DNA methylation and regulatory elements during chicken germline stem cell differentiation

Funding for Open Access provided by the UMD Libraries' Open Access Publishing Fund.The production of germ cells in vitro would open important new avenues for stem biology and human medicine, but the mechanisms of germ cell differentiation are not well understood. The chicken, as a great model for embryology and development, was used in this study to help us explore its regulatory mechanisms. In this study, we reported a comprehensive genome-wide DNA methylation landscape in chicken germ cells, and transcriptomic dynamics was also presented. By uncovering DNA methylation patterns on individual genes, some genes accurately modulated by DNA methylation were found to be associated with cancers and virus infection, e.g., AKT1 and CTNNB1. Chicken-unique markers were also discovered for identifying male germ cells. Importantly, integrated epigenetic mechanisms were explored during male germ cell differentiation, which provides deep insight into the epigenetic processes associated with male germ cell differentiation and possibly improves treatment options to male infertility in animals and humans

Cloning of neuraminidase (NA) gene and identification of its antiviral activity

Neuraminidase not only works as an antigen, inducing target-specific antibodies, but also plays a role of  enzyme activity and destroys the sialic acid receptor required by virus infection of the host cell surface which  protects the host from virus damage. In order to explore a new idea to use neuraminidase (NA) gene and  produce disease-resistant transgenic poultry, prokaryotic expression vector pGEX-NA was constructed to  make NA polyclone antibody. Eukaryotic expression vector pcDNA3.0-NA and pcDNA3.0/EGFP-NA was  constructed to reveal its subcelluar location by immunofluorescence and enhanced green fluorescent fusion  protein (EGFP). Chicken embryonic fibroblast (CEF) cells were transfected with pcDNA3.0-NA and selected by  G418 for two weeks, the transfected cells were challenged by Newcastle disease virus (NDV), the morphology of CEF cells were observed to detect the antiviral ability of NA gene. CEF cells were incubated by the cell  lysates extracted from the NIH 3T3 cells, which were transfected with pcDNA3.0-NA. The results show that  pGEX-NA could express NA protein in vitro and NA polyclone antibody worked very well; immunofluorescence and EGFP fusion protein revealed that NA protein located at the cytoplasm near the membrane; NDV-CEF  inhibition experiment showed the NA protein could resist and delayed CEF cells from NDV infection.Key words: Neuraminidase (NA), newcastle disease virus (NDV), antiviral activity, chicken embryonic fibroblast (CEF)

Induciranje pluripotentnih matičnih stanica upotrebom mRNA: učinak valproične kiseline, 5-azacitidina i askorbinske kiseline

In the bourgeoning fields of tissue engineering and regenerative medicine, induced pluripotent stem cells (iPSCs) technology with gene therapy are promising candidates for alternative stem cell source and cell transplantation. In this study, small molecules as anti-oxidant; ascorbic acid (ASA), histone deacetylase inhibitors (HDACi); Valproic acid (VPA), and DNA methyltransferase inhibitors (DNMTi); 5-Azacytidine (5-AzaC) were examined during the generation of murine iPSCs using mRNA of Yamanaka factors from mouse embryonic fibroblasts (MEFs). These modulators were selected based on their well-known effect on the epigenetic status and chromatin modification during early reprogramming. iPSC generation was performed by using synthesized mRNAs of Yamanaka factors Oct4, Sox2, c-Myc, and Klf4 (OSCK) as a standard reprogramming strategy. Both morphological changes and the expression level of the pluripotency markers were examined. 5-AzaC with 1 μM concentration has a slightly toxic effect on the cells, affecting its proliferation and growing efficiency. In contrast, the use of VPA or ASA led to a two-fold increase in the number of iPSC colonies. The iPSCs cultured with the addition of VPA or ASA showed a high expression of the tested pluripotency markers, with a significant increase, more than that of the cells cultured with the addition of 5-AzaC. These findings shed light on the role of ASA, VPA, and 5-AzaC during murine iPSCs generation using a mRNA reprogramming strategy.Ubrzani razvoj u područjima tkivnog inženjerstva i regenerativne medicine, potaknuo je tehnologiju pluripotentnih matičnih stanica (iPSCs) koja zajedno s genskom terapijom predstavlja obečavajući izvor matičnih odnosno transplantacijskih stanica. U ovom su radu, za vrijeme stvaranja mišjih iPSC-a upotrebom mRNA Yamanaka faktora od mišjih embrionalnih fibroblasta (MEF), istraženi učinci različitih modulatora: malih molekula kao antioksidansa, askorbinske kiseline (ASA), inhibitora histonske deacetilaze (HDACi), valproične kiseline (VPA), inhibitora DNA metiltransferaze (DNMTi) i 5-azacitidina (5-AzaC). Ovi su modulatori odabrani zbog njihova dobro poznatog učinka na epigenetski status i modifikaciju kromatina za vrijeme ranog reprogramiranja. Stvaranje iPSC-a postignuto je upotrebom sintetiziranih mRNA Yamanaka faktora Oct4, Sox2, c-Myc i Klf4 (OSCK). Istražene su i morfološke promjene i razina ekspresije markera pluripotencije. 5-AzaC s koncentracijom od 1 μM imao je mali toksičan učinak na stanice, utječući na proliferaciju i njihov rast. Nasuprot tome, upotreba VPA-a ili ASA-e dovela je do dvostrukog povećanja broja iPSC kolonija. iPSC kultura s dodatkom VPA-a ili ASA-e pokazala je visoku ekspresiju testiranih markera pluripotencije, sa znakovitim višom razinom u odnosu na stanice kojima je dodan 5-AzaC. Ovi rezultati rasvjetljuju ulogu ASA-e, VPA-a i 5-AzaC-a za vrijeme stvaranja mišjih iPSC-a primjenom strategije reprogramiranja mRNA

Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs) generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF) cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1) and Rex-1 (ZFP-42, zinc finger protein 42). Using embryonic stem cells (ESCs) conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs) using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF) cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations

Effects of cerium on the selective catalytic reduction activity and structural properties of manganese oxides supported on multi-walled carbon nanotubes catalysts

A series of manganese and cerium oxides supported on multi-walled carbon nanotubes (MWCNTs) catalysts for low-temperature NH3 selective catalytic reduction (SCR) of NOx were prepared by the pore volume impregnation method. The SCR activity of Mn-Ce/MWCNTs catalysts was compared with that of Mn/MWCNTs catalyst The effects of Ce were characterized by transmission electron microscopy, N-2 adsorption-desorption, H-2 temperature-programmed reduction, X-ray photoelectron spectroscopy and X-ray powder diffraction. The results show that the addition of cerium oxides could improve the SCR activity of Mn/MWCNTs catalysts. Mn-Ce/MWCNTs catalyst with a Ce/Mn ratio of 0.6 was found to have the highest activity. The addition of cerium oxides enhanced the dispersion of metal oxides on the MWCNTs. It could also increase the specific surface area and total pore volume, and decrease the average pore size of the catalysts. Ce would improve the concentration of oxygen and the valence of manganese. Furthermore, from the XRD results, it was obvious that the crystalline MnOx disappeared because of the introduction of Ce to the catalyst. MnOx mainly existed in an amorphous state or microcrystal structure in the Mn-Ce/MWCNTs catalysts. CeO2 was found to be the main phase for CeOx. (c) 2013, Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Published by Elsevier B.V. All rights reserved

Enhanced Catalytic Performance of Hierarchical MnOx/ZSM-5 Catalyst for the Low-Temperature NH3-SCR

A ZSM-5 zeolite with a hierarchical pore structure was synthesized by the desilication-recrystallization method using tetraethyl ammonium hydroxide (TEAOH) and cetyltrimethylammonium bromide (CTAB) as the desilication and structure-directing agents, respectively. The MnOx/ZSM-5 catalyst was synthesized by the ethanol dispersion method and applied for the low-temperature selective catalytic reduction of NOx with NH3. The results showed that NOx conversion of the hierarchical MnOx/ZSM-5 catalyst could reach 100% at about 120 °C and could be maintained in the temperature range of 120–240 °C with N2 selectivity over 90%. Furthermore, the hierarchical MnOx/ZSM-5catalyst presented better SO2 resistance performance than the traditional catalyst in the presence of 100 ppm SO2 at 120 °C. XRD, SEM, TEM, XPS, BET, NH3-TPD, and TG were applied to characterize the structural properties of the MnOx/ZSM-5 catalysts. These results showed that the MnOx/ZSM-5 catalyst had micropores (0.78 nm) and mesopores (3.2 nm) leading to a larger specific surface area, which improved the mass transfer of reactants and products while reducing the formation of sulfates. The better catalytic performance over hierarchical MnOx/ZSM-5 catalyst could be attributed to the higher concentration of Mn4+ and chemisorbed oxygen species and higher surface acidity. The improved SO2 resistance was related to the catalyst’s hierarchical pore structure

