prolong life-span. Heterologous organ transplantation using pig organs which are similar to human beings and easy to make mass-production has known as one of the alternatives. To ensure potential usage of the pig organ for transplantation application, it is essentially required to generate transgenic pig modifying immuno-related genes. Previously, we reported production of heterozygous α 1,3-galactosyltransferase (GalT) knock-out and human membrane cofactor protein (MCP) expressing pig (GalT-MCP/+), which is enforced for suppression of hyperacute and acute immunological rejection. In this study, we reported generation of homozygous pig (GalT-MCP/-MCP) by crossbreeding GalT-MCP/+ pigs. Two female founders gave birth to six of GalT-MCP/-MCP, and seven GalT-MCP/+ pigs. We performed quantitative real-time PCR, western blot, and flow cytometry analyses to confirm GalT and MCP expression. We showed that fibroblasts of the GalT-MCP/-MCP pig do not express GalT and its product Gal antigen, while efficiently express MCP. We also showed no expression of GalT, otherwise expression of MCP at heart, kidney, liver and pancreas of transgenic pig. Taken together, we suggest that the GalT-MCP/-MCP pig is a useful candidate to apply xenotransplantation study.

Primary hepatocytes (PHs) are considered the ‘gold standard’ in drug screening owing to their ability to express many drug-metabolizing enzymes and transporters. Culturing hepatocytes and maintaining their fate in vitro is a major issue since last decade. The main problem with in vitro hepatocytes culture is that they rapidly lose their hepatic morphology and liver-specific functions in culture. Herein, we isolated rat PHs, and cultured them in monolayers (2-D) and spheroids (3-D). The 2-D-cultured PHs exhibited elongated morphology, whereas the 3-D-cultured PHs exhibited spheroid morphology with gradual diameter decrease until 7 days. After 7 days of in vitro culture, PHs were analyzed for the expression of hepatic (Alb, Tf, and Afp) and apoptotic markers (Bax and Bcl2), and co-expression of CYP3A1 and Abumin after 2 and 7 days. Furthermore, in both cultures, PHs were induced with 3-methylcholanthrene (3-MC, Cyp1a-specific inducer) and dexamethasone (Cyp3a-specific inducer) for 48 and 72 h, respe...

Pigs have been extensively used as mediators of xenotransplantation research. Specifically, the Massachusetts General Hospital (MGH) miniature pig was developed to fix major histocompatibility antigens for use in xenotransplantation studies. We generated transgenic pigs for xenotransplantation using MGH pigs. However, it has not been studied yet whether these pigs show similarity of reproductive physiological characteristics to wild types of MGH miniature pig. In this study we analyzed the estrous cycles and pregnancy characteristics of wild type (WT) and transgenic MGH miniature pigs, which were α1,3-galactosyltransferase (GalT) heterozygous and homozygous knock-out, and membrane cofactor protein (MCP) inserted in its locus, GalT-MCP/+ and GalT-MCP/-MCP pigs. Estrous cycles of WT, GalT-MCP/+ and GalT-MCP/-MCP pigs were 20.9±0.74, 20.1±1.26, and 17.3±0.87 days, respectively, and periods of estrous were 3.2±0.10, 3.1±0.12, and 3.1±0.11 days. The periods of gestation of WT, GalT-MCP/+ and GalT-MCP/-MCP pigs were 114.2±0.37, 113.3±0.67, and 115.4±0.51 days, respectively. Litter sizes of WT, GalT-MCP/+ and GalT-MCP/-MCP pigs were 4.8±0.35, 4.8±1.11 and 3.0±0.32 respectively. There were no significant differences on estrous cycle, periods of estrous and gestation, and litter size among WT, GalT-MCP/+ and GalT-MCP/-MCP pigs, meaning that GalT knock-out and additional expression MCP of the MGH miniature pig did not effect on reproduction traits. These results provide relevant information to establish breeding system for MGH transgenic pig, and for propagation of GalT-MCP/-MCP pig to supply for xenotransplantation research.

One of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-human-primate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation. The transgenic cells were constructed an hCD73 expression vector under control porcine Icam2 promoter (pIcam2-hCD73) and established donor cell lines expressing hCD73. The numbers of transferred reconstructed embryos were 127 ± 18.9. The pregnancy and delivery rate of surrogates were 8/18 (44%) and 3/18 (16%). The total number of delivered cloned pigs were 10 (2 alive, 7 mummy, and 1 died after birth). Among them, three live hCD73-pigs were successfully delivered by Caesarean section, but one was dead after birth. The two hCD73 TG cloned pigs had normal reproductive ability. They mated with wild type (WT) MGH (Massachusetts General Hospital) female sows and produced totally 16 piglets. Among them, 5 piglets were identified as hCD73 TG pigs. In conclusion, we successfully generated the hCD73 transgenic cloned pigs and produced their litters by natural mating. It can be possible to use a mate for the production of multiple transgenic pigs such as α-1,3-galactosyltransferase knock-out /hCD46 for xenotransplantation.

