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The purpose of this study was to design and synthesize Palladium nanoparticles (PdNPs) using an environmentally friendly approach and evaluate the in vitro efficacy of PdNPs in human ovarian cancer A2780 cells. Ultraviolet-Visible (UV-Vis) spectroscopy was used to monitor the conversion of Pd(II) ions to Pd(0)NPs. X-ray diffraction (XRD) revealed the crystallinity of the as-synthesized PdNPs and Fourier transform infrared spectroscopy (FTIR) further confirmed the role of the leaf extract of Evolvulus alsinoides as a reducing and stabilizing agent for the synthesis of PdNPs. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) showed that the average size of the NPs was 5 nm. After a 24-h exposure to PdNPs, cell viability and light microscopy assays revealed the dose-dependent toxicity of the PdNPs. Furthermore, the dose-dependent cytotoxicity of the PdNPs was confirmed by lactate dehydrogenase (LDH), increased reactive oxygen species (ROS) generation, activation of PdNPs-induced autophagy, impairment of mitochondrial membrane potential (MMP), enhanced caspase-3 activity, and detection of TUNEL-positive cells. Our study demonstrates a single, simple, dependable and green approach for the synthesis of PdNPs using leaf extracts of Evolvulus alsinoides. Furthermore, the in vitro efficacy of PdNPs in human ovarian cancer cells suggests that it could be an effective therapeutic agent for cancer therapy.

Background The phenotypic changes that accompany differentiation of resident fibroblasts into myofibroblasts are important aspects of the wound healing process. Recent studies showed that peroxisome proliferator-activated receptor (PPAR) δ plays a critical role in wound healing. Objective To determine whether the nuclear receptor PPARδ can modulate the differentiation of human dermal fibroblasts (HDFs) into myofibroblasts. Methods These studies were undertaken in primary HDFs using Western blot analyses, small interfering (si)RNA-mediated gene silencing, reporter gene assays, chromatin immunoprecipitation (ChIP), migration assays, collagen gel contraction assays, and real-time PCR. Results Activation of PPARδ by GW501516, a specific ligand of PPARδ, specifically upregulated the myofibroblast marker α-smooth muscle actin (α-SMA) in a time- and concentration-dependent manner. This induction was significantly inhibited by the presence of siRNA against PPARδ, indicating that PPARδ is involved in myofibroblast transdifferentiation of HDFs. Ligand-activated PPARδ increased α-SMA promoter activity in a dual mode by directly binding a direct repeat-1 (DR1) site in the α-SMA promoter, and by inducing expression of transforming growth factor (TGF)-β, whose downstream effector Smad3 interacts with a Smad-binding element (SBE) in another region of the promoter. Mutations in these cis-elements totally abrogated transcriptional activation of the α-SMA gene by the PPARδ ligand; thus both sites represent novel types of PPARδ response elements. GW501516-activated PPARδ also increased the migration and contractile properties of HDFs, as demonstrated by Transwell and collagen lattice contraction assays, respectively. In addition, PPARδ-mediated upregulation of α-SMA was correlated with elevated expression of myofibroblast markers such as collagen I and fibronectin, with a concomitant reduction in expression of the epithelial marker E-cadherin. Conclusion PPARδ plays pivotal roles in wound healing by promoting fibroblast-to-myofibroblast differentiation via TGF-β/Smad3 signaling.

Predicting resistance or susceptibility to infectious diseases on the basis of the genotypes of animals regarding disease or immune related genes could be important in animal production. We evaluated the potential influence of the genetic polymorphisms of four immune related genes, porcine beta defensin 4, interferon-induced GTP-binding protein Mx1, natural resistance-associated macrophage protein 1 (Nramp1), and Toll-like receptor 2 (TLR2) on post-weaning piglet survivability in farms. We selected a single SNP that satisfied the criteria of non-synonymous substitutions and the minor allele frequency >0.1 from each gene, and performed PCR–RFLP. Distributions of allele and genotype frequencies of the SNPs were, in general, significantly different among the five breeds (Berkshire, Yorkshire, Duroc, Landrace, and Korean native pigs. Initially, 371 randomly collected Yorkshire × Landrace F1 piglets consisting of post-weaning survival (n = 185) and non-survival groups (n = 186) were genotyped for the selected SNPs. For the Nramp1 and TLR2 SNPs, genotype frequencies were significantly different between the two groups (P

Sangiliyandi Gurunathan, Jae Woong Han, Jung Hyun Park, Eunsu Kim, Yun-Jung Choi, Deug-Nam Kwon, Jin-Hoi Kim Department of Animal Biotechnology, Konkuk University, Seoul, Republic ofKorea Background: Graphene and graphene-based nanocomposites are used in various research areas including sensing, energy storage, and catalysis. The mechanical, thermal, electrical, and biological properties render graphene-based nanocomposites of metallic nanoparticles useful for several biomedical applications. Epithelial ovarian carcinoma is the fifth most deadly cancer in women; most tumors initially respond to chemotherapy, but eventually acquire chemoresistance. Consequently, the development of novel molecules for cancer therapy is essential. This study was designed to develop a simple, non-toxic, environmentally friendly method for the synthesis of reduced graphene oxide–silver (rGO–Ag) nanoparticle nanocomposites using Tilia amurensis plant extracts as reducing and stabilizing agents. The anticancer properties of rGO–Ag were evaluated in ovarian cancer cells. Methods: The synthesized rGO–Ag nanocomposite was characterized using various analytical techniques. The anticancer properties of the rGO–Ag nanocomposite were evaluated using a series of assays such as cell viability, lactate dehydrogenase leakage, reactive oxygen species generation, cellular levels of malonaldehyde and glutathione, caspase-3 activity, and DNA fragmentation in ovarian cancer cells (A2780). Results: AgNPs with an average size of 20 nm were uniformly dispersed on graphene sheets. The data obtained from the biochemical assays indicate that the rGO–Ag nanocomposite significantly inhibited cell viability in A2780 ovarian cancer cells and increased lactate dehydrogenase leakage, reactive oxygen species generation, caspase-3 activity, and DNA fragmentation compared with other tested nanomaterials such as graphene oxide, rGO, and AgNPs. Conclusion: T. amurensis plant extract-mediated rGO–Ag nanocomposites could facilitate the large-scale production of graphene-based nanocomposites; rGO–Ag showed a significant inhibiting effect on cell viability compared to graphene oxide, rGO, and silver nanoparticles. The nanocomposites could be effective non-toxic therapeutic agents for the treatment of both cancer and cancer stem cells. Keywords: graphene–silver nanocomposites, silver nanoparticles, ovarian cancer cells, cancer stem cells, cell viability, caspase-3

The genetic diversity of the major histocompatibility complex (MHC) class I molecules of pigs has not been well characterized. Therefore, the influence of MHC genetic diversity on the immune-related traits of pigs, including disease resistance and other MHC-dependent traits, is not well understood. Here, we attempted to develop an efficient method for systemic analysis of the polymorphisms in the epitope-binding region of swine leukocyte antigens (SLA) class I genes. We performed a comparative analysis of the last 92 bp of the 5' untranslated region (UTR) to the beginning of exon 4 of six SLA classical class I-related genes, SLA-1, -2, -3, -4, -5, and -9, from 36 different sequences. Based on this information, we developed a genomic polymerase chain reaction (PCR) and direct sequencing-based comprehensive typing method for SLA-2. We successfully typed SLA-2 from 400 pigs and 8 cell lines, consisting of 9 different pig breeds, and identified 49 SLA-2 alleles, including 31 previously reported alleles and 18 new alleles. We observed differences in the composition of SLA-2 alleles among different breeds. Our method can be used to study other SLA class I loci and to deepen our knowledge of MHC class I genes in pigs.