5-Azacytidine-Induced Cardiomyocyte Differentiation of Very Small Embryonic-Like Stem Cells

The use of stem cells in generating cell-based pacemaker therapies for bradyarrhythmia is currently being considered. Due to the propensity of stem cells to form tumors, as well as ethical issues surrounding their use, the seed cells used in cardiac biological pacemakers have limitations. Very small embryonic-like stem cells (VSELs) are a unique and rare adult stem cell population, which have the same structural, genetic, biochemical, and functional characteristics as embryonic stem cells without the ethical controversy. In this study, we investigated the ability of rat bone marrow- (BM-) derived VSELs to differentiate in vitro into cardiomyocytes by 5-Azacytidine (5-AzaC) treatment. The morphology of VSELs treated with 10 μM 5-AzaC increased in volume and gradually changed to cardiomyocyte-like morphology without massive cell death. Additionally, mRNA expression of the cardiomyocyte markers cardiac troponin-T (cTnT) and α-sarcomeric actin (α-actin) was significantly upregulated after 5-AzaC treatment. Conversely, stem cell markers such as Nanog, Oct-4, and Sox2 were continuously downregulated posttreatment. On day 14 post-5-AzaC treatment, the positive expression rates of cTnT and α-actin were 18.41±1.51% and 19.43±0.51%, respectively. Taken together, our results showed that rat BM-VSELs have the ability to differentiate into cardiomyocytes in vitro. These findings suggest that VSELs would be useful as seed cells in exploring the mechanism of biological pacemaker activity

Improving the Performance of Gd Addition on Catalytic Activity and SO₂ Resistance over MnO_x/ZSM-5 Catalysts for Low-Temperature NH₃-SCR

SO2 poisoning is a great challenge for the practical application of Mn-based catalysts in low-temperature selective catalytic reduction (SCR) reactions of NOx with NH3. A series of Gadolinium (Gd)-modified MnOx/ZSM-5 catalysts were synthesized via a citric acid–ethanol dispersion method and evaluated by low-temperature NH3-SCR. Among them, the GdMn/Z-0.3 catalyst with the molar ratio of Gd/Mn of 0.3 presented the highest catalytic activity, in which a 100% NO conversion could be obtained in the temperature range of 120–240 °C. Furthermore, GdMn/Z-0.3 exhibited good SO2 resistance compared with Mn/Z in the presence of 100 ppm SO2. The results of Brunauer–Emmett–Teller (BET), X-ray photoelectron spectroscopy (XPS), temperature-programmed reduction of H2 (H2-TPR) and temperature-programmed desorption of NH3 (NH3-TPD) illustrated that such catalytic performance was mainly caused by large surface area, abundant Mn4+ and surface chemisorbed oxygen species, strong reducibility and the suitable acidity of the catalyst. The in situ diffuse reflectance infrared Fourier transform spectra (DRIFTS) results revealed that the addition of Gd greatly inhibited the reaction between the SO2 and MnOx active sites to form bulk manganese sulfate, thus contributing to high SO2 resistance. Moreover, in situ DRIFTS experiments also shed light on the mechanism of low-temperature SCR reactions over Mn/Z and GdMn/Z-0.3, which both followed the Langmuir–Hinshelwood (L–H) and Eley–Rideal (E–R) mechanism

Study on the Function and Mechanism of Lin28B in the Formation of Chicken Primordial Germ Cells

Lin28A and Lin28B are two homologues of the same family of RNA binding proteins (RBPs). The function and molecular mechanism of Lin28A in the formation of primordial germ cells (PGCs) are very clear, but the related research on Lin28B is rarely reported. Here, we found that the overexpression of Lin28B can promote the formation of PGC in vivo. Furthermore, the overexpression of Lin28B also resulted in the inhibition of totipotency gene expression and upregulated the PGCs marker genes, and a significant increase in the number of PGCs in genital ridge, as detected by Periodic Acid-Schiff(PAS) staining. However, the inhibited Lin28B expression showed completely opposite results, which were confirmed on the PGC induction model in vitro. Mechanistically, we found that the overexpression of Lin28B can inhibit the maturation of let-7a-3p, and the results of high-throughput sequencing indicated that let-7a-3p was a negative regulator of the formation process of PGCs. Therefore, we conclude that our results determine that Lin28B participates in the formation of PGCs through let-7a-3p, which set a theoretical foundation for improving the function and mechanism of Lin28 family in the formation of PGCs