It is very difficult to get the information about semen quality analysis in transgenic pigs because of limited numbers and research facilities. Therefore, in the present study, we analyzed the semen quality of transgenic boars generated for xenotransplantation research. Briefly, the semen samples were collected from 5 homozygous α1,3-Galactosyltransferase knock-out (GalT-/-) transgenic boars and immediately transported to the laboratory. These semen samples were decupled with DPBS and conducted to analyze semen parameters by a computer-assisted semen analysis (CASA) system. The boar semen were examined all 12 parameters such as total motility (TM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), and hyperactivated (HYP), etc. In results, among the 5 GalT-/- boars, three boars (#134, 144, and 170) showed normal range of semen parameters, but #199 and 171 boars showed abnormal ranges of semen parameters according to standard ranges of semen parameters. Unfortunately, #171 boar showed azoospermia symptom with rare sperm counts in the original semen. Conclusively, assessment of semen parameters by CASA system is useful to pre-screening of reproductively healthy boar prior to natural mating and artificial insemination for multiplication and breeding.

Unlike mouse results, cloning efficiency of nuclear transfer from porcine induced pluripotent stem cells (piPSCs) is very low. The present study was performed to investigate the effect of cell cycle inhibitors on the cell cycle synchronization of piPSCs. piPSCs were generated using combination of six human transcriptional factors under stem cell culture condition. To examine the efficiency of cell cycle synchronization, piPSCs were cultured on a matrigel coated plate with stem cell media and they were treated with staurosporine (STA, 20 nM), daidzein (DAI, 100 μM), roscovitine (ROSC, 10 μM), or olomoucine (OLO, 200 μM) for 12 h. Flow Cytometry (FACs) data showed that piPSCs in control were in G1 (37.5±0.2%), S (34.0±0.6%) and G2/M (28.5±0.4%). The proportion of cells at G1 in DAI group was significantly higher than that in control, while STA, ROSC and OLO treatments could not block the cell cycle of piPSCs. Both of viability and apoptosis were affected by STA and ROSC treatment, but there were no significantly differences between control and DAI groups. Real-Time qPCR and FACs results revealed that DAI treatment did not affect the expression of pluripotent gene, Oct4. In case of OLO, it did not affect both of viability and apoptosis, but Oct4 expression was significantly decreased. Our results suggest that DAI could be used for synchronizing piPSCs at G1 stage and has any deleterious effect on survival and pluripotency sustaining of piPSCs.

In this study, we attempted to upgrade GT(−MCP/−MCP) pig genetically to express MCP at a higher level and additionally thrombomodulin (TBM), which have respective roles as a complement regulatory protein and a coagulation inhibitor. We constructed a dicistronic cassette consisting of codon-optimized MCP (mMCP) and TBM (m-pI2), designed for ubiquitous expression of MCP and endothelium specific expression of TBM. The cassette was confirmed to allow extremely increased MCP expression compared with non-modified MCP, and an endothelial-specific TBM expression. We thus transfected m-pI2 into ear-skin fibroblasts isolated from a GT(−MCP/−MCP) pig. By twice selection using magnetically activated cell sorting (MACS), and single-cell culture, we were able to obtain clones over 90% expressing MCP. The cells of a clone were provided as a donor for nuclear transfer resulting in the generation of a GT(−MCP/−MCP)/mMCP/TBM pig, which was confirmed to be carrying cells expressing MCP and functioning as an inhibitor against the cytotoxic effect of normal monkey serum, comparable with donor cells. Collectively, these results demonstrated an effective approach for upgrading transgenic pig, and we assumed that upgraded pig would increase graft survival. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-2091-z) contains supplementary material, which is available to authorized users.

The present study aimed to investigate the characteristics of mPEA15 expressing transgenic pig (TG pig) as a potential model for diabetes. Expression analysis confirmed the ubiquitous expression of mPEA15 in TG pigs at F4. Oral glucose tolerance test results showed that restoration of normal glucose levels was significantly delayed in the TG pigs when compared with that in the wild-type pigs (WT pigs). Primary skeletal muscle cells isolated from TG pigs demonstrated reduced glucose uptake and reduced GLUT4 translocation to the plasma membrane in response to insulin treatment. Combined, these results suggest that mPEA15 expressing pigs has a glucose intolerance and insulin resistance which are known to mediate the pathophysiology of type 2 diabetes mellitus. Thus, mPEA15 transgenic pigs would serve as a promising model for diabetes translational research. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-019-2021-0) contains supplementary material, which is available to authorized users.

The present study was aimed to investigate the effects of 3D8 scFv-secreting Probiotic Lactobacillus reuteri (L. reuteri) on growth performance, inflammatory responses, and intestinal microbial flora in chickens. To this end, a total of 14 healthy wild-type chickens were divided into two experimental groups. Each group was orally administrated with a daily dose of 109 colony-forming units (CFU) of 3D8 scFv-producing L. reuteri or wild-type (WT) for 35 days. Administration of L. reuteri/3D8 scFv significantly improved the body weight of chickens when compared to L. reuteri/WT group. The bacterial taxonomic composition of the fecal microbiota was determined by pyrosequencing of 16S rRNA gene amplicons. Firmicutes, Actinobacteria, and Proteobacteria were dominant phyla in two experimental groups. However, in 3D8 L. reuteri treatment groups at genus level, the Lactobacillus was highly abundant, being represented by 18.12%. In addition, serum levels of primary cytokines such as IL-6, IL-8, TNF-α, IFN-γ, IL-4, and IGF1 were markedly reduced in the probiotic L. reuteri 3D8 group. In summary, our results indicate that the administration of L. reuteri expressing 3D8 scFv has a modulatory effect on inflammatory responses, improves weight gain while not affecting the common microbial composition of the chicken intestine.