Weld overlay was first used in power plants in the US in the early 1980s as an interim method of repairing the welds of flawed piping joints. Weld overlaid piping joints in nuclear power plants must be examined periodically using ultrasonic examination technology. Portable phased array ultrasonic technology has recently become available. Currently, the application of preemptive weld overlays as a mitigation technique and/as a method to improve the examination surface condition for more complex configurations is becoming more common. These complex geometries may require several focused conventional transducers for adequate inspection of the overlay, the original weld, and the base material. Alternatively, Phased array ultrasonic probes can be used to generate several inspection angles simultaneously at various focal depths to provide better and faster coverage than that possible by conventional methods. Thus, this technology can increase the speed of examinations, save costs, and reduce radiation exposure. In this paper, we explain the general sequence of the inspection of weld overlay and the results of signal analysis for some PAUT (phased array ultrasonic testing) signals detected in on-site inspections.

Reprogramming is one of the most essential areas of research in stem cell biology. Despite this importance, the mechanism and correlates of reprogramming remain largely unknown. In this study, we investigated the cytoplasmic remodeling and changes in metabolism that occur during reprogramming and differentiation of pluripotent stem cells. Specifically, we examined the cellular organelles of three pluripotent stem cells, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and epiblast stem cells (EpiSCs), by electron microscopy. We found that the cellular organelles of primed pluripotent EpiSCs were more similar to those of naive pluripotent ESCs and iPSCs than somatic cells. EpiSCs, as well as ESCs and iPSCs, contain large nuclei, poorly developed endoplasmic reticula, and underdeveloped cristae; however, their mitochondria were still mature relative to the mitochondria of ESCs and iPSCs. Next, we differentiated these pluripotent stem cells into neural stem cells (NSCs) in vitro and compared the morphology of organelles. We found that the morphology of organelles of NSCs differentiated from ESCs, iPSCs, and EpiSCs was indistinguishable from brain-derived NSCs. Finally, we examined the changes in energy metabolism that accompanied mitochondrial remodeling during reprogramming and differentiation. We found that the glycolytic activity of ESCs and iPSCs was greater compared with EpiSCs, and that the glycolytic activity of EpiSCs was greater compared with NSCs differentiated from ESCs, iPSCs, and EpiSCs. These results suggest that a change in the cellular state is accompanied by dynamic changes in the morphology of cytoplasmic organelles and corresponding changes in energy metabolism.

The characterization of the genetic variations of major histocompatibility complex (MHC) is essential to understand the relationship between the genetic diversity of MHC molecules and disease resistance and susceptibility in adaptive immunity. We previously reported the development of high-resolution individual locus typing methods for three of the most polymorphic swine leukocyte antigens (SLA) class II loci, namely, SLA-DQA, SLA-DQB1, and SLA-DRB1. In this study, we extensively modified our previous protocols and developed a method for the simultaneous amplification of the three SLA class II genes and subsequent analysis of individual loci using direct sequencing. The unbiased and simultaneous amplification of alleles from the all three hyper-polymorphic and pseudogene containing genes such as MHC genes is extremely challenging. However, using this method, we demonstrated the successful typing of SLA-DQA, SLA-DQB1, and SLA-DRB1 for 31 selected individuals comprising 26 different SLA class II haplotypes which were identified from 700 animals using the single locus typing methods. The results were identical to the known genotypes from the individual locus typing. The new method has significant benefits over the individual locus typing, including lower typing cost, use of less biomaterial, less effort and fewer errors in handling large samples for multiple loci. We also extensively characterized the haplotypes of SLA class II genes and reported three new haplotypes. Our results should serve as a basis to investigate the possible association between polymorphisms of MHC class II and differences in immune responses to exogenous antigens.

Sangiliyandi Gurunathan, Jung Hyun Park, Jae Woong Han, Jin-Hoi KimDepartment of Animal Biotechnology, Konkuk University, Seoul, Republic ofKoreaBackground: Recently, the use of nanotechnology has been expanding very rapidly in diverse areas of research, such as consumer products, energy, materials, and medicine. This is especially true in the area of nanomedicine, due to physicochemical properties, such as mechanical, chemical, magnetic, optical, and electrical properties, compared with bulk materials. The first goal of this study was to produce silver nanoparticles (AgNPs) using two different biological resources as reducing agents, Bacillus tequilensis and Calocybe indica. The second goal was to investigate the apoptotic potential of the as-prepared AgNPs in breast cancer cells. The final goal was to investigate the role of p53 in the cellular response elicited by AgNPs.Methods: The synthesis and characterization of AgNPs were assessed by various analytical techniques, including ultraviolet-visible (UV-vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). The apoptotic efficiency of AgNPs was confirmed using a series of assays, including cell viability, leakage of lactate dehydrogenase (LDH), production of reactive oxygen species (ROS), DNA fragmentation, mitochondrial membrane potential, and Western blot.Results: The absorption spectrum of the yellow AgNPs showed the presence of nanoparticles. XRD and FTIR spectroscopy results confirmed the crystal structure and biomolecules involved in the synthesis of AgNPs. The AgNPs derived from bacteria and fungi showed distinguishable shapes, with an average size of 20 nm. Cell viability assays suggested a dose-dependent toxic effect of AgNPs, which was confirmed by leakage of LDH, activation of ROS, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in MDA-MB-231 breast cancer cells. Western blot analyses revealed that AgNPs induce cellular apoptosis via activation of p53, p-Erk1/2, and caspase-3 signaling, and downregulation of Bcl-2. Cells pretreated with pifithrin-alpha were protected from p53-mediated AgNPs-induced toxicity.Conclusion: We have demonstrated a simple approach for the synthesis of AgNPs using the novel strains B. tequilensis and C. indica, as well as their mechanism of cell death in a p53-dependent manner in MDA-MB-231 human breast cancer cells. The present findings could provide insight for the future development of a suitable anticancer drug, which may lead to the development of novel nanotherapeutic molecules for the treatment of cancers.Keywords: apoptosis, UV-vis spectroscopy, X-ray diffraction, ROS generation

Tubastatin A (TubA) is a highly selective histone deacetylase 6 (HDAC6) inhibitor. As expected, mouse germinal vesicle oocytes fail to extrude the first polar body following TubA treatment. However, a previous study demonstrated that homozygous Hdac6 knockout (KO) mice can be viable and fertile. Therefore, we asked whether TubA is indeed a specific inhibitor of HDAC6 activity. RNA-sequencing and in silico analysis demonstrated that the TubA-treated group presented significant changes in the expression of Hdac subfamily genes such as Hdac6, 10, and 11, and Sirtuin 2, 5, 6, and 7. Additionally, gene expression related to the p53, MAPK, Wnt, and Notch signaling pathways in the TubA-treated group were increased significantly; in contrast, gene expression related to metabolism, DNA replication, and oxidative phosphorylation was decreased significantly. Furthermore, gene expression related to cell cycle, cell structure, pyrimidine metabolism, pentose phosphate pathway, mitochondrial activation, proteasome pathway, RNA polymerase, DNA replication, cyclin-dependent kinase, nucleolar activity, and MI arrest were significantly decreased, indicating that TubA-induced abnormal meiotic maturation and oocyte senescence may be due to the combined effects of HDAC and Sirtuin inhibition, and not HDAC6 inhibition alone. Thus, we believed that this system could provide a model for monitoring the effects of TubA on mouse oocytes.

The Rag2 knockout (KO) mouse is one of the most popular immune compromised animal models used in biomedical research. The immune compromised state concurrently alters many signalling pathways and molecules, including miRNAs and mRNA transcripts that are involved in important biological processes. In addition, miRNAs and transcripts are interdependent, often forming a feedback loop; dysregulation in one might alter the expression of the other, and both participate in many physiological processes including immune regulation. Here, we describe a comprehensive dataset containing alterations in the expression of both miRNAs and mRNAs in Rag2 KO mice compared to their wild type counterparts. The miRNA and mRNA expression profiles were generated from total RNA using a miRNA expression microarray or a BeadChip microarray, respectively. Hence, this dataset will provide the groundwork for a comparative study of the miRNAs and mRNAs that are dysregulated in Rag2 KO mice. It is hoped that the data will illuminate how miRNAs mediate immune regulation, as well as the interaction between miRNAs and mRNAs in Rag2 KO mice.

Additional file 1. PCR amplification of hTERT from genomic DNA of immortalized PAM cells. The results of PCR (A) and reverse transcription (RT) PCR (B) are indicated. The names of the cell lines are indicated on top. “+”, positive control plasmid; “−”, negative control. GAPDH was used as the control for RT-PCR. Product sizes are indicated by the arrows.

G protein-coupled receptors (GPCRs) are a large class of transmembrane receptors categorized into five distinct families: rhodopsin, secretin, adhesion, glutamate, and frizzled. They bind and regulate 80% of all hormones and account for 20-50% of the pharmaceuticals currently on the market. Hundreds of GPCRs integrate and coordinate the functions of individual cells, mediating signaling between various organs. GPCRs are crucial players in tumor progression, adipogenesis, and inflammation. Several studies have also confirmed their central roles in embryonic development and stem cell maintenance. Recently, GPCRs have emerged as key players in the regulation of cell survival, proliferation, migration, and self-renewal in pluripotent (PSCs) and cancer stem cells (CSCs). Our study and other reports have revealed that the expression of many GPCRs is modulated during the generation of induced PSCs (iPSCs) or CSCs as well as during CSC sphere formation. These GPCRs may have crucial roles in the regulation of selfrenewal and other biological properties of iPSCs and CSCs. This review addresses the current understanding of the role of GPCRs in stem cell maintenance and somatic reprogramming to PSCs or CSCs. [BMB Reports 2015; 48(2): 68-80]

Chronic exposure to hydrophobic bile acids such as chenodeoxycholic acid (CDCA) and cholic acid (CA) in the liver during cholestasis causes hepatotoxicity and inflammatory response. However, the detailed mechanisms regarding the role of autophagy in cholestatic hepatotoxicity remain largely unknown. Here we determined autophagic clearance in livers of bile duct-ligated mice, in which bile acids accumulate, and in human hepatoma HepG2 cells treated with CDCA and CA. The accumulation of bile acids caused defective autophagic clearance, shown by the accumulation of insoluble p62 and ubiquitinated proteins and cell death accompanied by caspase-3 processing. Hepatocytes exposed to bile acids also showed the accumulation of autophagosomes via suppressed autophagy flux. Treatment of CDCA markedly suppressed Beclin-1 expression, which exhibits a higher cytotoxicity than CA. Moreover, pharmacological or genetic inhibition of autophagy enhanced bile acid-induced cell death. Finally, in vivo, bile duct ligation led to aberrant accumulation of p62 and ubiquitinated proteins in the liver. Our data demonstrate that inhibited autophagy is an essential component of liver injury during cholestasis.

The signaling of chemokine stromal cell-derived factor (SDF)-1 and its receptor C-X-C motif chemokine receptor 4 (CXCR4) is involved in the cellular proliferation, survival, and migration of various cell types. Although SDF-1/CXCR4 has been implicated in the maintenance of the spermatogonial population during mouse testis development, their expression patterns and functions in boar testis remain unclear. In the present study, the expression pattern of SDF-1 and CXCR4 was determined during pre-pubertal and post-pubertal stage boar testes and in vitro cultured porcine spermatogonial stem cells (pSSCs). The role of these proteins in colony formation in cultured pSSCs was also investigated. Interestingly, SDF-1 expression was observed in PGP 9.5-positve spermatogonia in all developing stages of boar testis; however, CXCR4 expression was only detected in spermatogonia from 5-day-old boar testis. In addition, SDF-1 and CXCR4 expression was observed in cultured pSSCs from 5-day-old boar testes, and inhibition of the CXCR4 receptor signaling pathway by AMD3100 significantly decreased the colony formation of pSSCs. These results suggest that SDF-1 and CXCR4 are useful markers for detecting stage-specific spermatogonia in boar testis. Our results reveal the role of the SDF-1/CXCR4 axis in pSSC in vitro culture.

Whereas stage-specific markers for spermatogonial cells have been well investigated in mouse, the specific markers of germ cells in the testis of domestic animals have not been well defined. Phosphoglycerate kinase (PGK), an enzyme that converts 1,3-bisphosphoglycerate and adenosine diphosphate to 3-phosphoglycerate and adenosine triphosphate, has two isozymes: PGK1 and PGK2. In mouse, PGK1 exists only during the early stages of spermatogenesis, and PGK2 is then expressed during the pachytene spermatocyte stage. In this study, we investigated the localization of PGK2 in the developing porcine testis, and compared the similarities and differences in its expression with that of the PGK2 in mouse. The PGK2 protein was found to be exclusively expressed in spermatids of the adult mouse testis, whereas PGK2-positive cells were observed in the prepubertal and postpubertal testes of pigs. Based on this result, we examined the expression of PGK2 in in vitro-cultured porcine undifferentiated spermatogonia and found it to be maintained in the cultured cells. To verify this result and identify the spermatogonial stem cell-like potential in recipient testes, PKH26 dye-stained PGK2-positive cells were transplanted into the testes of busulfan-treated immunodeficient mouse that had been depleted of both testicular germ cells and somatic cells. The transplanted cells colonized the recipient testis at 8 weeks post transplantation, and fluorescence microscopy identified the cells in the basement membranes of the seminiferous tubules of the injected mouse. Taken together, our results suggest that PGK2 is expressed differently in the testes of mouse and pigs according to developmental stage. This finding should contribute to the study of spermatogenesis and the production of transgenic domestic animals through in vitro spermatogonial sperm cell culture.

Dysregulated serum fatty acids are associated with a lipotoxic placental environment, which contributes to increased pregnancy complications via altered trophoblast invasion. However, the role of saturated and unsaturated fatty acids in trophoblastic autophagy has yet to be explored. Here, we demonstrated that prolonged exposure of saturated fatty acids interferes with the invasiveness of human extravillous trophoblasts. Saturated fatty acids (but not unsaturated fatty acids) inhibited the fusion of autophagosomes and lysosomes, resulting in the formation of intracellular protein aggregates. Furthermore, when the trophoblast cells were exposed to saturated fatty acids, unsaturated fatty acids counteracted the effects of saturated fatty acids by increasing degradation of autophagic vacuoles. Saturated fatty acids reduced the levels of the matrix metalloproteinases (MMP)-2 and MMP-9, while unsaturated fatty acids maintained their levels. In conclusion, saturated fatty acids induced decreased trophoblast invasion, of which autophagy dysfunction plays a major role.

Tubastatin A (Tub-A), a highly selective histone deacetylase 6 (HDAC6) inhibitor, has been widely used as a cytotoxic anticancer agent, or for the treatment of patients with asthma. However, the potential toxicity of Tub-A on oocyte maturation and asymmetric division is still unclear. Therefore, the present study was designed to examine the effect and potential regulatory role of Tub-A on the meiotic maturation of oocytes. We observed that Tub-A treatment induced an increased level of the acetylation of α-tubulin, and a failure of spindle migration and actin cap formation. Based on the spindle structure, most Tub-A treated oocytes were arrested in an MI-like or a GVBD-like stage and exhibited decondensed chromosomes in a dose dependent manner. Moreover, Tub-A treatment decreased the protein expression of mTOR, a factor responsible for spindle formation, and the expression of mDia1, an inhibitor of actin assembly, in an HDAC6 expression-dependent manner. Importantly, following Tub-A supplementation, most oocytes failed to extrude the first polar body, which indicates that these defects are closely linked to abnormal oocyte maturation. Taken together, our data demonstrates that HDAC6 is one of the essential factors for oocyte maturation and asymmetric division via the HDAC6/mTOR or mDia1 pathway in mice.