The present study was aimed to investigate the effects of 3D8 scFv-secreting Probiotic Lactobacillus reuteri (L. reuteri) on growth performance, inflammatory responses, and intestinal microbial flora in chickens. To this end, a total of 14 healthy wild-type chickens were divided into two experimental groups. Each group was orally administrated with a daily dose of 10(9) colony-forming units (CFU) of 3D8 scFv-producing L. reuteri or wild-type (WT) for 35 days. Administration of L. reuteri/3D8 scFv significantly improved the body weight of chickens when compared to L. reuteri/WT group. The bacterial taxonomic composition of the fecal microbiota was determined by pyrosequencing of 16S rRNA gene amplicons. Firmicutes, Actinobacteria, and Proteobacteria were dominant phyla in two experimental groups. However, in 3D8 L. reuteri treatment groups at genus level, the Lactobacillus was highly abundant, being represented by 18.12%. In addition, serum levels of primary cytokines such as IL-6, IL-8, TNF-α, IFN-γ, IL-4, and IGF1 were markedly reduced in the probiotic L. reuteri 3D8 group. In summary, our results indicate that the administration of L. reuteri expressing 3D8 scFv has a modulatory effect on inflammatory responses, improves weight gain while not affecting the common microbial composition of the chicken intestine.

Nucleotide metabolism in endothelium is variable between different species. Recent studies demonstrated that this variability could contribute coagulation dysfunction, even though organs of the alpha1,3-galactosyltransferase gene knockout pig were transplanted into the primate. CD73 (ecto-5’-nucelotidase) is an enzyme at cell surface catalyzing the hydrolysis of adenosine triphosphate to adenosine, which plays role on a substance for anti-inflammatory and anti-coagulant. Thus, overexpression of CD73 in endothelial cells of the pig is considered as an approach to reduce coagulopathy. In this study, we constructed a human CD73 expression vector under control of porcine Icam2 promoter (pIcam2-hCD73), which is expressed specifically at endothelial cells, and of CMV promoter as a control (CMV-CD73). First, we transfected the CMV-CD73 vector into HEK293 cells, and then confirmed CD73 expression at cell surface by flow cytometry analysis. Next, we transfected the pIcma2-CD73 and CMV-CD73 vectors into primary porcine fibroblasts and endothelial cells. Consequence was that the pIcma2-CD73 vector was expressed only at the porcine endothelial cells, meaning that the pIcam2 promoter lead to endothelial cell-specific expression of CD73in vitro. Finally, we nucleofected the pIcam2-hCD73 vector into passage 3 fibroblasts, and enforced hygromycin selection of 400mg/ml. We were able to obtain forty three colonies harboring pIcam2-CD73 to provide donor cells for transgenic cloned porcine production.

There is a growing interest in the application of primary hepatocytes for treatment of liver diseases in humans and for drug development. Several studies have focused on long-term survival and di-differentiation blocking of primary hepatocytes in an in vitro culture system. Therefore, the present study also aimed to optimize an in vitro culture system using primary rat hepatocytes. Primary rat hepatocytes from 6-week-old male Crl:CD rats were isolated using a modified two-step collagenase perfusion. Healthy 3.5 × 106 primary rat hepatocytes were seeded into a 2 dimensional (2D) culture in a 25T culture flask coated with collagen type I or into a 3D culture in a 125-ml spinner flask for 7 days. Production of plasma protein (ALB and TF), apoptosis (BAX and BCL2), and CYP (CYP3A1) related genes were compared between the 2D and 3D culture systems. The 3D culture system had an advantage over the 2D system because of the relatively high expression of ALB and low expression of BAX in the 3D system. However, the level of CYP3A1 did not improve in the 3D culture with and without the presence of a dexamethasone inducer. Therefore, 3D culture has an advantage for albumin production and primary rat hepatocyte survivability, but a low expression of CYP3A1 indicated that primary rat hepatocytes require a high?density culture for stress reduction by continuous flow.

Although piglets have been delivered by embryo transfer (ET) with in vitro produced (IVP) embryos and blastocysts, a success rate has still remained lower level. Unlike mouse, human, and bovine, it is difficult to a production of piglets by in vitro fertilization (IVF) because of an inappropriate in vitro culture (IVC) system in pig. Therefore, the present study was conducted to investigate whether minimized exposure time in IVC can improve the pregnancy and delivery rates of piglets. Immediately after IVM, the oocytes were denuded and co-incubated with freshly ejaculated boar semen for 3.5 to 4 hours at 38.5 °C under 5% CO2 in air. To avoid long-term exposure to in vitro state, we emitted IVC step after IVF. After that the presumptive zygotes were transferred into both oviducts of the surrogate on the same day or 1 day after the onset of estrus. Pregnancy was diagnosed on day 28 after ET and then was checked regularly every month by ultrasound examination. The 3 out of 4 surrogates were determined as pregnant (75%) and a total of 5 piglets (2 females and 3 males) were delivered at 118.3 ± 2.5 days of pregnancy period. In conclusion, a short-term exposure time may be an important factor in the production of IVP-derived piglets. It can be apply to the in vitro production system of transgenic pig by IVF, cloning, and pronuclear microinjection methods.