We demonstrate that OCT4 expression is regulated by germ cell nuclear factor (GCNF) via its interactions with three nuclear receptor (NR) binding sites within OCT4 promoter conserved regions (CRs) in human embryonic carcinoma (EC) NCCIT cells. OCT4 expression is gradually reduced during the retinoic acid-induced differentiation, while GCNF temporarily increased after 2 days and then significantly decreased. In addition, OCT4 expression is significantly reduced by overexpression of exogenous GCNF, but increased by GCNF shRNA-mediated knockdown. The transcriptional activity of OCT4 is significantly inhibited by dose-dependent overexpression of GCNF. While mutants at each of the NR binding sites retain the repressive effects of GCNF on OCT4 promoter activity, the repressive effect was completely eliminated in the reporter construct with all binding sites mutated even in the presence of GCNF. Furthermore, the transcriptional activity of native minimal promoter (CR1-Luc) containing the first NR binding site was significantly reduced by GCNF overexpression, while the mutant retained basal activity to some extent. Next, an exogenous minimal ti promoter-inserted CR2 reporter construct containing the second and third NR binding sites (CR2-ti-Luc) was co-transfected with GCNF expression vector. The transcriptional activity of CR2-ti-Luc was significantly decreased by GCNF overexpression, while mutation of both binding sites retained the transcriptional activity of the reporter construct. Binding assays confirmed the direct interaction of GCNF with all three NR binding sites cooperatively. Taken together, GCNF acts as a transcriptional repressor in the regulation of OCT4 gene expression through cooperative interaction with three NR binding elements in pluripotent NCCIT cells.

In this study, we sought to identify novel antimicrobial peptides (AMPs) in Python bivittatus through bioinformatic analyses of publicly available genome information and experimental validation. In our analysis of the python genome, we identified 29 AMP-related candidate sequences. Of these, we selected five cathelicidin-like sequences and subjected them to further in silico analyses. The results showed that these sequences likely have antimicrobial activity. The sequences were named Pb-CATH1 to Pb-CATH5 according to their sequence similarity to previously reported snake cathelicidins. We predicted their molecular structure and then chemically synthesized the mature peptide for three putative cathelicidins and subjected them to biological activity tests. Interestingly, all three peptides showed potent antimicrobial effects against Gram-negative bacteria but very weak activity against Gram-positive bacteria. Remarkably, ΔPb-CATH4 showed potent activity against antibiotic-resistant clinical isolates and also was observed to possess very low hemolytic activity and cytotoxicity. ΔPb-CATH4 also showed considerable serum stability. Electron microscopic analysis indicated that ΔPb-CATH4 exerts its effects via toroidal pore preformation. Structural comparison of the cathelicidins identified in this study to previously reported ones revealed that these Pb-CATHs are representatives of a new group of reptilian cathelicidins lacking the acidic connecting domain. Furthermore, Pb-CATH4 possesses a completely different mature peptide sequence from those of previously described reptilian cathelicidins. These new AMPs may be candidates for the development of alternatives to or complements of antibiotics to control multidrug-resistant pathogens.

Spermatogenesis is initiated from spermatogonial stem cells (SSCs), which are derived from gonocytes. Although some rodent SSC markers have been investigated, other species- and developmental stage-specific markers of spermatogonia have not been identified. The objective of this study was to characterize the expression of undifferentiated embryonic cell transcription factor 1 (UTF1) gene as a potential marker for spermatogonia and SSCs in the boar testis. In boar testis tissue at pre-pubertal stages (tissues collected at 5, 30, and 60 days of age), UTF1 gene expression was detected in almost all spermatogonia cells that expressed a protein gene product 9.5 (PGP9.5), and immunocytochemical analysis of isolated total testicular cells showed that 91.14% of cells staining for PGP9.5 also stained for UTF1. However, in boar testis tissue at pubertal and post-pubertal stages (tissues collected at 90, 120, 150, and 180 days of age), UTF1 was not detected in all PGP9.5-positive cells in the basement membrane. While some PGP9.5-positive cells stained for UTF1, other cells stained only for PGP9.5 or UTF1. PGP9.5, UTF1, and NANOG was assessed in in vitro cultures of pig SSCs (pSSCs) from testes collected at 5 days of age. The relative amounts of PGP9.5, NANOG, and UTF1 mRNA were greater in pSSC colonies than in testis and muscle tissue. Thus, the UTF1 gene is expressed in PGP9.5-positive spermatogonia cells of pigs at 5 days of age, and its expression is maintained in cultured pSSC colonies, suggesting that UTF1 is a putative marker for early-stage spermatogonia in the pre-pubertal pig testis. These findings will facilitate the study of spermatogenesis and applications in germ cell research.

Summary Background Changes in skin connective tissues mediated by ultraviolet (UV) radiation have been suggested to cause the skin wrinkling normally associated with premature aging of the skin. Recent investigations have shown that peroxisome proliferator-activated receptor (PPAR) δ plays multiple biological roles in skin homeostasis. Objective We attempted to investigate whether PPARδ modulates elastin protein levels and secretion of matrix metalloproteinase (MMP)-2 in UVB-irradiated human dermal fibroblasts (HDFs) and mouse skin. Methods These studies were undertaken in primary HDFs or HR-1 hairless mice using Western blot analyses, small interfering (si)RNA-mediated gene silencing, and Fluorescence microscopy. Results In HDFs, UVB irradiation induced increased secretion of MMP-2 and reduced levels of elastin. Activation of PPARδ by GW501516, a ligand specific for PPARδ, markedly attenuated UVB-induced MMP-2 secretion with a concomitant increase in the level of elastin. These effects were reduced by the presence of siRNAs against PPARδ or treatment with GSK0660, a specific inhibitor of PPARδ. Furthermore, GW501516 elicited a dose- and time-dependent increase in the expression of elastin. Modulation of MMP-2 secretion and elastin levels by GW501516 was associated with a reduction in reactive oxygen species (ROS) production in HDFs exposed to UVB. Finally, in HR-1 hairless mice, administration of GW501516 significantly reduced UVB-induced MMP-2 expression with a concomitant increase in elastin levels, and these effects were significantly reduced by the presence of GSK0660. Conclusion Our results suggest that PPARδ-mediated modulation of MMP-2 secretion and elastin expression may contribute to the maintenance of skin integrity by inhibiting ROS generation.

Spermatogenesis is a process by which haploid cells differentiate from germ cells in the seminiferous tubules of the testes. TLE3, a transcriptional co-regulator that interacts with DNA-binding factors, plays a role in the development of somatic cells. However, no studies have shown its role during germ cell development in the testes. Here, we examined TLE3 expression in the testes during spermatogenesis. TLE3 was highly expressed in mouse testes and was dynamically regulated in different cell types of the seminiferous tubules, spermatogonia, spermatids, and Sertoli cells, but not in the spermatocytes. Interestingly, TLE3 was not detected in Sertoli cells on postnatal day 7 (P7) but was expressed from P10 onward. The microarray analysis showed that the expression of numerous genes changed upon TLE3 knockdown in a Sertoli cell line TM4. These include 1597 up-regulated genes and 1452 down-regulated genes in TLE3-knockdown TM4 cells. Ingenuity Pathway Analysis (IPA) showed that three factors were up-regulated and two genes were down-regulated upon TLE3 knockdown in TM4 cells. The abnormal expression of the three factors is associated with cellular malfunctions such as abnormal differentiation and Sertoli cell formation. Thus, TLE3 is differentially expressed in Sertoli cells and plays a crucial role in regulating cell-specific genes involved in the differentiation and formation of Sertoli cells during testicular development.

Ovarian cancer incidence continues to increase at an alarming rate. Although various therapeutic approaches exist for ovarian cancer, they have limitations, including undesired side effects. Therefore, nanoparticle (NP)-mediated therapy may be a viable, biocompatible, and suitable alternative. To the best of our knowledge, no comprehensive analysis has been undertaken on the cytotoxicity and cellular pathways involved in ovarian cancer cells, particularly SKOV3 cells. Here, we investigated the effect of palladium NPs (PdNPs) and the molecular mechanisms and cellular pathways involved in ovarian cancer. We assayed cell viability, proliferation, cytotoxicity, oxidative stress, DNA damage, and apoptosis and performed an RNA-Seq analysis. The results showed that PdNPs elicited concentration-dependent decreases in cell viability and proliferation and induced increasing cytotoxicity at increasing concentrations, as determined by leakage of lactate dehydrogenase, increased levels of reactive oxygen species and malondialdehyde, and decreased levels of antioxidants like glutathione and superoxide dismutase. Furthermore, our study revealed that PdNPs induce mitochondrial dysfunction by altering mitochondrial membrane potential, reducing adenosine triphosphate levels, inducing DNA damage, and activating caspase 3, all of which significantly induced apoptosis in SKOV3 cells following PdNPs treatment. Gene ontology (GO) term analysis of PdNPs-exposed SKOV3 cells showed various dysregulated pathways, particularly nucleosome assembly, telomere organization, and rDNA chromatin silencing. When genes downregulated by PdNPs were applied to GO term enrichment analysis, nucleosome assembly was the top-ranked biological pathway. We also provide evidence for an association between PdNPs exposure and multiple layers of epigenetic transcriptional control and establish a molecular basis for NP-mediated apoptosis. These findings provide a foundation, potential targets, and novel insights into the mechanism underlying toxicity and pathways in SKOV3 cells, and open new avenues to identify novel targets for ovarian cancer treatment.

Metal nanoparticles and the combination of metal nanoparticles with graphene oxide are widely used in environmental, agriculture, textile, and therapeutic applications. The effect of graphene oxide&ndash, green platinum nanoparticles (GO-PtNPs) on human prostate cancer cells (LNCaP) is unclear. Therefore, this study aimed to synthesize a nanocomposite of GO-PtNPs and evaluate their effect on prostate cancer cells. Herein, we synthesized GO-PtNPs using vanillin and characterized GO-PtNPs. GO-PtNP cytotoxicity in LNCaP cells was demonstrated by measuring cell viability and proliferation. Both decreased in a dose-dependent manner compared to that by GO or PtNPs alone. GO-PtNP cytotoxicity was confirmed by increased lactate dehydrogenase release and membrane integrity loss. Oxidative stress induced by GO-PtNPs increased malondialdehyde, nitric oxide, and protein carbonyl contents. The effective reactive oxygen species generation impaired the cellular redox balance and eventually impaired mitochondria by decreasing the membrane potential and ATP level. The cytotoxicity to LNCaP cells was correlated with increased expression of proapoptotic genes (p53, p21, Bax, Bak, caspase 9, and caspase 3) and decreased levels of antiapoptotic genes (Bcl2 and Bcl-xl). Activation of the key regulators p53 and p21 inhibited the cyclin-dependent kinases Cdk2 and Cdk4, suggesting that p53 and p21 activation in GO-PtNP-treated cells caused genotoxic stress and apoptosis. The increased expression of genes involved in cell cycle arrest and DNA damage and repair, and increased levels of 8-oxo-deoxyguanosine and 8-oxoguanine suggested that GO-PtNPs potentially induce oxidative damage to DNA. Thus, GO-PtNPs are both cytotoxic and genotoxic. LNCaP cells appear to be more susceptible to GO-PtNPs than to GO or PtNPs. Therefore, GO-PtNPs have potential as an alternate and effective cancer therapeutic agent. Finally, this work shows that the combination of graphene oxide with platinum nanoparticles opens new perspectives in cancer therapy. However further detailed mechanistic studies are required to elucidate the molecular mechanism of GO-PtNPs induced cytotoxicity in prostate cancer.

We investigated the role of peroxisome proliferator-activated receptor (PPAR) δ on angiotensin (Ang) II-induced activation of matrix metalloproteinase (MMP)-2 in vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand for PPARδ, attenuated Ang II-induced activation of MMP-2 in a concentration-dependent manner. GW501516 also inhibited the generation of reactive oxygen species in VSMCs treated with Ang II. A marked increase in the mRNA levels of tissue inhibitor of metalloproteinase (TIMP)-2 and -3, endogenous antagonists of MMPs, was also observed in GW501516-treated VSMCs. These effects were markedly reduced in the presence of siRNAs against PPARδ, indicating that the effects of GW501516 are PPARδ dependent. Among the protein kinases inhibited by GW501516, suppression of phosphatidylinositol 3-kinase/Akt signaling was shown to have the greatest effect on activation of MMP-2 in VSMCs treated with Ang II. Concomitantly, GW501516-mediated inhibition of MMP-2 activation in VSMCs treated with Ang II was associated with the suppression of cell migration to levels approaching those in cells not exposed to Ang II. Thus, activation of PPARδ confers resistance to Ang II-induced degradation of the extracellular matrix by upregulating expression of its endogenous inhibitor TIMP and thereby modulating cellular responses to Ang II in vascular cells.

The synthesis of graphene nanosheets from graphene oxide is an interesting area of nanobiotechnology because graphene-based nanomaterials have potential applications in the biomedical field. In this study, we developed a green, rapid, and simple method for the synthesis of graphene from graphene oxide, which uses the mitochondrial polypeptide humanin as a reducing agent. Graphene was prepared via one-step reduction of graphene oxide under mild conditions in an aqueous solution, and the resulting substance was characterized using a range of analytical procedures. UV-vis absorption spectroscopy confirmed the reduction of graphene oxide to graphene. Fourier transform infrared spectroscopy was used to study the changes in the surface functionalities, and X-ray diffraction was used to investigate the crystal structure of graphene. High resolution scanning electron microscopy and atomic force microscopy were also employed to investigate the morphologies of the synthesized grapheme, and Raman spectroscopy was used to evaluate its single- and multi-layer properties. The results described here suggest that the potent reducing agent humanin may be used as a substitute for hydrazine during graphene synthesis, thereby providing a safe, biocompatible and green method for the efficient deoxygenation of graphene oxide that can be used for large-scale production and biomedical applications.

The objective of this study was to establish a porcine spermatogonial germ cell (pSGC) line and develop an in vitro culture system. Isolated total testicular cells (TTCs) from 5-day-old porcine testes were primary cultured at 31, 34, and 37°C. Although the time of colony appearance was delayed at 31°C, strong alkaline phosphatase staining, expressions of pluripotency marker genes such as OCT4, NANOG, and THY1, and the gene expressions of the undifferentiated germ cell markers PLZF and protein gene product 9.5 (PGP9.5) were identified compared to 34 and 37°C. Cell cycle analysis for both pSGC and feeder cells at the three temperatures revealed that more pSGCs were in the G2/M phase at 31°C than 37°C at the subculture stage. In vitro, pSGCs could stably maintain undifferentiated germ cell and stem cell characteristics for over 60days during culture at 31°C. Xenotransplantation of pSGCs to immune deficient mice demonstrated a successful colonization and localization on the seminiferous tubule basement membrane in the recipient testes. In conclusion, pSGCs from neonatal porcine were successfully established and cultured for long periods under a low temperature culture environment in vitro.

We described a green, cost effective and rapid method for synthesizing anisotropic AgNPs using a novel bacterium called Escherichia fergusoni. Furthermore, synthesized AgNPs were characterized by various analytical techniques. The present study demonstrates the efficiency of biologically synthesized AgNPs as a cytotoxic agent against MCF-7 cells and also this study investigates possible molecular mechanisms underlying the cytotoxic effects of AgNPs. AgNPs showed dose dependent cytotoxicity against MCF-7 cells through activation of the lactate dehydrogenase (LDH), reactive oxygen species (ROS) generation and eventually leading to induction of apoptosis which was further confirmed through resulting nuclear fragmentation.