Pigs have been used as a good research model for xenotransplantation. Several groups have generated porcine-induced pluripotent stem cells (piPSCs) from differentiated somatic cells. Transgenic pigs with the alpha1,3-galactosyltransferase gene-knockout (GalT-KO) could successfully govern hyper acute rejection of organ transplants into primates. Thus, GalT-KO piPSCs could be a powerful cell resource for agricultural and biomedical applications. This study was performed to generate iPSCs from GalT-KO pigs and characterize their properties. We successfully generated a GalT-KO iPSC from a genetically modified pig using double alpha1,3-galactosyltransferase knockout alterations. Similar to mouse embryonic stem cells, the GalT-KO piPSCs were positive for classical pluripotency markers: POU5F1, NANOG, SOX2 and SSEA1, and were negative for: SSEA3, TRA-1-60 and TRA-1-81. Furthermore, these cells could form an embryoid body that differentiated into three germ layers in vitro, and were highly proliferative u...

Background: Corneal allotransplantation is a well-known technique to treat corneal blindness. However, the problem of organ scarcity in transplantology has become profound. One promising alternative could be xenograft using pig as an organ donor. Full thickness corneal xenotransplantation needs total immunosuppression. This study is an investigation of the efficacy of α1,3-ga lactosyltransferase gene-knockout (GTKO) transgenic pig-to-nonhuman primate lamellar corneal transplantation with minimal immunosuppression. Methods: We conducted 10 lamellae corneal xenotransplantation between 2016 and 2019. Clinically acceptable graft size (diam eter 7.5 mm, thickness 500 um). The dexamethasone subconjunctival injection (1.5 mg/0.3 mL) was administered for minimal immunosuppression immediately after surgery and eye drops of 0.5% levofloxacin and 1% prednisolone acetate were applied four times a day for 1 week, gradually tapered. No eye drops were added after two months. Three cases are alive without graft rejection. We examined the remaining seven cases for the histopathological features and Immunologic profiles. Results: As a result, three of the ten xenografts survival is significantly longer 1,239, 589, and 316 days. Corneal opacity result ed in graft failure, and terminated in seven cases. Rejected grafts showed extensive polymorphic cellular infiltration, different degrees of epithelial layer irregular attenuation, stromal neovascularization, and inflammatory cell infiltrations including lym phocytes, plasma cells, eosinophils, neutrophils. Stromal irregularity, fibrosis and edema are observed in two of seven cases resulting in a single case of sub epithelial detachment. Immunologic profiles of the recipients with rejected grafts shows minimal increase in anti-Gal antibody IgG and IgM but increase in anti-Gal IgG is seen in one case. Four cases have different systemic in flammatory conditions with regards to plasma C3a and D-dimer levels. The anterior stromal surface of the graft showed epitheli al nests and fibrous proliferation. Conclusions: The GTKO transgenic pig to NHP lamella xeno corneal transplantation could be a promising substitute for human corneal allograft. Lamella xeno corneal transplantation may be a feasible option with minimal immunosuppression.

The anti-inflammatory effects of oridonin (Ordn) have been well established in previous studies. However, the apoptotic effects of Ordn on oral cancer cells have not yet been evaluated, at least to the best of our knowledge. The aim of this study was to examine the apoptotic activity of Ordn in oral squamous cell carcinoma cells and to eluciudate the underlying mechanisms. For this purpose, we employed experimental techniques, such as MTT assay, DAPI staining, soft agar assay, flow cytometry and western blot analysis. Our results revealed that Ordn suppressed oral cancer cell proliferation and soft agar colony formation, while it induced reactive oxygen species (ROS)-dependent apoptosis in a dose or time-dependent manner. The generation of ROS was detected in HN22 and HSC4 cells treated with Ordn and the use of the free radical scavenger, N-acetyl-L-cysteine, almost blocked Ordn-induced apoptosis. The phosphorylation of JNK and p38 mitogen-activated protein kinase (MAPK) was manifested in the Ordn-treated cells. Furthermore, Ordn induced the apoptosis of oral cancer cells through the mitochondrial-dependent pathway, involving the loss of mitochondrial membrane potential, the release of cytochrome c, the induction of poly(ADP-Ribose) polymerase (PARP) cleavage, alterations in the ratios of apoptotic proteins and the activation of the caspase cascade. Taken together, these findings indicate that Ordn induces the apoptosis of oral cancer cells probably via ROS-mediated JNK/p38 MAPK and mitochondrial pathways; thus, Ordn may have potential for use in the treatment of oral cancer.