A green and simple approach is described for the large scale synthesis of reduced graphene oxide (rGO). The transition of graphene oxide (GO) into graphene was confirmed using various analytical techniques. Raman spectroscopy data indicate the partial removal of oxygen-containing functional groups from the surface of GO and formation of graphene. X-ray diffraction (XRD) was used to investigate the crystallinity of graphene nanosheets. The antibacterial activity of GO and rGO was evaluated using cell viability, reactive oxygen species (ROS) production and DNA fragmentation assays. The results suggest that GO and rGO possessed an excellent antimicrobial activity against Escherichia coli.

To confirm the beneficial effects of alpha (1,2)-fucosyltransferase (FUT1) M307 (A) on piglet survival on commercial farms, we performed PCR-RFLP analysis of FUT1 M307 in successfully marketed (n = 245) and disease affected/deceased pigs during weaning (n = 252) at a commercial farm. We also evaluated the FUT1 genotypes of 190 healthy pigs from three different genetic backgrounds. The distribution of genotypes differed between the successfully marketed and disease affected/deceased pig groups. The frequency of the A allele, associated with resistance to edema and post-weaning diarrhea, was higher in the post-weaning survival group (0.21) than in the non-survival group (0.16, P 0.05). The odds ratio for piglet survival between AA and GG genotypes was 1.98; thus, piglet survival for individuals with the AA genotype was almost two-fold greater than for GG individuals. The FUT1 gene polymorphism can be used as an effective marker for selection programs to improve post-weaning piglet survival.

This study investigated the effects of two different activation regimens on the developmental potential of somatic cell nuclear transfer (SCNT) embryos and postnatal survivability of the cloned piglets. In vitro matured oocytes were enucleated and reconstructed with porcine fetal fibroblasts. On the basis of the activation regimen used, the reconstructed porcine embryos were allocated into two groups: Group 1-simultaneous electrical pulses and activation group (SFA group); and Group 2-electrical fusion without calcium followed by electrical pulses with calcium after colcemid and cytochalasin B treatment for 5h (DA group). Embryonic development in both SFA and DA groups was determined at day 6 of culture in NSCU-23 medium. To investigate the post-implantation development after the two activation methods, embryos were cultured for 1 day and then transferred into the oviducts of estrus-synchronized recipients. DA group had significantly (p0.05) higher cleavage rates than SFA group. However, the developmental rate to the blastocyst stage and the mean cell number of blastocysts did not differ (p0.05) between SFA and DA groups. Moreover, the pregnancy rate of SFA group was not significantly different compared to DA group. A total of 20 cloned piglets (SFA group-8 live piglets, DA group-11 live piglets and one stillborn) were obtained in the present study. The birth weight of the cloned piglets (live births) did not differ (p0.05) between the two groups. Furthermore, no difference was observed in the postnatal survival rates of the cloned piglets obtained using two different activation regimens. These results suggest that the timing of artificial activation and additional chemical treatments do not affect the developmental rate of porcine SCNT embryos. Remarkably, the pregnancy rate and postnatal survivability of the cloned piglets did not vary between SFA and DA groups.

Graphene has attracted a great deal of scientific interest due to its unique properties and its various prospective applications. However, the use of graphene in technological purposes depends on the development of suitable methodologies for its mass production. Several environmentally safe methods have been emerging for the production of graphene from graphite oxide which are based on the solvothermal and the electrochemical processes. Herein, we developed a green, simple and cost effective approach to produce graphene by using an Escherichia fergusoni as a reducing agent. This reduction method avoids the use of environmentally harmful reagents such as hydrazine and hydrazine hydrate. The resulting reduced graphene oxide (rGO) was characterized by a wide range of analytical techniques. UV-Vis spectra of the samples confirm the transition of graphene oxide (GO) into graphene. Fourier transform infra-red spectroscopy (FT-IR) spectrum of the rGO indicates a significant reduction of the intensity of all oxygen containing moieties suggesting efficient conversion of GO to rGO. X-ray diffraction (XRD) was used to investigate the crystalline of graphene nanosheets, whereas high resolution scanning electron microscopy (SEM) and atomic force microscopy (AFM) were employed to investigate the morphologies of prepared graphene. Raman spectroscopy images provide clear evidence for the formation of graphene. All these findings clearly indicate that GO could be reduced by simple, eco-friendly method by using E. fergusoni to produce water dispersible graphene.

Several biomarkers are routinely used clinically for predicting preterm labor; however, these factors are either nonspecific or detected too late. Here, we performed protein profiles in preterm- and term-derived human umbilical cord by using 2DE. Approximately 200 different proteins were identified between preterm- and term-delivered umbilical cords. Among them, 48 proteins were identified. A comparison of preterm proteome to that of term proteome revealed potential candidates for biomarkers, such as hypoxia-inducible proteins, phosphorylated heat-shock protein 27 (HSP27), transgelin, vimentin, and transferrin that are specific to preterm umbilical cords. Especially, HSP27 in preterm-derived umbilical cords shows a significant increase in the mono- and tetra-phosphorylation. The real importance of all of HSP27 phosphorylation as well as hypoxia-inducible factor 1alpha, and glyceraldehyde 3-phosphate dehydrogenase require further validation in vitro and in vivo; nevertheless, we believe that they could represent promising diagnostic targets for detection of sudden early delivery. In conclusion, the results of the current study may provide important insights into the molecular mechanisms underlying umbilical cord development.

Spermatogenesis, the complex process of sperm cell development including mitotic cell division and meiosis, relies on spermatogonial stem cells (SSCs). While markers for developing germ cells have been well investigated in mice, developmental stage-specific markers of germ cells in domestic animals have not been identified. Sal-like protein 4 (SALL4) is known as a putative marker for undifferentiated spermatogonia in rodents; however, its expression in domestic animals has not been investigated. The objective of this study was to characterize the expression of SALL4 in the developmental stages of boar testes and SSCs. Interestingly, all SALL4-expressing cells responded positively to PGP9.5, which is known as a spermatogonia marker in boar testes, while some PGP9.5-positive cells did not express SALL4 in pre-pubertal boar testes. At this stage, the expression of SALL4 was observed in GFRα1-positive cells, and its expression was maintained in cultured pSSCs in vitro, suggesting that SALL4 is a marker of early-stage boar spermatogonia that express GFRα1 in pre-pubertal testes. Additionally, SALL4 expression was observed in c-Kit-positive but not in PGP9.5- or SCP3-positive cells in post-pubertal testes. In conclusion, SALL4 is expressed in early undifferentiated spermatogonia in pre-pubertal boar testes and in primary spermatocytes in post-pubertal boar testes. Therefore, SALL4 can be used as a stage-specific marker of developing germ cells in boar testes.

Many diseases, including myocardial infarction, autoimmune disease, viral diseases, neurodegenerative diseases, and cancers, are frequently diagnosed with aberrant expression of microRNAs (miRNAs) and their allied pathways. This indicates the crucial role of miRNAs in maintaining biological and physiological processes. miR-7641 is a miRNA whose role in disease has not been fully investigated. In the present study, we investigated the expression pattern of miR-7641 and its target genes in different cancer cells, as well as in clinical cancer patients. Our data confirmed RPS16 and TNFSF10 as two direct targets of miR-7641, while gene expression study showed that a group of genes are also deregulated by miR-7641, including many ribosomal proteins that are frequently co-expressed with RPS16 in breast cancer. Direct inhibition of miR-7641 using a locked nucleic acid upregulated the expression of its target genes, sensitized cancer cells, and enhanced the efficiency of therapeutic agents such as doxorubicin. In addition, inhibition of miR-7641 boosted doxorubicin-mediated apoptosis of cancer cells via upregulation of apoptotic molecules Caspase 9 (CAS9) and poly ADP ribose polymerase (PARP) and downregulation of anti-apoptotic molecule BCL2. Thus, miR-7641 might be a clinically important cancer biomarker. Inhibition of miR-7641 expression could be an efficient treatment strategy for clinical patients.

Several β2-microglobulin (B2M) -bound protein complexes undertake key roles in various immune system pathways, including the neonatal Fc receptor (FcRn), cluster of differentiation 1 (CD1) protein, non-classical major histocompatibility complex (MHC), and well-known MHC class I molecules. Therefore, the duplication of B2M may lead to an increase in the biological competence of organisms to the environment. Based on the pig genome assembly SSC10.2, a segmental duplication of ~45.5 kb, encoding the entire B2M protein, was identified in pig chromosome 1. Through experimental validation, we confirmed the functional duplication of the B2M gene with a completely identical coding sequence between two copies in pigs. Considering the importance of B2M in the immune system, we performed the phylogenetic analysis of B2M duplication in ten mammalian species, confirming the presence of B2M duplication in cetartioldactyls, like cattle, sheep, goats, pigs and whales, but non-cetartiodactyl species, like mice, cats, dogs, horses, and humans. The density of long interspersed nuclear element (LINE) at the edges of duplicated blocks (39 to 66%) was found to be 2 to 3-fold higher than the average (20.12%) of the pig genome, suggesting its role in the duplication event. The B2M mRNA expression level in pigs was 12.71 and 7.57 times (2-ΔΔCt values) higher than humans and mice, respectively. However, we were unable to experimentally demonstrate the difference in the level of B2M protein because species specific anti-B2M antibodies are not available. We reported, for the first time, the functional duplication of the B2M gene in animals. The identification of partially remaining duplicated B2M sequences in the genomes of only cetartiodactyls indicates that the event was lineage specific. B2M duplication could be beneficial to the immune system of pigs by increasing the availability of MHC class I light chain protein, B2M, to complex with the proteins encoded by the relatively large number of MHC class I heavy chain genes in pigs. Further studies are necessary to address the biological meaning of increased expression of B2M.

Silver nanoparticles (AgNPs) have been used as an antimicrobial and disinfectant agents. However, there is limited information about antitumor potential. Therefore, this study focused on determining cytotoxic effects of AgNPs on MDA-MB-231 breast cancer cells and its mechanism of cell death. Herein, we developed a green method for synthesis of AgNPs using culture supernatant ofBacillus funiculus, and synthesized AgNPs were characterized by various analytical techniques such as UV-visible spectrophotometer, particle size analyzer, and transmission electron microscopy (TEM). The toxicity was evaluated using cell viability, metabolic activity, and oxidative stress. MDA-MB-231 breast cancer cells were treated with various concentrations of AgNPs (5 to 25 μg/mL) for 24 h. We found that AgNPs inhibited the growth in a dose-dependent manner using MTT assay. AgNPs showed dose-dependent cytotoxicity against MDA-MB-231 cells through activation of the lactate dehydrogenase (LDH), caspase-3, reactive oxygen species (ROS) generation, eventually leading to induction of apoptosis which was further confirmed through resulting nuclear fragmentation. The present results showed that AgNPs might be a potential alternative agent for human breast cancer therapy.

Scientific Reports 6: Article number: 33784; published online: 28 September 2016; updated: 21 November 2016.

Growth and differentiation factor 3 (GDF3), a mammalian-specific transforming growth factor β ligand, and OCT4, one of key stem cell transcription factors, are expressed in testicular germ cell tumors (TGCTs) as well as pluripotent stem cells. To understand the molecular mechanism by which OCT4 and GDF3 function in tumorigenesis as well as stemness, we investigated the transcriptional regulation of GDF3 mediated by OCT4 in human embryonic carcinoma (EC) NCCIT cells, which are pluripotent stem cells of TGCTs. GDF3 and OCT4 was highly expressed in undifferentiated NCCIT cells and then significantly decreased upon retinoic acid-induced differentiation in a time-dependent manner. Moreover, GDF3 expression was reduced by short hairpin RNA-mediated knockdown of OCT4 and increased by OCT4 overexpression, suggesting that GDF3 and OCT4 have a functional relationship in pluripotent stem cells. A promoter-reporter assay revealed that the GDF3 promoter (-1721-Luc) activity was significantly activated by OCT4 in a dose-dependent manner. Moreover, the minimal promoter (-183-Luc) was sufficient for OCT4-mediated transcriptional activation and provided a potential binding site for the direct interaction with OCT4. Collectively, this study provides the evidence about the regulatory mechanism of GDF3 mediated by OCT4 in pluripotent EC cells.

Objective The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. Methods Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts. Results The competence to develop into blastocysts was not significantly different between the sexes. The mean cell number of female and male cloned blastocysts obtained by in vivo culture (143.8±10.5 to 159.2±14.8) was higher than that of in vitro culture of somatic cell nuclear transfer (SCNT) groups (31.4±8.3 to 33.4±11.1). After embryo transfer, 5 pregnant gilts from each treatment delivered 15 female and 22 male piglets. The average birth weight of the cloned piglets, gestation length, and the postnatal survival rates were not significantly different (p

Spermatogenesis begins at puberty and continues throughout a male's life. This process is initiated and maintained by spermatogonial stem cells in the seminiferous tubules, and these cells produce haploid spermatozoa. Markers of male germ cells have been fully identified in rodents, including mice and rats, but not in canines. To characterize the canine male germ cells, histological and immunohistochemical analyses were performed, using prepubertal (1-3-month-old), early pubertal (4-month-old), and postpubertal (7-month-old) dog testes. Expression of protein gene product 9.5 (PGP9.5), deleted in azoospermia-like (DAZL), synaptonemal complex protein (SCP3), tyrosine-protein kinase Kit (C-kit), and acrosin was confirmed by immunohistochemical analysis. PGP9.5 and DAZL were detected in spermatogonia and co-localized near the basement membrane of seminiferous tubules. Some SCP3-positive cells expressed PGP9.5 but not C-kit, and most of these cells were located near the basement membrane. C-kit is a marker of differentiated spermatogenic cells. In addition, acrosin was detected in C-kit-positive spematocytes and mature spermatozoa, whereas C-kit was detected in Sertoli cells in all stages of canine testis development. We suggest that male germ cell markers detected in other species are conserved in canines. PGP9.5, DAZL, SCP3, and acrosin expressions were conserved among various species, but C-kit expression varied. This study might facilitate the identification of stage-specific canine germ cell markers and cellular mechanisms of spermatogenesis.

Embryonic body-like colony formation is a unique pattern in male germ cell cultures, including spermatogonial stem cells. However, detailed information of the colony formation has not yet been sufficiently reported in male germ cell culture. To elucidate the formation of germ cell-derived colony (GDC), glial cell-derived neurotrophic factor receptor alpha-1 (GFRα-1)-positive pig germ cells were isolated using an immunomagnetic cell isolation method and labeled with red- or green-fluorescent dye. In GDC culture, red-fluorescent-labeled germ cells were evenly distributed in the wells from day 1 to 4, and they clustered together at the time of GDC formation on day 6. Interestingly, feeder cells migrated to the site of colony formation as spermatogonia carriers. Furthermore, when freshly prepared green-labeled GFRα-1-positive germ cells were added, mixed-fluorescent dye (red and green) colonies were observed. On bromodeoxyuridine (BrdU) treatment, 58% ± 3.13% of germ cells were positive to protein gene product 9.5 but negative to BrdU cells. Immunocytochemistry and reverse transcription-polymerase chain reaction results showed that cultured GDC cells were positive to stem cell- and pig germ cell-specific marker genes. In conclusion, in vitro formation of GDCs is mainly dependent on the aggregation of single germ cells as well as on the slow proliferation of germ cells.