Background Licochalconce (LC) H is an artificial compound in the course of synthesizing LCC in 2013. So far, few studies on the effects of LCH have been found in the literature. Despite progress in treatment modalities for oral cancer, the cure from cancer has still limitations. Purpose The effects of LCH were investigated on human oral squamous cell carcinoma (OSCC) cells to elucidate its mechanisms. Study Design We explored the mechanism of action of LCH by which it could have effects on JAK2/STAT3 signaling pathway. Methods To confirm LCH anti-cancer effect, analyzed were MTT assay, DAPI staining, soft agar, kinase assay, molecular docking simulation, flow cytometry and Western blotting analysis. Results According to docking and molecular dynamics simulations, the predicted pose of the complex LCH and JAK2 seems reasonable and LCH is strongly bound to active JAK2 with opened activation loop. The LCH inhibitor is surrounded by specific ATP-binding pocket in which it is stabilized by forming hydrogen bonds and hydrophobic interactions. It is shown that LCH plays as a competitive inhibitor in an active state of JAK2. LCH caused a dose-dependent decrease in phosphorylation of JAK2 and STAT3. More interestingly, LCH suppressed JAK2 kinase activity in vitro by its direct binding to the JAK2. LCH significantly inhibited the JAK2/STAT3 signaling pathway, causing the down-regulation of target genes such as Bcl-2, survivin, cyclin D1, p21 and p27. In addition, LCH inhibited cell proliferation and colony formation of OSCC cells in a dose- and time-dependent manner, as well as induction of cell apoptosis through extrinsic and intrinsic pathway. The induction of apoptosis in OSCC cells by LCH was evident in the increased production of ROS, loss of mitochondrial membrane potential, release of cyto c, variation of apoptotic proteins and activation of caspase cascade. Conclusion LCH not only induces apoptosis in OSCC cells through the JAK/STAT3 signaling pathway but also inhibits cell growth. It is proposed that LCH has a promising use for the chemotherapeutic agent of oral cancer.

Diabetes mellitus, the most common metabolic disorder, is divided into two types: type 1 and type 2. The essential treatment of type 1 diabetes, caused by immune-mediated destruction of β-cells, is transplantation of the pancreas; however, this treatment is limited by issues such as the lack of donors for islet transplantation and immune rejection. As an alternative approach, stem cell therapy has been used as a new tool. The present study revealed that bone marrowderived mesenchymal stromal cells (BM-MSCs) could be transdifferentiated into pancreatic cells by the insertion of a key gene for embryonic development of the pancreas, the pancreatic and duodenal homeobox factor 1 (PDX1). To avoid immune rejection associated with xenotransplantation and to develop a new cell-based treatment, BM-MSCs from α-1,3-galactosyltransferase knockout (GalT KO) pigs were used as the source of the cells. Transfection of the EGFP-hPDX1 gene into GalT KO pig-derived BM-MSCs was performed by electroporation. Cells were evaluated for hPDX1 expression by immunofluorescence and RT-PCR. Transdifferentiation into pancreatic cells was confirmed by morphological transformation, immunofluorescence, and endogenous pPDX1 gene expression. At 3~4 weeks after transduction, cell morphology changed from spindle-like shape to round shape, similar to that observed in cuboidal epithelium expressing EGFP. Results of RT-PCR confirmed the expression of both exogenous hPDX1 and endogenous pPDX1. Therefore, GalT KO pig-derived BM-MSCs transdifferentiated into pancreatic cells by transfection of hPDX1. The present results are indicative of the therapeutic potential of PDX1-expressing GalT KO pig-derived BM-MSCs in β-cell replacement. This potential needs to be explored further by using in vivo studies to confirm these findings.

Cloned pigs produced by somatic cell nuclear transfer (SCNT) are important as a potential alternative source of organs. Although SCNT has created new possibilities for targeted gene modification, the successful cloning of pigs is rare. Here, we successfully conducted serial SCNT for three generations. We determined that the piglet genome was inherited from donor cell nuclei using microsatellite analysis of each generation. The methylation of differentially methylated regions (DMRs) in H19 was gradually reduced over the three generations of serial SCNT. By contrast, methylation of the insulin-like growth factor 2 (Igf2) DMR increased in the F1 generation, compared to the F0, and remained at the higher level in the F2 and F3 generations. The methylation patterns of housekeeping genes such as GAPDH and β-actin were unchanged in the serially cloned pigs. In addition, expression levels of H19 and Igf2 were variable for each generation of serial SCNT piglets, but there was no clear relationship between the meth...

Most studies of xenografts have been carried out with complex immunosuppressive regimens to prevent immune rejection; however, such treatments may be fatal owing to unknown causes. Here, we performed immune molecular profiling following anti-CD154 monoclonal antibody (mAb) treatment in heterotopic abdominal cardiac xenografts from α-1,3-galactosyltransferase-knockout pigs into cynomolgus monkeys to elucidate the mechanisms mediating the undesirable fatal side effects of immunosuppressive agents. Blood samples were collected from healthy monkeys as control and then at 2 days after xenograft transplantation and just before humane euthanasia; 94 genes related to the immune system were analyzed. The basic immunosuppressive regimen included cobra venom factor, anti-thymocyte globulin, and rituximab, with and without anti-CD154 mAbs. The maintenance therapy was followed with tacrolimus, MMF, and methylprednisolone. The number of upregulated genes was initially decreased on Day 2 (-/+ anti-CD154 mAb, 22/13) and then increased before euthanasia in recipients treated with anti-CD154 mAbs (-/+ anti-CD154 mAb, 30/37). The number of downregulated genes was not affected by anti-CD154 mAb treatment. Additionally, the number of upregulated genes increased over time for both groups. Interestingly, treatment with anti-CD154 mAbs upregulated coagulation inducers (CCL2/IL6) before euthanasia. In conclusion, immunosuppressive regimens used for cardiac xenografting affected upregulation of 6 inflammation genes (CXCL10, MPO, MYD88, NLRP3, TNFα, and TLR1) and downregulation of 8 genes (CCR4, CCR6, CD40, CXCR3, FOXP3, GATA3, STAT4, and TBX21).

To compensate for the critical shortage of human organs for allotransplantation, xenotransplantation studies using genetically modified pigs are being performed in Korea. Two types of pigs that are used are α1,3-galactosyltransferase gene knockout (GalT KO) pigs and GalT KO+hCD46 (human complement regulatory protein) pigs. The present study measured the gestation time, birth weight, daily growth rate, and heart weight of both kinds of transgenic minipigs. The gestation period for both types of pigs was 117∼119 days. There was no difference in the body weight of GalT KO (—/+) and GalT KO (—/—) piglets, but GalT KO+hCD46 (—hCD46+/+) pigs were significantly heavier at birth than were GalT KO+hCD46 (—hCD46+/—hCD46+) pigs. During the first 10 weeks of life, the daily weight gain of GalT KO+hCD46 (—hCD46+/—CD46+) piglets, which are considered the optimal type for xenotransplantation, was 0.19 kg. The weight of hearts from GalT KO piglets up to two months of age was affected more by body weight than by age. Transgenic pigs showed no differences in gestation period or reproductive ability compared with normal pigs. These results comprise basic data that may be used in xenotransplantation studies and transgenic animal production in Korea.

BACKGROUND AND PURPOSE Reactive oxygen species (ROS) are potent regulators of stem cell behaviour; however, their physiological significance as regards MMP-mediated regulation of the motility of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) has not been characterized. In the present study, we investigated the role of hydrogen peroxide (H2O2) and associated signalling pathways in promoting UCB-MSCs motility.

Endoplasmic reticulum (ER) stress suppresses osteoblast differentiation. Activating transcription factor (ATF) 3, a member of the ATF/cAMP response element-binding protein family of transcription factors, is induced by various stimuli including cytokines, hormones, DNA damage, and ER stress. However, the role of ATF3 in osteoblast differentiation has not been elucidated. Treatment with tunicamycin (TM), an ER stress inducer, increased ATF3 expression in the preosteoblast cell line, MC3T3-E1. Overexpression of ATF3 inhibited bone morphogenetic protein 2-stimulated expression and activation of alkaline phosphatase (ALP), an osteogenic marker. In addition, suppression of ALP expression by TM treatment was rescued by silencing of ATF3 using shRNA. Taken together, these data indicate that ATF3 is a novel negative regulator of osteoblast differentiation by specifically suppressing ALP gene expression in preosteoblasts.

Pigs are used as potential donor animals for xenotransplantation. However, porcine endogenous retrovirus (PERV), shown to infect both human and non-human primate (NHP) cells in vitro, presents a risk of transmission to humans in xenotransplantation. In this study, we analyzed PERV transmission in various organs after pig-to-NHP xenotransplantation. We utilized pig-to-NHP xenotransplant tissue samples obtained using two types of transgenic pigs from the National Institute of Animal Science (NIAS, Republic of Korea), and examined them for the existence of PERV genes in different organs via PCR and RT-PCR with specific primers. To determine PERV insertion into chromosomes, inverse PCR using PERV long terminal repeat (LTR) region-specific primers was conducted. The PERV gene was not detected in NHP organs in cardiac xenotransplantation but detected in NHP bladders in renal xenotransplantation. The insertion experiment confirmed that PERVs originate from porcine donor cells rather than integrated provirus in the NHP chromosome. We also demonstrate the presence of pig cells in the NHP bladder after renal xenotransplantation using specific-porcine mitochondrial DNA gene PCR. The PERV sequence was detected in the bladder of NHPs after renal xenotransplantation by porcine cell-microchimerism but did not integrate into the NHP chromosome.