An enigmatic question exists concerning the pro- or anti-cancer status of mesenchymal stem cells (MSCs). Despite growing interest, this question remains unanswered, and the debate became intensified with new evidences backing each side. Here, we showed that human adipose MSC (hAMSC)-derived conditioned medium (CM) exhibited inhibitory effects on A2780 human ovarian cancer cells by blocking the cell cycle, and activating mitochondria-mediated apoptosis signalling. Explicitly, we demonstrated that exosomes, an important biological component of hAMSC-CM, could restrain proliferation, wound-repair and colony formation ability of A2780 and SKOV-3 cancer cells. Furthermore, hAMSC-CM-derived exosomes induced apoptosis signalling by upregulating different pro-apoptotic signalling molecules, such as BAX, CASP9, and CASP3, as well as downregulating the anti-apoptotic protein BCL2. More specifically, cancer cells exhibited reduced viability following fresh or protease-digested exosome treatment; however, treatment with RNase-digested exosomes could not inhibit the proliferation of cancer cells. Additionally, sequencing of exosomal RNAs revealed a rich population of microRNAs (miRNAs), which exhibit anti-cancer activities by targeting different molecules associated with cancer survival. Our findings indicated that exosomal miRNAs are important players involved in the inhibitory influence of hAMSC-CM towards ovarian cancer cells. Therefore, we believe that these comprehensive results will provide advances concerning ovarian cancer research and treatment.

Clinical applications of induced pluripotent stem cells (iPSCs) require development of technologies for the production of "footprint-free" (gene integration-free) iPSCs, which avoid the potential risk of insertional mutagenesis in humans. Previously, several studies have shown that mRNA transfer can generate "footprint-free" iPSCs, but these studies did not use a delivery vehicle and thus repetitive daily transfection was required because of mRNA degradation. Here, we report an mRNA delivery system employing graphene oxide (GO)-polyethylenimine (PEI) complexes for the efficient generation of "footprint-free" iPSCs. GO-PEI complexes were found to be very effective for loading mRNA of reprogramming transcription factors and protection from mRNA degradation by RNase. Dynamic suspension cultures of GO-PEI/RNA complexes-treated cells dramatically increased the reprogramming efficiency and successfully generated rat and human iPSCs from adult adipose tissue-derived fibroblasts without repetitive daily transfection. The iPSCs showed all the hallmarks of pluripotent stem cells including expression of pluripotency genes, epigenetic reprogramming, and differentiation into the three germ layers. These results demonstrate that mRNA delivery using GO-PEI-RNA complexes can efficiently generate "footprint-free" iPSCs, which may advance the translation of iPSC technology into the clinical settings.

Solid tumors are frequently associated with resistance to chemotherapy because the fraction of hypoxic tumor cells is substantial. To understand the underlying mechanism of hypoxia on silver nanoparticle (AgNPs)-induced apoptosis, the expression of hypoxia-inducible factor (HIF)-1α, a hallmark of hypoxia, was measured in the presence and absence of AgNPs. The results showed that HIF-1α expression was upregulated after AgNPs treatment under both hypoxic and normoxic conditions. Cell viability assays showed that AgNPs promoted cell death in cancer cells but not in non-cancer cells, as cancer cells are slightly more acidic than normal cells. However, reactive oxygen species generation induced by AgNPs in lung cancer cells caused high susceptibility to oxidative stress, whereas pre-exposure to hypoxia blocked AgNPs-induced oxidative stress. Notably, HIF-1α inhibited AgNPs-induced mitochondria-mediated apoptosis by regulating autophagic flux through the regulation of ATG5, LC3-II, and p62. Further, cell viability after treatment of cancer cells with AgNPs under hypoxic conditions was lower in HIF-1α siRNA-transfected cells than in control siRNA-transfected cells, indicating that HIF-1α knockdown enhances hypoxia induced decrease in cell viability. Our results suggest that hypoxia-mediated autophagy may be a mechanism for the resistance of AgNPs-induced apoptosis and that strategies targeting HIF-1α may be used for cancer therapy.

The availability of simple, robust, and cost-effective methods for the large-scale production of bacteriotoxic peptides such as antimicrobial peptides (AMPs) is essential for basic and pharmaceutical research. However, the production of bacteriotoxic proteins has been difficult due to a high degree of toxicity in bacteria and proteolytic degradation. In this study, we inserted AMPs into the Green fluorescent protein (GFP) in a loop region and expressed them as insoluble proteins in high yield, circumventing the inherent toxicity of AMP production in Escherichia coli. The AMPs inserted were released by cyanogen bromide and purified by chromatography. We showed that highly potent AMPs such as Protegrin-1, PMAP-36, Buforin-2, and Bactridin-1 are produced in high yields and produced AMPs showed similar activities compared to chemically synthesized AMPs. We increased the yield more than two-fold by inserting three copies of Protegrin-1 in the GFP scaffold. The immunogold electron micrographs showed that the expressed Protegrin-1 in the GFP scaffold forms large and small size aggregates in the core region of the inclusion body and become entirely nonfunctional, therefore not influencing the proliferation of E. coli. Our novel method will be applicable for diverse bacteriotoxic peptides which can be exploited in biomedical and pharmaceutical researches.

Bax inhibitor-1 (BI-1), a member of the BI-1 family of integral membrane proteins, was originally identified as an inhibitor of stress-induced cell death in mammalian cells. Previous studies have shown that the withdrawal of leukemia inhibitory factor (LIF) results in differentiation of the majority of mouse embryonic stem (mES) cells into various cell lineages, while some ES cells die within 3 days. Thus, to investigate the function of BI-1 in ES cell survival and neuronal differentiation, we generated mES cell lines that overexpress BI-1 or a carboxy-terminal BI-1ΔC mutant. Overexpression of BI-1 in mES cells significantly increased cell viability and resistance to apoptosis induced by LIF withdrawal, while the control vector or BI-1ΔC-overexpressing mES cells had no effect. Moreover, overexpression of BI-1 produced significant inhibition of the p38 mitogen-activated protein kinases (MAPK) pathway in response to LIF withdrawal, while activity of the extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) MAPK pathway was increased. Interestingly, we found that BI-1-overexpressing cells showed higher expression levels of neuroectodermal markers (Otx1, Lmx1b, En1, Pax2, Wnt1, Sox1, and Nestin) and greater neuronal differentiation efficiency than control or BI-1ΔC-overexpressing mES cells did. Considering these findings, our results indicated that BI-1-modulated MAPK activity plays a key role in protecting mES cells from LIF-withdrawal-induced apoptosis and in promoting their differentiation toward neuronal lineages.

Genomic reprogramming factors in the cytoplasm of germinal vesicle (GV) stage oocytes have been shown to improve the efficiency of producing cloned mouse offspring through the exposure of nuclei to a GV cytoplasmic extract prior to somatic cell nuclear transfer (SCNT) to enucleated oocytes. Here, we developed an extract of GV stage pig oocytes (GVcyto-extract) to investigate epigenetic reprogramming events in treated somatic cell nuclei. This extract induced differentiation-associated changes in fibroblasts, resulting in cells that exhibit pluripotent stem cell-like characteristics and that redifferentiate into three primary germ cell layers both in vivo and in vitro. The GVcyto-extract treatment induced large numbers of high-quality SCNT-generated blastocysts, with methylation and acetylation of H3-K9 and expression of Oct4 and Nanog at levels similar to in vitro fertilized embryos. Thus, GVcyto-extract could elicit differentiation plasticity in treated fibroblasts, and SCNT-mediated reprogramming reset the epigenetic state in treated cells more efficiently than in untreated cells. In summary, we provide evidence for the generation of stem-like cells from differentiated somatic cells by treatment with porcine GVcyto-extract.