Ursolic acid (UA) a bio-active ingredient found in a variety of fruits and vegetables, and it has potent antioxidant activity. However, the role of UA in mouse embryonic stem (ES) cells is poorly understood. This study investigated the functional role of UA in regulating the development of mouse ES cells under hypoxia. Hypoxia did not exert a significant effect on the undifferentiated state of mouse ES cells. However, it induced reactive oxygen species (ROS) generation and increased the level of lactate dehydrogenase (LDH) production at 48 h of hypoxic exposure. Conversely, oxidative stress induced by hypoxia was significantly inhibited by UA () pretreatment. Hypoxia significantly decreased cell survival and the level of [] thymidine incorporation, both of which recovered following pretreatment of UA. In addition, UA decreased the apoptotic effect of hypoxia by attenuating caspase-3 cleavage or by recovering cellular inhibition of the apoptotic protein (cIAP)-2 and Bcl-2 expression. We further found that UA decreased senescence-associated beta-galactosidase activity. We suggest that UA is a natural antioxidant and one of the functional modulators of hypoxia-induced survival, apoptosis, proliferation, and aging in mouse ES cells.

Xenotransplantation of pig organs into primates leads to hyperacute rejection (HAR). Functional ablation of the pig α 1,3-galactosyltransferase (GalT) gene, which abrogates expression of the Gal α 1-3Gal β 1-4GlcNAc-R (Gal) antigen, which inhibits HAR. However, antigens other than Gal may induce immunological rejection by their cognate antibody responses. Ultimately, overexpression of complement regulatory proteins reduces acute humoral rejection by non-Gal antibodies when GalT is ablated. In this study, we developed a vector-based strategy for ablation of GalT function and concurrent expression of membrane cofactor protein (MCP, CD46). We constructed an MCP expression cassette (designated as MCP-IRESneo) and inserted between the left and the right homologous arms to target exon 9 of the GalT gene. Nucleofection of porcine ear skin fibroblasts using the U-023 and V-013 programs resulted in high transfection efficiency and cell survival. We identified 28 clones in which the MCP-IRESneo vector had been successfully targeted to exon 9 of the GalT gene. Two of those clones, with apparent morphologically mitotic fibroblast features were selected through long-term culture. GalT gene expression was downregulated in these 2 clones. Importantly, MCP was shown to be efficiently expressed at the cell surface and to efficiently protect cell lysis against normal human complement serum attack in vitro.

Massachusetts General Hospital miniature pigs (MGH minipigs) have been established for organ transplantation studies across the homozygous major histocompatibility complex, but cloning efficiency of MGH minipigs is extremely low. This study was designed to increase the productivity of MGH minipigs by nuclear transfer of post-thaw donor cells after 1 h co-incubation with roscovitine. The MGH minipig cells were genetically modified with GT KO (alpha1,3-galactosyltransferase knock-out) and hCD46 KI (human CD46 knock-in) and used as donor cells. The GT KO/hCD46 KI donor cells were cultured for either 3 days (control group) or 1 h after thawing with 15 μM roscovitine (experimental group) prior to the nuclear transfer. The relative percentage of the transgenic donor cells that entered into G0/G1 was 93.7 % (±2.54). This was different from the donor cells cultured for 1 h with the roscovitine-treated group (84.6 % ±4.6) (P

Delphinidin, gallic acid, betulinic acid, and ursolic acid, which are bio-active ingredients in a variety of fruits, vegetables, and herbs, have potent antioxidant activity and various biological activities. However, it is not clear whether these bio-active ingredients can significantly contribute to the protection of embryonic stem (ES) cells from hypoxia-induced apoptosis. In the present study, hypoxia-induced ES cells apoptosis with time, which were abrogated by pretreatment with all ingredients. Hypoxia-induced ROS generation was blocked by pretreatment with all ingredients in a dose-dependent manner, with the maximum ROS scavenging effect observed for delphinidin. Hypoxia increased phosphorylation of JNK and NF-κB were blocked by pretreatment of delphinidin as well as NAC. Hypoxia decreased phosphorylation of Akt(thr308) and (ser473); these decreases were reversed by pretreatment with delphinidin or NAC. However, Akt inhibition did not affect NF-κB phosphorylation. Delphinidin attenuated the hypoxia-induced increase in Bax, cleaved caspase-9, cleaved caspase-3, and decrease in Bcl-2, which were diminished by pretreatment of Akt inhibitor. Hypoxia induced Bax translocation from the cytosol to mitochondria. Furthermore, hypoxia induced mitochondria membrane potential loss and cytochrome c release in cytosol, which were blocked by delphinidin pretreatment. Hypoxia induced cleavage of procaspase-9 and procaspase-3 which were blocked by delphinidin or SP600125, but Akt inhibitor abolished the protection effect of delphinidin. Moreover, inhibition of JNK and NF-κB abolished hypoxia-induced ES cell apoptosis and inhibition of Akt attenuated delphinidin-induced blockage of apoptosis. The results indicate that delphinidin can prevent hypoxia-induced apoptosis of ES cells through the inhibition of JNK and NF-κB phosphorylation, and restoration of Akt phosphorylation.

Soon after RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease opened a new era of targeted genome editing, the CRISPR/Cas9 platform began to be extensively used to modify genes in various types of cells and organisms. However, successful CRISPR/Cas9-mediated insertion/deletion (indel) mutation remains to be demonstrated in avian cell lines. The objective of this study was to design a poultry-specific CRISPR/Cas9 system to efficiently introduce targeted deletion mutation in chromosomes of the quail muscle clone 7 (QM7) cell line using a customized quail CRISPR vector. In this study, two avian-specific promoters, quail 7SK (q7SK) promoter and CBh promoter, the hybrid form of cytomegalovirus and chicken β-actin promoters, were cloned into a CRISPR vector for the expression of guide RNA and Cas9 protein, respectively. Then, guide RNA, which was designed to target 20-base pair (bp) nucleotides in the quail melanophilin (MLPH) locus, was ligated to the modified CRISPR vector and transfected to QM7 cells. Our results showed multiple indel mutations in the quail MLPH locus in nearly half of the alleles being tested, suggesting the high efficiency of the system for targeted gene modification. The new CRISPR vector developed from this study has the potential application to generate knockout avian cell lines and knockout poultry.

Kahweol, a diterpene molecule, has antiproliferative effects on several types of human cancer cells, but whether it has apoptotic effect in non-small cell lung cancer (NSCLC) is not known. To explore this possibility, we incubated cells from two NSCLC cell lines, NCI-H358 and NCI‑H1299, with different concentrations of kahweol and used the MTS assay, DAPI staining, propidium iodide staining, Annexin V staining, immunocytochemical test, and western blot analysis to characterize this molecule and the signaling pathway underlying its effects. The kahweol-treated cells showed significantly decreased cell viability, increased nuclear condensation, and an increased number of Annexin V-positive NSCLC cells. Suppression of basic transcription factor 3 (BTF3) was followed by apoptosis induced by kahweol via the ERK-mediated signaling pathway in a dose- and time-dependent manner. In addition, kahweol modulated the protein expression of BTF3 genes involved in cell-cycle regulation and apoptosis-related proteins, resulting in apoptotic cell death. Our results collectively indicated that kahweol inhibited the proliferation of NSCLC cells through ERK-mediated signaling pathways and the downregulation of BTF3.

The use of chlorella as an immune stimulant to enhance nonspecific host defense mechanisms or as an antimicrobial to inhibit bacterial growth has been reported. Thus, the aim of the present study was to clarify the effect of recombinant chlorella supplementation on growth performance, meat quality, and the blood profile, excreta microflora, and nutrient digestibility in broilers. A total of 375 one-day-old ROSS 308 broilers (male and female) were allotted to 5 dietary treatments using 5 cages with 15 chicks per cage. Treatments were: 1) NC, basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) PC1, 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) T1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). The broilers in the T2 treatment groups showed higher body weight gain (BGW) by 2.55% (P 0.01) and lower feed conversion ratio (FCR) by 2.75% (P 0.05) compared with those fed the control NC treatment group. Moreover, the blood contents of blood urea nitrogen (BUN), creatinine, and IgA in the broilers of the T2 treatment group were significantly increased by 28.12, 23.07, and 29.72%, respectively -more than those found in the broilers of the NC treatment group (P 0.01). In contrast, the LDL/C in the blood from the animals in the T2 treatment group was significantly decreased by 23.23% - more than that in the blood from the NC broilers (P 0.05). Based on these results, we suggest that the dietary supplementation of broilers with recombinant chlorella could improve their growth performance, increase the concentration of IgA and apparently metabolizable nitrogen in the blood, and decrease ammonia emissions. Therefore, our findings have important implications for the effect of recombinant chlorella supplementation through increasing the concentration of IgA and the level of metabolizable nitrogen.

Esculetin, a coumarin derivative, is a phenolic compound isolated from Artemisia capillaris, Citrus limonia, and Euphorbia lathyris. Although it has been reported to have anti-inflammatory, anti-oxidant, and anti-proliferative activities in several human cancers, its anti-proliferative activity against non-small-cell lung carcinoma (NSCLC) and the molecular mechanisms involved have not been adequately elucidated. In this study, we used two NSCLC cell lines (NCI-H358 and NCI-H1299) to investigate the anti-proliferative activity and apoptotic effect of esculetin. Our data showed that esculetin-treated cells exhibited reduced proliferation and apoptotic cell morphologies. Intriguingly, the transcription factor specificity protein 1 (Sp1) was significantly suppressed by esculetin in a dose- and time-dependent manner. Furthermore, the levels of p27 and p21, two key regulators of the cell cycle, were up-regulated by the esculetin-mediated down-regulation of Sp1; the level of a third cell-cycle regulator, survivin, was decreased, resulting in caspase-dependent apoptosis. Therefore, we conclude that esculetin could be a potent anti-proliferative agent in patients with NSCLC.

This study aimed at investigating whether a porcine follicular fluid (pFF) supplementation positively affects the characteristics of donor cells and the developmental competence of porcine cloned embryos. Ear fibroblast cells (donor cell) from an Massachusetts General Hospital miniature pig were cultured in different culture methods: (1) Dulbecco's modified Eagle's medium (DMEM)+10% FBS (Control); (2) DMEM+0.5% FBS (SS); and (3) DMEM+10% FBS+10% pFF (pFF) for 72 h. In each conditioned medium, the concentrations of 4 amino acids (Thr, Glu, Pro, and Val) in the pFF group were significantly different from those in the control group (p

Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A→T), 16062 (T→C), and 16135 (G→A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor’s mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction.