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Sample records for early porcine embryonic

  1. Toward Development of Pluripotent Porcine Stem Cells by Road Mapping Early Embryonic Development

    DEFF Research Database (Denmark)

    Petkov, Stoyan; Freude, Kristine; Mashayekhi-Nezamabadi, Kaveh

    2017-01-01

    The lack in production of bona fide porcine pluripotent stem cells has definitely been hampered by a lack of research into porcine embryo development. Embryonic development in mammals is the extraordinary transition of a single-celled fertilized zygote into a complex fetus, which occurs...... in the uterus of the maternal adult during the early stages of gestation. Biomedical pig models could serve as genetic backgrounds for establishment of embryonic stem cells (ESCs) or other pluripotent stem cells (such as iPSC), which may be used to model and study diseases in vitro. This chapter provides...... insight into the current knowledge of pluripotent states in the developing pig embryo and the current status in establishment of bona fide porcine ESC (pESC) and piPSCs. It reflects the potential causes underlying the difficulty in establishing pluripotent stem cells and reviews recent data on global...

  2. Porcine embryonic stem cells

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane

    2008-01-01

    The development of porcine embryonic stem cell lines (pESC) has received renewed interest given the advances being made in the production of immunocompatible transgenic pigs. However, difficulties are evident in the production of pESCs in-vitro. This may largely be attributable to differences...

  3. Fe(III Is Essential for Porcine Embryonic Development via Mitochondrial Function Maintenance.

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    Ming-Hui Zhao

    Full Text Available Iron is an important trace element involved in several biological processes. The role of iron in porcine early embryonic development remains unknown. In the present study, we depleted iron (III, Fe3+ with deferoxamine (DFM, a specific Fe3+ chelator, in cultured porcine parthenotes and monitored embryonic development, apoptosis, mitochondrial membrane potential, and ATP production. Results showed biphasic function of Fe3+ in porcine embryo development. 0.5 μM DFM obviously increased blastocyst formation (57.49 ± 2.18% vs. control, 43.99 ± 1.72%, P < 0.05 via reduced (P < 0.05 production of reactive oxygen species (ROS, further increased mitochondrial membrane potential and ATP production in blastocysts (P < 0.05. 0.5 μM DFM decreased mRNA expression of Caspase 3 (Casp3 and increased Bcl-xL. However, results showed a significant reduction in blastocyst formation in the presence of 5.0 μM DFM compared with the control group (DFM, 21.62 ± 3.92% vs. control, 43.99 ± 1.73%, P < 0.05. Fe3+ depletion reduced the total (DFM, 21.10 ± 8.78 vs. control, 44.09 ± 13.65, P < 0.05 and increased apoptotic cell number (DFM, 11.10 ± 5.24 vs. control, 2.64 ± 1.43, P < 0.05 in the blastocyst. An obvious reduction in mitochondrial membrane potential and ATP level after 5.0 μM DFM treatment was observed. Co-localization between mitochondria and cytochrome c was reduced after high concentration of DFM treatment. In conclusion, Fe3+ is essential for porcine embryonic development via mitochondrial function maintenance, but redundant Fe3+ impairs the function of mitochondria.

  4. Porcine pluripotency cell signaling develops from the inner cell mass to the epiblast during early development

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane; Christensen, Josef; Gao, Yu

    2009-01-01

      The signaling mechanisms regulating pluripotency in porcine embryonic stem cells and embryos are unknown. In this study, we characterize cell signaling in the in-vivo porcine inner cell mass and later-stage epiblast. We evaluate expression of OCT4, NANOG, SOX2, genes within the JAK/STAT pathway...... pluripotency in human embryonic stem cells is detectable in the porcine epiblast, but not in the inner cell mass. Copyright (c) 2009 Wiley-Liss, Inc.......  The signaling mechanisms regulating pluripotency in porcine embryonic stem cells and embryos are unknown. In this study, we characterize cell signaling in the in-vivo porcine inner cell mass and later-stage epiblast. We evaluate expression of OCT4, NANOG, SOX2, genes within the JAK/STAT pathway...... (LIF, LIFR, GP130), FGF pathway (bFGF, FGFR1, FGFR2), BMP pathway (BMP4), and downstream-activated genes (STAT3, c-Myc, c-Fos, and SMAD4). We discovered two different expression profiles exist in the developing porcine embryo. The D6 porcine blastocyst (inner cell mass stage) is devoid...

  5. Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes.

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    Morovic, Martin; Strejcek, Frantisek; Nakagawa, Shoma; Deshmukh, Rahul S; Murin, Matej; Benc, Michal; Fulka, Helena; Kyogoku, Hirohisa; Pendovski, Lazo; Fulka, Josef; Laurincik, Jozef

    2017-12-01

    It is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)). Our results show that the re-injection of isolated oocyte nucleoli, either from the pig (P + P) or mouse (P + M), into previously enucleolated and subsequently matured porcine oocytes rescues their development after parthenogenetic activation and some of these develop up to the blastocyst stage (P + P, 11.8%; P + M, 13.5%). In nucleolus re-injected 8-cell and blastocyst stage embryos the number of nucleoli labeled with C23 in P + P and P + M groups was lower than in control (non-manipulated) group. UBF was localized in small foci within the nucleoli of blastocysts in control and P + P embryos, however, in P + M embryos the labeling was evenly distributed in the nucleoplasm. The TEM and autoradiographic evaluations showed the formation of functional nucleoli and de novo rRNA synthesis at the 8-cell stage in both, control and P + P group. In the P + M group the formation of comparable nucleoli was delayed. In conclusion, our results indicate that the mouse nucleolus can rescue embryonic development of enucleolated porcine oocytes, but the localization of selected nucleolar proteins, the timing of transcription activation and the formation of the functional nucleoli in NuTE compared with control group show evident aberrations.

  6. Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

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    Li, Dong; Secher, Jan Ole Bertelsen; Juhl, Morten

    2017-01-01

    Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells...... are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast...... populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore...

  7. Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure.

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    Jeon, Yubyeol; Nam, Yeong-Hee; Cheong, Seung-A; Kwak, Seong-Sung; Lee, Eunsong; Hyun, Sang-Hwan

    2016-08-25

    Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation.

  8. Function of JARID2 in bovines during early embryonic development

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    Yao Fu

    2017-12-01

    Full Text Available Histone lysine modifications are important epigenetic modifications in early embryonic development. JARID2, which is a member of the jumonji demethylase protein family, is a regulator of early embryonic development and can regulate mouse development and embryonic stem cell (ESC differentiation by modifying histone lysines. JARID2 can affect early embryonic development by regulating the methylation level of H3K27me3, which is closely related to normal early embryonic development. To investigate the expression pattern of JARID2 and the effect of JARID2-induced H3K27 methylation in bovine oocytes and early embryonic stages, JARID2 mRNA expression and localization were detected in bovine oocytes and early embryos via qRT-PCR and immunofluorescence in the present study. The results showed that JARID2 is highly expressed in the germinal vesicle (GV, MII, 2-cell, 4-cell, 8-cell, 16-cell and blastocyst stages, but the relative expression level of JARID2 in bovine GV oocytes is significantly lower than that at other oocyte/embryonic stages (p < 0.05, and JARID2 is expressed primarily in the nucleus. We next detected the mRNA expression levels of embryonic development-related genes (OCT4, SOX2 and c-myc after JARID2 knockdown through JARID2-2830-siRNA microinjection to investigate the molecularpathwayunderlying the regulation of H3K27me3 by JARID2 during early embryonic development. The results showed that the relative expression levels of these genes in 2-cell embryos weresignificantly higher than those in the blastocyst stage, and expression levels were significantly increased after JARID2 knockdown. In summary, the present study identified the expression pattern of JARID2 in bovine oocytes and at each early embryonic stage, and the results suggest that JARID2 plays a key role in early embryonic development by regulating the expression of OCT4, SOX2 and c-myc via modification of H3K27me3 expression. This work provides new data for improvements in the

  9. Characterization, expression profiles, intercellular distribution and association analysis of porcine PNAS-4 gene with production traits

    NARCIS (Netherlands)

    Mo, D.L.; Zhu, Z.M.; Pas, te M.F.W.; Li, X.Y.; Yang, S.L.; Wang, H.; Wang, H.L.; Li, K.

    2008-01-01

    Background - In a previous screen to identify differentially expressed genes associated with embryonic development, the porcine PNAS-4 gene had been found. Considering differentially expressed genes in early stages of muscle development are potential candidate genes to improve meat quality and

  10. Lipid Supplement in the Cultural Condition Facilitates the Porcine iPSC Derivation through cAMP/PKA/CREB Signal Pathway

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    Wei Zhang

    2018-02-01

    Full Text Available Large numbers of lipids exist in the porcine oocytes and early embryos and have the positive effects on their development, suggesting that the lipids may play an important role in pluripotency establishment and maintenance in pigs. However, the effects of lipids and their metabolites, such as fatty acids on reprogramming and the pluripotency gene expression of porcine-induced pluripotent stem cells (iPSCs, are unclear. Here, we generated the porcine iPSCs that resemble the mouse embryonic stem cells (ESCs under lipid and fatty-acid-enriched cultural conditions (supplement of AlbuMAX. These porcine iPSCs show positive for the ESCs pluripotency markers and have the differentiation abilities to all three germ layers, and importantly, have the capability of aggregation into the inner cell mass (ICM of porcine blastocysts. We further confirmed that lipid and fatty acid enriched condition can promote the cell proliferation and improve reprogramming efficiency by elevating cAMP levels. Interestingly, this lipids supplement promotes mesenchymal–epithelial transition (MET through the cAMP/PKA/CREB signal pathway and upregulates the E-cadherin expression during porcine somatic cell reprogramming. The lipids supplement also makes a contribution to lipid droplets accumulation in the porcine iPSCs that resemble porcine preimplantation embryos. These findings may facilitate understanding of the lipid metabolism in porcine iPSCs and lay the foundation of bona fide porcine embryonic stem cell derivation.

  11. Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system.

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    Nakayama, Asuka; Sato, Masahiro; Shinohara, Mariko; Matsubara, Shyuichiro; Yokomine, Takaaki; Akasaka, Eri; Yoshida, Mitsutoshi; Takao, Sonshin

    2007-01-01

    Porcine embryonic fibroblasts (PEF) are important as donor cells for nuclear transfer for generation of genetically modified pigs. In this study, we determined an optimal protocol for transfection of PEF with the Amaxa Nucleofection system, which directly transfers DNA into the nucleus of cells, and compared its efficiency with conventional lipofection and electroporation. Cell survival and transfection efficiency were assessed using dye-exclusion assay and a green fluorescent protein (GFP) reporter construct, respectively. Our optimized nucleofection parameters yielded survival rates above 60%. Under these conditions, FACS analysis demonstrated that 79% of surviving cells exhibited transgene expression 48 h after nucleofection when program U23 was used. This efficiency was higher than that of transfection of PEFs with electroporation (ca. 3-53%) or lipofection (ca. 3-8%). Transfected cells could be expanded as stably transgene-expressing clones over a month. When porcine nuclear transfer (NT) was performed using stable transformant expressing GFP as a donor cell, 5-6% of reconstituted embryos developed to blastocysts, from which 30-50% of embryos exhibited NT-embryo-derived green fluorescence. Under the conditions evaluated, nucleofection exhibited higher efficiency than conventional electroporation and lipofection, and may be a useful alternative for generation of genetically engineered pigs through nuclear transfer.

  12. Progress, problems and prospects of porcine pluripotent stem cells

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    Hanning WANG,Yangli PEI,Ning LI,Jianyong HAN

    2014-02-01

    Full Text Available Pluripotent stem cells (PSCs, including embryonic stem cells (ESCs and induced PSCs (iPSCs, can differentiate into cells of the three germ layers, suggesting that PSCs have great potential for basic developmental biology research and wide applications for clinical medicine. Genuine ESCs and iPSCs have been derived from mice and rats, but not from livestock such as the pig─an ideal animal model for studying human disease and regenerative medicine due to similarities with human physiologic processes. Efforts to derive porcine ESCs and iPSCs have not yielded high-quality PSCs that can produce chimeras with germline transmission. Thus, exploration of the unique porcine gene regulation network of preimplantation embryonic development may permit optimization of in vitro culture systems for raising porcine PSCs. Here we summarize the recent progress in porcine PSC generation as well as the problems encountered during this progress and we depict prospects for generating porcine naive PSCs.

  13. In vitro culture and characterization of putative porcine embryonic germ cells derived from domestic breeds and yucatan mini pig embryos at days 20-24 of gestation

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    Petkov, Stoyan Gueorguiev; Marks, Hendrik; Klein, Tino

    2011-01-01

    Embryonic germ cells (EGC) are cultured pluripotent cells derived from primordial germ cells (PGC). This study explored the possibility of establishing porcine EGC from domestic breeds and Yucatan mini pigs using embryos at Days 17-24 of gestation. In vitro culture of PGC from both pooled...

  14. Impact of nutritional stress on early embryonic survival

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    Sukanta Mondal

    2015-09-01

    Full Text Available Background: Low reproductive efficiency is the most critical problem faced by the livestock industry across the globe. Early embryonic loss is one the major cause of poor reproductive efficiency resulting in delayed pregnancy, fewer calves born, reduced milk production, slower genetic progress and substantial financial loss to the beef or dairy industry. The establishment of pregnancy results from the interaction between the embryo and the dam and is the culmination of a series of events initiated with development of the follicle and gametes. Among numerous internal and external factors nutrition has the potency to alter the micro-environment of the oocyte and the embryo, making it more hostile to optimal fertilization and pre-implantation embryonic growth. Understanding the impact of nutritional stress on oocyte function, embryo development and reciprocal signaling networks between the embryo and uterus will lead to alleviation of the problems of early embryonic mortality.

  15. Ultrasonographic appearance of early embryonic mortality in buffalo (Bubalus bubalis

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    Giuseppe Catone

    2010-01-01

    Full Text Available Embryonic mortality is one of the main causes responsible of the decline in fertility that occurs in buffaloes during periods of increasing daylight length (out sexual breeding season. Transrectal ultrasonography for pregnancy diagnosis offers some advantages over palpation per rectum: earlier diagnosis of pregnancy/non-pregnancy, determination of embryo/fetus viability, reduction of misdiagnosis, and reduction of .potential. iatrogenic embryo/fetal attrition. Non pregnant buffaloes on Day 25 after AI showed higher Resistive Index (RI (P<0.05 and Pulsatility Index (P=0.07 values, registered on CL on Days 10 after AI, compared to pregnant buffaloes. RI values were significantly higher (P=0.02 in non pregnant buffaloes also on Day 45 after AI. Colour Doppler sonography could be used to gain specific information relating to the ovarian blood flow in predicting early embryonic loss and to describe the ultrasonographic features of early embryonic death in buffaloes.

  16. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo.

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    Binghua Xue

    Full Text Available Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.

  17. Confinement and clearance of OCT4 in the porcine embryo at stereomicroscopically defined stages around gastrulation

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    Vejlsted, Morten; Offenberg, Hanne Kjær; Thorup, Flemming

    2006-01-01

    was selectively observed in the epiblast. A prominent crescent-shaped thickening at the posterior region of the embryonic disk marked the first polarization within this structure reflecting incipient cell ingression. Following differentiation of the epiblast, clearance of OCT4 from the three germ layers......In the areas of developmental biology and embryonic stem cell research, reliable molecular markers of pluripotency and early lineage commitment are sparse in large animal species. In this study, we present morphological and immunohistochemical findings on the porcine embryo in the period around...... gastrulation, days 8-17 postinsemination, introducing a steromicroscopical staging system in this species. In embryos at the expanding hatched blastocyst stage, OCT4 is confined to the inner cell mass. Following detachment of the hypoblast, and formation of the embryonic disk, this marker of pluripotency...

  18. Virtual reality imaging techniques in the study of embryonic and early placental health.

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    Rousian, Melek; Koster, Maria P H; Mulders, Annemarie G M G J; Koning, Anton H J; Steegers-Theunissen, Régine P M; Steegers, Eric A P

    2018-04-01

    Embryonic and placental growth and development in the first trimester of pregnancy have impact on the health of the fetus, newborn, child and even the adult. This emphasizes the importance of this often neglected period in life. The development of three-dimensional transvaginal ultrasonography in combination with virtual reality (VR) opens the possibility of accurate and reliable visualization of embryonic and placental structures with real depth perception. These techniques enable new biometry and volumetry measurements that contribute to the knowledge of the (patho)physiology of embryonic and early placental health. Examples of such measurements are the length of complex structures like the umbilical cord, vitelline duct, limbs and cerebellum or the volume of the whole embryo and brain cavities. Moreover, for the first time, embryos can now be staged in vivo (Carnegie stages) and vasculature volumes of both the embryo and the early placenta can be measured when VR is combined with power Doppler signals. These innovative developments have already been used to study associations between periconceptional maternal factors, such as age, smoking, alcohol use, diet and vitamin status, and embryonic and early placental growth and development. Future studies will also focus on the identification of abnormal embryonic and early placental development already in the earliest weeks of pregnancy, which provides opportunities for early prevention of pregnancy complications. Copyright © 2018 IFPA, Elsevier Ltd. Published by Elsevier Ltd.. All rights reserved.

  19. Melatonin prevents postovulatory oocyte aging and promotes subsequent embryonic development in the pig.

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    Wang, Tao; Gao, Ying-Ying; Chen, Li; Nie, Zheng-Wen; Cheng, Wei; Liu, Xiaoyan; Schatten, Heide; Zhang, Xia; Miao, Yi-Liang

    2017-06-26

    Oxidative stress is known as a major contributing factor involved in oocyte aging, which negatively affects oocyte quality and development after fertilization. Melatonin is an effective free radical scavenger and its metabolites AFMK and AMK are powerful detoxifiers that eliminate free radicals. In this study, we used porcine oocytes to test the hypothesis that melatonin could scavenge free radicals produced during oocyte aging, thereby maintaining oocyte quality. We compared reactive oxygen species levels, apoptosis levels, mitochondrial membrane potential ratios, total glutathione contents and expression levels in fresh, aged and melatonin-treated aged porcine oocytes and observed the percentage of blastocyst formation following parthenogenetic activation. We found that melatonin could effectively maintain the morphology of oocytes observed in control oocytes, alleviate oxidative stress, markedly decrease early apoptosis levels, retard the decline of mitochondrial membrane potential and significantly promote subsequent embryonic development in oocytes aged for 24 hr in vitro . These results strongly suggest that melatonin can prevent postovulatory oocyte aging and promote subsequent embryonic development in the pig, which might find practical applications to control oocyte aging in other mammalian species including humans to maintain the quality of human oocytes when performing clinical assisted reproductive technology.

  20. Transcriptome Landscapes of Mammalian Embryonic Cells

    NARCIS (Netherlands)

    Brinkhof, B.

    2015-01-01

    This thesis describes research on gene expression profiles from different embryonic stages and cell types to identify genes involved in pluripotency or differentiation in bovine and porcine cells. The results are compared with data from other mammals. RNA expression profiles of morula and blastocyst

  1. A Nestin-cre transgenic mouse is insufficient for recombination in early embryonic neural progenitors

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    Huixuan Liang

    2012-09-01

    Nestin-cre transgenic mice have been widely used to direct recombination to neural stem cells (NSCs and intermediate neural progenitor cells (NPCs. Here we report that a readily utilized, and the only commercially available, Nestin-cre line is insufficient for directing recombination in early embryonic NSCs and NPCs. Analysis of recombination efficiency in multiple cre-dependent reporters and a genetic mosaic line revealed consistent temporal and spatial patterns of recombination in NSCs and NPCs. For comparison we utilized a knock-in Emx1cre line and found robust recombination in NSCs and NPCs in ventricular and subventricular zones of the cerebral cortices as early as embryonic day 12.5. In addition we found that the rate of Nestin-cre driven recombination only reaches sufficiently high levels in NSCs and NPCs during late embryonic and early postnatal periods. These findings are important when commercially available cre lines are considered for directing recombination to embryonic NSCs and NPCs.

  2. Combinatorial binding in human and mouse embryonic stem cells identifies conserved enhancers active in early embryonic development.

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    Jonathan Göke

    2011-12-01

    Full Text Available Transcription factors are proteins that regulate gene expression by binding to cis-regulatory sequences such as promoters and enhancers. In embryonic stem (ES cells, binding of the transcription factors OCT4, SOX2 and NANOG is essential to maintain the capacity of the cells to differentiate into any cell type of the developing embryo. It is known that transcription factors interact to regulate gene expression. In this study we show that combinatorial binding is strongly associated with co-localization of the transcriptional co-activator Mediator, H3K27ac and increased expression of nearby genes in embryonic stem cells. We observe that the same loci bound by Oct4, Nanog and Sox2 in ES cells frequently drive expression in early embryonic development. Comparison of mouse and human ES cells shows that less than 5% of individual binding events for OCT4, SOX2 and NANOG are shared between species. In contrast, about 15% of combinatorial binding events and even between 53% and 63% of combinatorial binding events at enhancers active in early development are conserved. Our analysis suggests that the combination of OCT4, SOX2 and NANOG binding is critical for transcription in ES cells and likely plays an important role for embryogenesis by binding at conserved early developmental enhancers. Our data suggests that the fast evolutionary rewiring of regulatory networks mainly affects individual binding events, whereas "gene regulatory hotspots" which are bound by multiple factors and active in multiple tissues throughout early development are under stronger evolutionary constraints.

  3. Expression of early transcription factors Oct-4, Sox-2 and Nanog by porcine umbilical cord (PUC matrix cells

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    Schultz Bruce

    2006-02-01

    Full Text Available Abstract Background Three transcription factors that are expressed at high levels in embryonic stem cells (ESCs are Nanog, Oct-4 and Sox-2. These transcription factors regulate the expression of other genes during development and are found at high levels in the pluripotent cells of the inner cell mass. The downregulation of these three transcription factors correlates with the loss of pluripotency and self-renewal, and the beginning of subsequent differentiation steps. The roles of Nanog, Oct-4 and Sox-2 have not been fully elucidated. They are important in embryonic development and maintenance of pluripotency in ESCs. We studied the expression of these transcription factors in porcine umbilical cord (PUC matrix cells. Methods Cells were isolated from Wharton's jelly of porcine umbilical cords (PUC and histochemically assayed for the presence of alkaline phosphatase and the presence of Nanog, Oct-4 and Sox-2 mRNA and protein. PCR amplicons were sequenced and compared with known sequences. The synthesis of Oct-4 and Nanog protein was analyzed using immunocytochemistry. FACS analysis was utilized to evaluate Hoechst 33342 dye-stained cells. Results PUC isolates were maintained in culture and formed colonies that express alkaline phosphatase. FACS analysis revealed a side population of Hoechst dye-excluding cells, the Hoechst exclusion was verapamil sensitive. Quantitative and non-quantitative RT-PCR reactions revealed expression of Nanog, Oct-4 and Sox-2 in day 15 embryonic discs, PUC cell isolates and porcine fibroblasts. Immunocytochemical analysis detected Nanog immunoreactivity in PUC cell nuclei, and faint labeling in fibroblasts. Oct-4 immunoreactivity was detected in the nuclei of some PUC cells, but not in fibroblasts. Conclusion Cells isolated from PUC express three transcription factors found in pluripotent stem cell markers both at the mRNA and protein level. The presence of these transcription factors, along with the other

  4. Activation of the Aryl Hydrocarbon Receptor Interferes with Early Embryonic Development

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    Manolis Gialitakis

    2017-11-01

    Full Text Available The transcriptional program of early embryonic development is tightly regulated by a set of well-defined transcription factors that suppress premature expression of differentiation genes and sustain the pluripotent identity. It is generally accepted that this program can be perturbed by environmental factors such as chemical pollutants; however, the precise molecular mechanisms remain unknown. The aryl hydrocarbon receptor (AHR is a widely expressed nuclear receptor that senses environmental stimuli and modulates target gene expression. Here, we have investigated the AHR interactome in embryonic stem cells by mass spectrometry and show that ectopic activation of AHR during early differentiation disrupts the differentiation program via the chromatin remodeling complex NuRD (nucleosome remodeling and deacetylation. The activated AHR/NuRD complex altered the expression of differentiation-specific genes that control the first two developmental decisions without affecting the pluripotency program. These findings identify a mechanism that allows environmental stimuli to disrupt embryonic development through AHR signaling.

  5. Effect of Kisspeptin on the Developmental Competence and Early Transcript Expression in Porcine Oocytes Parthenogenetically Activated with Different Methods

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    Islam M. Saadeldin

    2018-01-01

    Full Text Available Recent studies showed the modulatory effect of kisspeptin (KP on calcium waves through the cell membrane and inside the cell. Spermatozoon can induce similar ooplasmic calcium oscillations at fertilization to trigger meiosis II. Here, we evaluated the effect of KP supplementation with 6-dimethylaminopurine (6-DMAP for 4 h on embryonic development after oocyte activation with single electric pulse, 5 µM ionomycin, or 8% ethanol. Compared to control nonsupplemented groups, KP significantly improved embryo developmental competence electric- and ethanol-activated oocytes in terms of cleavage (75.3% and 58.6% versus 64% and 48%, respectively, p<0.05 and blastocyst development (31.3% and 10% versus 19.3% and 4%, respectively, p<0.05. MOS expression was increased in electrically activated oocytes in presence of KP while it significantly reduced CCNB1 expression. In ionomycin treated group, both MOS and CCNB1 showed significant increase with no difference between KP and control groups. In ethanol-treated group, KP significantly reduced CCNB1 but no effect was observed on MOS expression. The early alterations in MOS and CCNB1 mRNA transcripts caused by KP may explain the significant differences in the developmental competence between the experimental groups. Kisspeptin supplementation may be adopted in protocols for porcine oocyte activation through electric current and ethanol to improve embryonic developmental competence.

  6. Breaking down pluripotency in the porcine embryo reveals both a premature and reticent stem cell state in the inner cell mass and unique expression profiles of the naive and primed stem cell states.

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    Hall, Vanessa Jane; Hyttel, Poul

    2014-09-01

    To date, it has been difficult to establish bona fide porcine embryonic stem cells (pESC) and stable induced pluripotent stem cells. Reasons for this remain unclear, but they may depend on inappropriate culture conditions. This study reports the most insights to date on genes expressed in the pluripotent cells of the porcine embryo, namely the inner cell mass (ICM), the trophectoderm-covered epiblast (EPI), and the embryonic disc epiblast (ED). Specifically, we reveal that the early porcine ICM represents a premature state of pluripotency due to lack of translation of key pluripotent proteins, and the late ICM enters a transient, reticent pluripotent state which lacks expression of most genes associated with pluripotency. We describe a unique expression profile of the porcine EPI, reflecting the naive stem cell state, including expression of OCT4, NANOG, CRIPTO, and SSEA-1; weak expression of NrOB1 and REX1; but very limited expression of genes in classical pathways involved in regulating pluripotency. The porcine ED, reflecting the primed stem cell state, can be characterized by the expression of OCT4, NANOG, SOX2, KLF4, cMYC, REX1, CRIPTO, and KLF2. Further cell culture experiments using inhibitors against FGF, JAK/STAT, BMP, WNT, and NODAL pathways on cell cultures derived from day 5 and 10 embryos reveal the importance of FGF, JAK/STAT, and BMP signaling in maintaining cell proliferation of pESCs in vitro. Together, this article provides new insights into the regulation of pluripotency, revealing unique stem cell states in the different porcine stem cell populations derived from the early developing embryo.

  7. Early gene regulation of osteogenesis in embryonic stem cells

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    Kirkham, Glen R.

    2012-01-01

    The early gene regulatory networks (GRNs) that mediate stem cell differentiation are complex, and the underlying regulatory associations can be difficult to map accurately. In this study, the expression profiles of the genes Dlx5, Msx2 and Runx2 in mouse embryonic stem cells were monitored over a 48 hour period after exposure to the growth factors BMP2 and TGFβ1. Candidate GRNs of early osteogenesis were constructed based on published experimental findings and simulation results of Boolean and ordinary differential equation models were compared with our experimental data in order to test the validity of these models. Three gene regulatory networks were found to be consistent with the data, one of these networks exhibited sustained oscillation, a behaviour which is consistent with the general view of embryonic stem cell plasticity. The work cycle presented in this paper illustrates how mathematical modelling can be used to elucidate from gene expression profiles GRNs that are consistent with experimental data. © 2012 The Royal Society of Chemistry.

  8. Transcriptomic profiling of bovine IVF embryos revealed candidate genes and pathways involved in early embryonic development

    Directory of Open Access Journals (Sweden)

    Yandell Brian S

    2010-01-01

    Full Text Available Abstract Background Early embryonic loss is a large contributor to infertility in cattle. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. The objective of this study was to identify genes differentially expressed between blastocysts and degenerative embryos at early stages of development. Results Using microarrays, genome-wide RNA expression was profiled and compared for in vitro fertilization (IVF - derived blastocysts and embryos undergoing degenerative development up to the same time point. Surprisingly similar transcriptomic profiles were found in degenerative embryos and blastocysts. Nonetheless, we identified 67 transcripts that significantly differed between these two groups of embryos at a 15% false discovery rate, including 33 transcripts showing at least a two-fold difference. Several signaling and metabolic pathways were found to be associated with the developmental status of embryos, among which were previously known important steroid biosynthesis and cell communication pathways in early embryonic development. Conclusions This study presents the first direct and comprehensive comparison of transcriptomes between IVF blastocysts and degenerative embryos, providing important information for potential genes and pathways associated with early embryonic development.

  9. Cortical Morphogenesis during Embryonic Development Is Regulated by miR-34c and miR-204

    DEFF Research Database (Denmark)

    Veno, Morten T.; Veno, Susanne T.; Rehberg, Kati

    2017-01-01

    The porcine brain closely resembles the human brain in aspects such as development and morphology. Temporal miRNA profiling in the developing embryonic porcine cortex revealed a distinct set of miRNAs, including miR-34c and miR-204, which exhibited a highly specific expression profile across...

  10. Spatio-temporal regulation of circular RNA expression during porcine embryonic brain development

    DEFF Research Database (Denmark)

    Venø, Morten T; Hansen, Thomas B; Venø, Susanne T

    2015-01-01

    BACKGROUND: Recently, thousands of circular RNAs (circRNAs) have been discovered in various tissues and cell types from human, mouse, fruit fly and nematodes. However, expression of circRNAs across mammalian brain development has never been examined. RESULTS: Here we profile the expression of circ......RNA in five brain tissues at up to six time-points during fetal porcine development, constituting the first report of circRNA in the brain development of a large animal. An unbiased analysis reveals a highly complex regulation pattern of thousands of circular RNAs, with a distinct spatio-temporal expression...... are functionally conserved between mouse and human. Furthermore, we observe that "hot-spot" genes produce multiple circRNA isoforms, which are often differentially expressed across porcine brain development. A global comparison of porcine circRNAs reveals that introns flanking circularized exons are longer than...

  11. Ovarian activity and early embryonic development in the rusty bat ...

    African Journals Online (AJOL)

    The reproductive pattern of the female rusty bat, Pipistrellus rusticus, was investigated by means of a histological examination of the ovarian follicles as well as early embryonic development. Bats were collected from two localities in Limpopo Province. Female rusty bats are seasonal monestrous breeders, initiating ...

  12. Early Embryonic Heart Rate in Normal Pregnancies In Memory of ...

    African Journals Online (AJOL)

    To determine the appearance and development of embryonic heart rate a total of n = 317 Nigerian pregnant women were studied in the very early pregnancy from 23 – 56 days from the onset of last menstrual period (LMP). All pregnancies had a subsequent successful outcome. Transvaginal ultrasonography was ...

  13. Dual effects of fluoxetine on mouse early embryonic development

    International Nuclear Information System (INIS)

    Kim, Chang-Woon; Choe, Changyong; Kim, Eun-Jin; Lee, Jae-Ik; Yoon, Sook-Young; Cho, Young-Woo; Han, Sunkyu; Tak, Hyun-Min; Han, Jaehee; Kang, Dawon

    2012-01-01

    Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50 μM) for different durations. When late 2-cells were incubated with 5 μM fluoxetine for 6 h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetine (5 μM) over 24 h showed a reduction in blastocyst formation. The addition of fluoxetine (5 μM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K + channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ∼ 30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating. Highlights: ► Short-term exposure of 2-cells to fluoxetine enhances mouse blastocyst formation. ► The enhancive effect of fluoxetine is resulted from CaMKII activation

  14. Dual effects of fluoxetine on mouse early embryonic development

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chang-Woon [Department of Physiology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751 (Korea, Republic of); Department of Obstetrics and Gynecology, Samsung Changwon Hospital, Sungkyunkwan University, Changwon 630-723 (Korea, Republic of); Choe, Changyong [National Institute of Animal Science, RDA, Cheonan 330-801 (Korea, Republic of); Kim, Eun-Jin [Department of Physiology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751 (Korea, Republic of); Lee, Jae-Ik [Department of Obstetrics and Gynecology, Gyeongsang National University Hospital, Jinju 660-702 (Korea, Republic of); Yoon, Sook-Young [Fertility Center of CHA Gangnam Medical Center, CHA University, Seoul 135-081 (Korea, Republic of); Cho, Young-Woo; Han, Sunkyu; Tak, Hyun-Min; Han, Jaehee [Department of Physiology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751 (Korea, Republic of); Kang, Dawon, E-mail: dawon@gnu.ac.kr [Department of Physiology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751 (Korea, Republic of)

    2012-11-15

    Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50 μM) for different durations. When late 2-cells were incubated with 5 μM fluoxetine for 6 h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetine (5 μM) over 24 h showed a reduction in blastocyst formation. The addition of fluoxetine (5 μM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K{sup +} channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ∼ 30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating. Highlights: ► Short-term exposure of 2-cells to fluoxetine enhances mouse blastocyst formation. ► The enhancive effect of fluoxetine is resulted from Ca

  15. Targeted DNA Methylation Analysis by High Throughput Sequencing in Porcine Peri-attachment Embryos

    OpenAIRE

    MORRILL, Benson H.; COX, Lindsay; WARD, Anika; HEYWOOD, Sierra; PRATHER, Randall S.; ISOM, S. Clay

    2013-01-01

    Abstract The purpose of this experiment was to implement and evaluate the effectiveness of a next-generation sequencing-based method for DNA methylation analysis in porcine embryonic samples. Fourteen discrete genomic regions were amplified by PCR using bisulfite-converted genomic DNA derived from day 14 in vivo-derived (IVV) and parthenogenetic (PA) porcine embryos as template DNA. Resulting PCR products were subjected to high-throughput sequencing using the Illumina Genome Analyzer IIx plat...

  16. Regulation of bone morphogenetic proteins in early embryonic development

    Science.gov (United States)

    Yamamoto, Yukiyo; Oelgeschläger, Michael

    2004-11-01

    Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

  17. [Establishment of sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo].

    Science.gov (United States)

    Jiang, Hua; Feng, You-Ji; Xie, Yi; Han, Jin-Lan; Wang, Zack; Chen, Tong

    2008-10-14

    To establish a sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo. Human embryonic stem were (hESCs) were cultured on the mouse embryo fibroblasts and then were induced to differentiate to form three-dimensional EB. The hEBs were cultured in media containing various angiogenesis-related factors: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), endostatin, angiostatin, and platelet factor (PF)-4 of different concentrations for 3 days to observe the sprouting of the hEBs. 3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate labeled acetylated low density lipoprotein (Dil-AcLDL) was added onto the hEBs foe 4 h Immunofluorescence assay was used to observe if Dil-AcLDL was absorbed and if CD31 was expressed so as to determine the existence of embryonic endothelial cells in the sprouting structures. The ideal culturing condition was analyzed. The differentiated EBs formed sprouting structures in the collagen I matrix containing VEGF and FGF. The sprouts among individual EBs were able to link to each other and form vascular network-like structures. In the presence of VEGF and FGF, the sprouts branching from the EBs assimilated Dil-AcLDL, expressed CD31 and formed a 3-dimensional cylindrical organization. The concentrations of growth factors ideally stimulating sprouting growth were 100 ng/ml of VEGF and 50 ng/ml of FGF. The networks among the EBs were abolished by the angiostatin, endostatin, and PF4. The sprouting from hEBs accumulates embryonic endothelial cells and the sprouting network-like structures are indeed endothelial in nature. Inducing of sprouting EBs is an ideal model that mimics early embryonic vasculogenesis in humans.

  18. Pluripotent Stem Cell Studies Elucidate the Underlying Mechanisms of Early Embryonic Development

    Directory of Open Access Journals (Sweden)

    Lingyu Li

    2011-03-01

    Full Text Available Early embryonic development is a multi-step process that is intensively regulated by various signaling pathways. Because of the complexity of the embryo and the interactions between the germ layers, it is very difficult to fully understand how these signals regulate embryo patterning. Recently, pluripotent stem cell lines derived from different developmental stages have provided an in vitro system for investigating molecular mechanisms regulating cell fate decisions. In this review, we summarize the major functions of the BMP, FGF, Nodal and Wnt signaling pathways, which have well-established roles in vertebrate embryogenesis. Then, we highlight recent studies in pluripotent stem cells that have revealed the stage-specific roles of BMP,FGF and Nodal pathways during neural differentiation. These findings enhance our understanding of the stepwise regulation of embryo patterning by particular signaling pathways and provide new insight into the mechanisms underlying early embryonic development.

  19. Functional verification of a porcine myostatin propeptide mutant.

    Science.gov (United States)

    Ma, Dezun; Jiang, Shengwang; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Xiao, Gaojun; Yang, Jinzeng; Cui, Wentao

    2015-10-01

    Myostatin is a member of TGF-β superfamily that acts as a key negative regulator in development and growth of embryonic and postnatal muscles. In this study, the inhibitory activities of recombinant porcine myostatin propeptide and its mutated form (at the cleavage site of metalloproteinases of BMP-1/TLD family) against murine myostatin was evaluated in vivo by intraperitoneal injection into mice. Results showed that both wild type and mutated form of porcine propeptide significantly inhibited myostatin activity in vivo. The average body weight of mice receiving wild type propeptide or its mutated form increased by 12.5 % and 24.14%, respectively, compared to mice injected with PBS, implying that the in vivo efficacy of porcine propeptide mutant is greater than its wild type propeptide. Transgenic mice expressing porcine myostatin propeptide mutant were generated to further verify the results obtained from mice injected with recombinant porcine propeptide mutant. Compared with wild type (non-transgenic) mice, relative weight of gastrocnemius, rectusfemoris, and tibialis anterior increased by 22.14 %, 34.13 %, 25.37%, respectively, in transgenic male mice, and by 19.90 %, 42.47 %, 45.61%, respectively, in transgenic female mice. Our data also demonstrated that the mechanism by which muscle growth enhancement is achieved by these propeptides is due to an increase in fiber sizes, not by an increase in number of fiber cells.

  20. Characterization, expression profiles, intracellular distribution and association analysis of porcine PNAS-4 gene with production traits

    Directory of Open Access Journals (Sweden)

    Wang Heng

    2008-06-01

    Full Text Available Abstract Background In a previous screen to identify differentially expressed genes associated with embryonic development, the porcine PNAS-4 gene had been found. Considering differentially expressed genes in early stages of muscle development are potential candidate genes to improve meat quality and production efficiency, we determined how porcine PNAS-4 gene regulates meat production. Therefore, this gene has been sequenced, expression analyzed and associated with meat production traits. Results We cloned the full-length cDNA of porcine PNAS-4 gene encoding a protein of 194 amino acids which was expressed in the Golgi complex. This gene was mapped to chromosome 10, q11–16, in a region of conserved synteny with human chromosome 1 where the human homologous gene was localized. Real-time PCR revealed that PNAS-4 mRNA was widely expressed with highest expression levels in skeletal muscle followed by lymph, liver and other tissues, and showed a down-regulated expression pattern during prenatal development while a up-regulated expression pattern after weaning. Association analysis revealed that allele C of SNP A1813C was prevalent in Chinese indigenous breeds whereas A was dominant allele in Landrace and Large White, and the pigs with homozygous CC had a higher fat content than those of the pigs with other genotypes (P Conclusion Porcine PNAS-4 protein tagged with green fluorescent protein accumulated in the Golgi complex, and its mRNA showed a widespread expression across many tissues and organs in pigs. It may be an important factor affecting the meat production efficiency, because its down-regulated expression pattern during early embryogenesis suggests involvement in increase of muscle fiber number. In addition, the SNP A1813C associated with fat traits might be a genetic marker for molecular-assisted selection in animal breeding.

  1. Molecular cloning and characterization of porcine Na⁺/K⁺-ATPase isoforms α1, α2, α3 and the ATP1A3 promoter.

    Directory of Open Access Journals (Sweden)

    Carina Henriksen

    Full Text Available Na⁺/K⁺-ATPase maintains electrochemical gradients of Na⁺ and K⁺ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na⁺/K⁺-ATPase exists in four different isoforms (α1-α4 encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na⁺/K⁺-ATPase mutations, we have determined the porcine coding sequences of the α1-α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na⁺ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na⁺ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by

  2. Embryonic stem cells in pig and cattle

    DEFF Research Database (Denmark)

    Maddox-Hyttel, Poul; Wolf, Xenia Asbæk; Rasmussen, Mikkel Aabech

    2007-01-01

    Porcine and bovine cell lines derived from the inner cell mass (ICM) or epiblasts of blastocysts have been maintained over extended periods of time and characterized by morphology, identification of some stem cell markers and, in few cases, by production of chimaeric offspring. However, germ line...... transmission in chimaeras has never been obtained. Due to this incomplete characterization of the cell lines, the expression embryonic stem (ES)-like cells is presently used in pig and cattle. The ICM or epiblast can be isolated from the blastocyst by whole blastocyst culture, mechanical isolation......, or immunosurgery, and they are generally cultured on feeder cells. The resulting ES-like cells may be differentiated in vivo by chimaera and teratoma formation or in vitro by embryoid body formation and monolayer induction. It is likely that more well characterized and stable porcine and bovine ES cell lines...

  3. Importance of the pluripotency factor LIN28 in the mammalian nucleolus during early embryonic development.

    Science.gov (United States)

    Vogt, Edgar J; Meglicki, Maciej; Hartung, Kristina Ilka; Borsuk, Ewa; Behr, Rüdiger

    2012-12-01

    The maternal nucleolus is required for proper activation of the embryonic genome (EGA) and early embryonic development. Nucleologenesis is characterized by the transformation of a nucleolar precursor body (NPB) to a mature nucleolus during preimplantation development. However, the function of NPBs and the involved molecular factors are unknown. We uncover a novel role for the pluripotency factor LIN28, the biological significance of which was previously demonstrated in the reprogramming of human somatic cells to induced pluripotent stem (iPS) cells. Here, we show that LIN28 accumulates at the NPB and the mature nucleolus in mouse preimplantation embryos and embryonic stem cells (ESCs), where it colocalizes with the nucleolar marker B23 (nucleophosmin 1). LIN28 has nucleolar localization in non-human primate (NHP) preimplantation embryos, but is cytoplasmic in NHP ESCs. Lin28 transcripts show a striking decline before mouse EGA, whereas LIN28 protein localizes to NPBs at the time of EGA. Following knockdown with a Lin28 morpholino, the majority of embryos arrest between the 2- and 4-cell stages and never develop to morula or blastocyst. Lin28 morpholino-injected embryos arrested at the 2-cell stage were not enriched with nucleophosmin at presumptive NPB sites, indicating that functional NPBs were not assembled. Based on these results, we propose that LIN28 is an essential factor of nucleologenesis during early embryonic development.

  4. TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos.

    Science.gov (United States)

    Cao, Zubing; Hong, Renyun; Ding, Biao; Zuo, Xiaoyuan; Li, Hui; Ding, Jianping; Li, Yunsheng; Huang, Weiping; Zhang, Yunhai

    2017-01-01

    The poor efficiency of animal cloning is mainly attributed to the defects in epigenetic reprogramming of donor cells' chromatins during early embryonic development. Previous studies indicated that inhibition of histone deacetylases or methyltransferase, such as G9A, using Trichostatin A (TSA) or BIX-01294 significantly enhanced the developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos. However, potential mechanisms underlying the improved early developmental competence of SCNT embryos exposed to TSA and BIX-01294 are largely unclear. Here we found that 50 nM TSA or 1.0 μM BIX-01294 treatment alone for 24 h significantly elevated the blastocyst rate (P TSA treatment alone significantly reduced H3K9me2 level at the 4-cell stage, which is comparable with that in in vivo and in vitro fertilized counterparts. However, only co-treatment significantly decreased the levels of 5mC and H3K9me2 in trophectoderm lineage and subsequently increased the expression of OCT4 and CDX2 associated with ICM and TE lineage differentiation. Altogether, these results demonstrate that co-treatment of TSA and BIX-01294 enhances the early developmental competence of porcine SCNT embryos via improvements in epigenetic status and protein expression.

  5. Insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 mediate TGF-β- and myostatin-induced suppression of proliferation in porcine embryonic myogenic cell cultures

    International Nuclear Information System (INIS)

    Kamanga-Sollo, E.; Pampusch, M.S.; White, M.E.; Hathaway, M.R.; Dayton, W.R.

    2005-01-01

    We have previously shown that cultured porcine embryonic myogenic cells (PEMC) produce both insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 and secrete these proteins into their media. Exogenously added recombinant porcine (rp) IGFBP-3 and rpIGFBP-5 act via IGF-dependent and IGF-independent mechanisms to suppress proliferation of PEMC cultures. Furthermore, immunoneutralization of endogenous IGFBP-3 and IGFBP-5 in the PEMC culture medium results in increased DNA synthesis rate suggesting that endogenous IGFBP-3 and IGFBP-5 suppress PEMC proliferation. TGF-β superfamily members myostatin and TGF-β 1 have also been shown to suppress proliferation of myogenic cells, and treatment of cultured PEMC with either TGF-β 1 or myostatin significantly (P 1 and myostatin. Here, we show that immunoneutralization of IGFBP-5 also significantly (P 1 or myostatin-treated PEMC cultures restores Long-R3-IGF-I-stimulated DNA synthesis rates to 90% of the levels observed in control cultures receiving no TGF-β 1 or myostatin treatment (P 1 or myostatin-treated PEMC cultures, phosphosmad2 levels in these cultures were not affected. These findings strongly suggest that IGFBP-3 and IGFBP-5 affect processes downstream from receptor-mediated Smad phosphorylation that facilitate the ability of TGF-β and myostatin to suppress proliferation of PEMC

  6. Disruption of the Sec24d gene results in early embryonic lethality in the mouse.

    Directory of Open Access Journals (Sweden)

    Andrea C Baines

    Full Text Available Transport of newly synthesized proteins from the endoplasmic reticulum (ER to the Golgi is mediated by the coat protein complex COPII. The inner coat of COPII is assembled from heterodimers of SEC23 and SEC24. Though mice with mutations in one of the four Sec24 paralogs, Sec24b, exhibit a neural tube closure defect, deficiency in humans or mice has not yet been described for any of the other Sec24 paralogs. We now report characterization of mice with targeted disruption of Sec24d. Early embryonic lethality is observed in mice completely deficient in SEC24D, while a hypomorphic Sec24d allele permits survival to mid-embryogenesis. Mice haploinsufficient for Sec24d exhibit no phenotypic abnormality. A BAC transgene containing Sec24d rescues the embryonic lethality observed in Sec24d-null mice. These results demonstrate an absolute requirement for SEC24D expression in early mammalian development that is not compensated by the other three Sec24 paralogs. The early embryonic lethality resulting from loss of SEC24D in mice contrasts with the previously reported mild skeletal phenotype of SEC24D deficiency in zebrafish and restricted neural tube phenotype of SEC24B deficiency in mice. Taken together, these observations suggest that the multiple Sec24 paralogs have developed distinct functions over the course of vertebrate evolution.

  7. Gene function in early mouse embryonic stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Campbell Pearl A

    2007-03-01

    Full Text Available Abstract Background Little is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5 undergoing undirected differentiation into embryoid bodies (EBs over a period of two weeks. Results We identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1, our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2 that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set. Conclusion Our analysis profiles for the first time gene expression at a very early stage of m

  8. Molecular Cloning and Characterization of Porcine Na+/K+-ATPase Isoforms α1, α2, α3 and the ATP1A3 Promoter

    DEFF Research Database (Denmark)

    Henriksen, Carina; Kjaer-Sorensen, Kasper; Einholm, Anja Pernille

    2013-01-01

    Na+/K+-ATPase maintains electrochemical gradients of Na+ and K+ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na+/K+-ATPase exists in four different isoforms (α1–α4) encoded by different genes. With a view to future use of pig as an animal model...... of the arginine with the C-terminus, stabilizing one of the Na+ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue....... Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which...

  9. Retinoic acid synthesis and functions in early embryonic development

    Directory of Open Access Journals (Sweden)

    Kam Richard Kin Ting

    2012-03-01

    Full Text Available Abstract Retinoic acid (RA is a morphogen derived from retinol (vitamin A that plays important roles in cell growth, differentiation, and organogenesis. The production of RA from retinol requires two consecutive enzymatic reactions catalyzed by different sets of dehydrogenases. The retinol is first oxidized into retinal, which is then oxidized into RA. The RA interacts with retinoic acid receptor (RAR and retinoic acid X receptor (RXR which then regulate the target gene expression. In this review, we have discussed the metabolism of RA and the important components of RA signaling pathway, and highlighted current understanding of the functions of RA during early embryonic development.

  10. Early first trimester maternal ‘high fish and olive oil and low meat’ dietary pattern is associated with accelerated human embryonic development

    NARCIS (Netherlands)

    Parisi, Francesca; Rousian, Melek; Steegers-Theunissen, Régine P.M.; Koning, Anton H.J.; Willemsen, Sten P.; Vries, de Jeanne H.M.; Cetin, Irene; Steegers, Eric A.P.

    2018-01-01

    Background/objectives: Maternal dietary patterns were associated with embryonic growth and congenital anomalies. We aim to evaluate associations between early first trimester maternal dietary patterns and embryonic morphological development among pregnancies with non-malformed outcome.

  11. Characterization of the onset of embryonic control and early development in the bovine embryo

    International Nuclear Information System (INIS)

    Barnes, F.L.

    1988-01-01

    Bovine embryos were used to determine if morphological and molecular features of early development are similar to in vivo recovered bovine embryos and to determine at what level early bovine development is regulated. Radiolabeling of IVP embryos and in vivo recovered embryos with 35 S-methionine for SDS-polyacrylamide gel electrophoresis reveals that these embryos are equivalent. Few differences in protein profiles are observed between 1- and early 4-cell embryos. A change in protein profiles begins at the mid 4-cell stage and continues into the 8-cell stage. Few differences in protein profiles are observed between 1- and early 4-cell embryos. A change in protein profiles begins at the mid 4-cell stage and continues into the 8-cell stage. Few differences in protein profiles are observed between late 8-cells and morulae. This transition is α-amanitin sensitive therefore due to de novo embryonic transcription. Embryonic transcription is partially responsible for terminating the post-transcriptionally regulated period of early bovine development. Argentophillic nucleolar organizing regions (Ag-NORs) indicate onset of nucleolar activation. Ag-NORs were absent in 2- and 4-cell IVP embryos and rarely occurred in 8-cell IVP embryos cultured in vitro. IVP 1- and 2-cell embryos cultured to blastocysts in sheep oviducts demonstrated Ag-NORs. Thus the lack of nucleolar activation of IVP embryos cultured in vitro is culture induced between the 2- and 8-cell stage

  12. DOT1L inhibitor improves early development of porcine somatic cell nuclear transfer embryos

    DEFF Research Database (Denmark)

    Tao, Jia; Zhang, Yu; Zuo, Xiaoyuan

    2017-01-01

    Incomplete epigenetic reprogramming of the genome of donor cells causes poor early and full-term developmental efficiency of somatic cell nuclear transfer (SCNT) embryos. Previous research indicate that inhibition of the histone H3 K79 methyltransferase DOT1L, using a selective pharmacological...... inhibitor EPZ004777 (EPZ), significantly improved reprogramming efficiency during the generation of mouse induced pluripotent stem cells. However, the roles of DOT1L in porcine nuclear transfer-mediated cellular reprogramming are not yet known. Here we showed that DOT1L inhibition via 0.5 nM EPZ treatment...

  13. Assessment of porcine-induced pluripotent stem cells by in vivo assays

    DEFF Research Database (Denmark)

    Secher, Jan Ole Bertelsen; Freude, Karla Kristine; Petkov, Stoyan Gueorguiev

    Concerted efforts have been expended in deriving porcine induced pluripotent stem cells (piPSC) which are envisaged to more faithfully mimic human physiology than existing rodent-derived iPSC lines. While initial piPSC lines, first generated in 2009, exhibit the majority of hallmarks displayed by i......, human and murine episomal reprogramming approaches lead to integration of such transgenes. Thirdly, current culturing conditions fail to support the maintenance of either porcine embryonic stem cells (pESC) or piPSC. Lastly, piPSC are unable to reproducibly contribute to chimeric embryos as demonstrated......PSCs derived from other mammalian species, this is not without some caveats. Firstly, all existing piPSC-like cells are afflicted by insufficient activation of endogenous pluripotency genes. Secondly and associated with this, lack of silencing of exogenous pluripotency genes is a general drawback: in contrast...

  14. Early embryonic development and transplantation in tree shrews

    Directory of Open Access Journals (Sweden)

    Lan-Zhen YAN

    2016-07-01

    Full Text Available As a novel experimental animal model, tree shrews have received increasing attention in recent years. Despite this, little is known in regards to the time phases of their embryonic development. In this study, surveillance systems were used to record the behavior and timing of copulations; embryos at different post-copulation stages were collected and cultured in vitro; and the developmental characteristics of both early-stage and in vitro cultured embryos were determined. A total of 163 females were collected following effective copulation, and 150 were used in either unilateral or bilateral oviduct embryo collections, with 307 embryos from 111 females obtained (conception rate=74%. Among them, 237 embryos were collected from 78 females, bilaterally, i.e., the average embryo number per female was 3.04; 172 fertilized eggs collected from 55 females, bilaterally, were cultured for 24-108 h in vitro for developmental observations; finally, 65 embryos from 23 bilateral cases and 70 embryos from 33 unilateral cases were used in embryo transplantation.

  15. How the embryonic chick brain twists

    OpenAIRE

    Chen, Zi; Guo, Qiaohang; Dai, Eric; Forsch, Nickolas; Taber, Larry A.

    2016-01-01

    During early development, the tubular embryonic chick brain undergoes a combination of progressive ventral bending and rightward torsion, one of the earliest organ-level left–right asymmetry events in development. Existing evidence suggests that bending is caused by differential growth, but the mechanism for the predominantly rightward torsion of the embryonic brain tube remains poorly understood. Here, we show through a combination of in vitro experiments, a physical model of the embryonic m...

  16. Spatio-temporal regulation of ADAR editing during development in porcine neural tissues

    DEFF Research Database (Denmark)

    Venø, Morten Trillingsgaard; Bramsen, Jesper Bertram; Bendixen, Christian

    2012-01-01

    Editing by ADAR enzymes is essential for mammalian life. Still, knowledge of the spatio-temporal editing patterns in mammals is limited. By use of 454 amplicon sequencing we examined the editing status of 12 regionally extracted mRNAs from porcine developing brain encompassing a total of 64...... putative ADAR editing sites. In total 24 brain tissues, dissected from up to five regions from embryonic gestation day 23, 42, 60, 80, 100 and 115, were examined for editing....

  17. Porcine UCHL1: genomic organization, chromosome localization and expression analysis

    DEFF Research Database (Denmark)

    Larsen, Knud; Madsen, Lone Bruhn; Bendixen, Christian

    2012-01-01

    to and protection from Parkinson’s disease. Here we report cloning, characterization, expression analysis and mapping of porcine UCHL1. The UCHL1 cDNA was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The porcine cDNA codes...... in developing porcine embryos. UCHL1 transcript was detected as early as 40 days of gestation. A significant decrease in UCHL1 transcript was detected in basal ganglia from day 60 to day 115 of gestation...

  18. Suppression of decidual cell response induced by tributyltin chloride in pseudopregnant rats: a cause of early embryonic loss

    Energy Technology Data Exchange (ETDEWEB)

    Harazono, A.; Ema, M. [National Inst. of Health Sciences, Osaka Branch (Japan)

    2000-12-01

    In our previous studies, tributyltin chloride (TBTC1) at doses of 16.3 mg/kg and above caused implantation failure (preimplantation embryonic loss) and postimplantation embryonic loss in rats following administration on gestational day (GD) 0 through GD 3 and GD 4 through GD 7, respectively. This study was designed to assess the effects of TBTC1 on uterine function as a cause of early embryonic loss in pseudopregnant rats. TBTC1 was given orally to pseudopregnant rats at doses of 4.1, 8.1, 16.3 and 32.5 mg/kg on pseudopregnant day (PPD) 0 to PPD 3 or 8.1, 16.3, 32.5 and 65.1 mg/kg on PPD 4 to PPD 7. The decidual cell response was induced by bilateral scratch trauma on PPD 4. The uterine weight on PPD 9 served as an index of uterine decidualization. Uterine weight and serum progesterone levels on PPD 9 were significantly decreased after administration of TBTC1 at doses of 16.3 mg/kg and above on PPD 0 to PPD 3 or PPD 4 to PPD 7. Administration of TBTC1 at doses of 8.1 mg/kg and above on PPD 0 to 3 also significantly decreased serum progesterone levels on PPD 4. TBTC1 had no effect on ovarian weight and number of corpora lutea. It can be concluded that TBTC1 suppresses the uterine decidual cell response and decreases progesterone levels, and these effects are responsible for early embryonic loss due to TBTC1 exposure. (orig.)

  19. Early embryonic chromosome instability results in stable mosaic pattern in human tissues.

    Directory of Open Access Journals (Sweden)

    Hasmik Mkrtchyan

    Full Text Available The discovery of copy number variations (CNV in the human genome opened new perspectives on the study of the genetic causes of inherited disorders and the aetiology of common diseases. Here, a single-cell-level investigation of CNV in different human tissues led us to uncover the phenomenon of mitotically derived genomic mosaicism, which is stable in different cell types of one individual. The CNV mosaic ratios were different between the 10 individuals studied. However, they were stable in the T lymphocytes, immortalized B lymphoblastoid cells, and skin fibroblasts analyzed in each individual. Because these cell types have a common origin in the connective tissues, we suggest that mitotic changes in CNV regions may happen early during embryonic development and occur only once, after which the stable mosaic ratio is maintained throughout the differentiated tissues. This concept is further supported by a unique study of immortalized B lymphoblastoid cell lines obtained with 20 year difference from two subjects. We provide the first evidence of somatic mosaicism for CNV, with stable variation ratios in different cell types of one individual leading to the hypothesis of early embryonic chromosome instability resulting in stable mosaic pattern in human tissues. This concept has the potential to open new perspectives in personalized genetic diagnostics and can explain genetic phenomena like diminished penetrance in autosomal dominant diseases. We propose that further genomic studies should focus on the single-cell level, to better understand the aetiology of aging and diseases mediated by somatic mutations.

  20. The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

    DEFF Research Database (Denmark)

    Morovic, Martin; Murin, Matej; Strejcek, Frantisek

    2016-01-01

    in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early......One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription....... In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly...

  1. Physiology and Endocrinology Symposium: The current status of heat shock in early embryonic survival and reproductive efficiency

    Science.gov (United States)

    The Physiology and Endocrinology Symposium entitled “The Current Status of Heat Shock in Early Embryonic Survival and Reproductive Efficiency” was held at the Joint ADSA-CSAS-AMPA-WSAS-ASAS Meeting in Phoenix, Arizona, July 15 to 19, 2012. In recent years, data has accumulated suggesting a role for...

  2. Embryonic vaccines against cancer: an early history.

    Science.gov (United States)

    Brewer, Bradley G; Mitchell, Robert A; Harandi, Amir; Eaton, John W

    2009-06-01

    Almost 100 years have passed since the seminal observations of Schöne showing that vaccination of animals with fetal tissue would prevent the growth of transplantable tumors. Many subsequent reports have affirmed the general idea that immunologic rejection of transplantable tumors, as well as prevention of carcinogenesis, may be affected by vaccination with embryonic/fetal material. Following a decade of intense research on this phenomenon during approximately 1964-1974, interest appears to have waned. This earlier experimental work may be particularly pertinent in view of the rising interest in so-called cancer stem cells. We believe that further work - perhaps involving the use of embryonic stem cells as immunogens - is warranted and that the results reviewed herein support the concept that vaccination against the appearance of cancers of all kinds is a real possibility.

  3. Temporal repression of endogenous pluripotency genes during reprogramming of porcine induced pluripotent stem cells

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane; Christensen, Marianne; Rasmussen, Mikkel Aabech

    2012-01-01

    Porcine induced pluripotent stem cells (piPSCs) have the capacity to differentiate in vitro and in vivo and form chimeras. However, the lack of transgene silencing of exogenous DNA integrated into the genome and the inability of cells to proliferate in the absence of transgene expression...... pluripotency in the pig. This may help to explain the difficulties in producing stable piPSCs and bona fide embryonic stem cell lines in this species....... transgenes on the expression of the porcine endogenous pluripotency machinery. Endogenous and exogenous gene expression of OCT4, NANOG, SOX2, KLF4, and cMYC was determined at passages 5, 10, 15, and 20, both in cells cultured at 1¿µg/mL doxycycline or 4¿µg/mL doxycycline. Our results revealed that endogenous...

  4. Stepwise development of hematopoietic stem cells from embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kenji Matsumoto

    Full Text Available The cellular ontogeny of hematopoietic stem cells (HSCs remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit(+CD41(+CD45(- phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. We propose an HSC developmental model in which pre-HSCs and embryonic HSCs sequentially give rise to adult types of HSCs in a stepwise manner.

  5. Early embryonic failure: Expression and imprinted status of candidate genes on human chromosome 21

    Energy Technology Data Exchange (ETDEWEB)

    Sherman, L.S.; Bennett, P.R.; Moore, G.E. [Queen Charlotte`s and Chelsea Hospital, London (United Kingdom)

    1994-09-01

    Two cases of maternal uniparental (hetero)disomy for human chromosome 21 (mUPD21) have been identified in a systematic search for UPD in 23 cases of early embryonic failure (EEF). Bi-parental origin of the other chromosome pairs was confirmed using specific VNTR probes or dinucleotide repeat analysis. Both maternally and paternally derived isochromosomes 21q have previously been identified in two individuals with normal phenotypes. Full UPD21 has a different mechanism of origin than uniparental isochromosome 21q and its effect on imprinted genes and phenotypic outcome will therefore not necessarily be the same. EEF associated with mUPD21 suggests that developmentally important genes on HSA 21 may be imprinted such that they are only expressed from either the maternally or paternally derived alleles. We have searched for monoallelic expression of candidate genes on HSA 21 in human pregnancy (CBS, IFNAR, COL6A1) using intragenic DNA polymorphisms. These genes were chosen either because their murine homologues lie in imprinted regions or because they are potentially important in embryogenesis. Once imprinted candidate genes have been identified, their methylation status and expression in normal, early embryonic failure and uniparental disomy 21 pregnancies will be studied. At the same time, a larger number of cases of EEF are being examined to further investigate the incidence of UPD21 in this group.

  6. Time-Series Interactions of Gene Expression, Vascular Growth and Hemodynamics during Early Embryonic Arterial Development.

    Directory of Open Access Journals (Sweden)

    Selda Goktas

    Full Text Available The role of hemodynamic forces within the embryo as biomechanical regulators for cardiovascular morphogenesis, growth, and remodeling is well supported through the experimental studies. Furthermore, clinical experience suggests that perturbed flow disrupts the normal vascular growth process as one etiology for congenital heart diseases (CHD and for fetal adaptation to CHD. However, the relationships between hemodynamics, gene expression and embryonic vascular growth are poorly defined due to the lack of concurrent, sequential in vivo data. In this study, a long-term, time-lapse optical coherence tomography (OCT imaging campaign was conducted to acquire simultaneous blood velocity, pulsatile micro-pressure and morphometric data for 3 consecutive early embryonic stages in the chick embryo. In conjunction with the in vivo growth and hemodynamics data, in vitro reverse transcription polymerase chain reaction (RT-PCR analysis was performed to track changes in transcript expression relevant to histogenesis and remodeling of the embryonic arterial wall. Our non-invasive extended OCT imaging technique for the microstructural data showed continuous vessel growth. OCT data coupled with the PIV technique revealed significant but intermitted increases in wall shear stress (WSS between first and second assigned stages and a noticeable decrease afterwards. Growth rate, however, did not vary significantly throughout the embryonic period. Among all the genes studied, only the MMP-2 and CASP-3 expression levels remained unchanged during the time course. Concurrent relationships were obtained among the transcriptional modulation of the genes, vascular growth and hemodynamics-related changes. Further studies are indicated to determine cause and effect relationships and reversibility between mechanical and molecular regulation of vasculogenesis.

  7. Early embryonic androgen exposure induces transgenerational epigenetic and metabolic changes.

    Science.gov (United States)

    Xu, Ning; Chua, Angela K; Jiang, Hong; Liu, Ning-Ai; Goodarzi, Mark O

    2014-08-01

    Androgen excess is a central feature of polycystic ovary syndrome (PCOS), which affects 6% to 10% of young women. Mammals exposed to elevated androgens in utero develop PCOS-like phenotypes in adulthood, suggesting fetal origins of PCOS. We hypothesize that excess androgen exposure during early embryonic development may disturb the epigenome and disrupt metabolism in exposed and unexposed subsequent generations. Zebrafish were used to study the underlying mechanism of fetal origins. Embryos were exposed to androgens (testosterone and dihydrotestosterone) early at 26 to 56 hours post fertilization or late at 21 to 28 days post fertilization. Exposed zebrafish (F0) were grown to adults and crossed to generate unexposed offspring (F1). For both generations, global DNA methylation levels were examined in ovaries using a luminometric methylation assay, and fasting and postprandial blood glucose levels were measured. We found that early but not late androgen exposure induced changes in global methylation and glucose homeostasis in both generations. In general, F0 adult zebrafish exhibited altered global methylation levels in the ovary; F1 zebrafish had global hypomethylation. Fasting blood glucose levels were decreased in F0 but increased in F1; postprandial glucose levels were elevated in both F0 and F1. This androgenized zebrafish study suggests that transient excess androgen exposure during early development can result in transgenerational alterations in the ovarian epigenome and glucose homeostasis. Current data cannot establish a causal relationship between epigenetic changes and altered glucose homeostasis. Whether transgenerational epigenetic alteration induced by prenatal androgen exposure plays a role in the development of PCOS in humans deserves study.

  8. Initial embryology and pluripotent stem cells in the pig - the quest for establishing the pig as a model for cell therapy

    DEFF Research Database (Denmark)

    Secher, Jan; Callesen, Henrik; Freude, Karla Kristine

    2016-01-01

    genetically modified pigs emerged. Over the past years, renewed interest in porcine PSCs has sparked activities in deriving in particular porcine induced pluripotent stem cells to develop the pig as a faithful model for studying the potentials and risks associated with induced pluripotent stem cell......The quest for porcine pluripotent stem cells (PSCs) was initiated in the early 90s. Initially, it was the intention to benefit from these cells for production of genetically modified pigs using homologous recombination followed by derivation of chimeric offspring; a technology that has been used...... to produce genetically modified mice since the mid-80s. However, no convincing reports on the generation of bona fide porcine embryonic stem cells or embryonic germ cells resulted from these activities, and with the advent of somatic cell nuclear transfer during the late 90s, alternative methods for creating...

  9. Pro-apoptotic Effect of Pifithrin-α on Preimplantation Porcine Fertilized Embryo Development

    Directory of Open Access Journals (Sweden)

    Brendan Mulligan

    2012-12-01

    Full Text Available The aim of this study was to investigate the impact of a reported p53 inhibitor, pifithrin-α (PFT-α, on preimplantation porcine in vitro fertilized (IVF embryo development in culture. Treatment of PFT-α was administered at both early (0 to 48 hpi, and later stages (48 to 168 hpi of preimplantation development, and its impact upon the expression of five genes related to apoptosis (p53, bak, bcl-xL, p66Shc and caspase3, was assessed in resulting d 7 blastocysts, using real-time quantitative PCR. Total cell numbers, along with the number of apoptotic nuclei, as detected by the in situ cell death detection assay, were also calculated on d 7 in treated and non-treated control embryos. The results indicate that PFT-α, when administered at both early and later stages of porcine IVF embryo development, increases the incidence of apoptosis in resulting blastocysts. When administered at early cleavage stages, PFT-α treatment was shown to reduce the developmental competence of porcine IVF embryos, as well as reducing the quality of resulting blastocysts in terms of overall cell numbers. In contrast, at later stages, PFT-α administration resulted in marginally increased blastocyst development rates amongst treated embryos, but did not affect cell numbers. However, PFT-α treatment induced apoptosis and apoptotic related gene expression, in all treated embryos, irrespective of the timing of treatment. Our results indicate that PFT-α may severely compromise the developmental potential of porcine IVF embryos, and is a potent apoptotic agent when placed into porcine embryo culture media. Thus, caution should be exercised when using PFT-α as a specific inhibitor of p53 mediated apoptosis, in the context of porcine IVF embryo culture systems.

  10. Diagnostic investigation of porcine periweaning failure-to-thrive syndrome: lack of compelling evidence linking to common porcine pathogens.

    Science.gov (United States)

    Huang, Yanyun; Gauvreau, Henry; Harding, John

    2012-01-01

    Porcine periweaning failure-to-thrive syndrome (PFTS), an increasingly recognized syndrome in the swine industry of North America, is characterized by the anorexia of nursery pigs noticeable within 1 week of weaning, and progressive loss of body condition and lethargy during the next 1-2 weeks. Morbidity caused by PFTS is moderate, but case fatality is high. The etiology of PFTS is presently unknown and may include infectious agent(s), noninfectious factors, or both. PFTS was identified in a high health status farm with good management in early 2007. A diagnostic investigation was undertaken to identify the pathological lesions of, and infectious agents associated with, pigs demonstrating typical clinical signs. Affected (PFTS-SICK) and unaffected (PFTS-HLTHY) pigs from an affected farm, and unaffected pigs from 2 unaffected farms, were examined. The most prevalent lesions in PFTS-SICK pigs were superficial lymphocytic fundic gastritis, atrophic enteritis, superficial colitis, lymphocytic and neutrophilic rhinitis, mild nonsuppurative meningoencephalitis, and thymic atrophy. Rotavirus A and Betacoronavirus 1 (Porcine hemagglutinating encephalomyelitis virus) were identified only in PFTS-SICK pigs, but the significance of the viruses is uncertain because PFTS is not consistent with the typical presentation following infection by these pathogens. Porcine reproductive and respiratory syndrome virus, Porcine circovirus-2, Influenza A virus, Alphacoronavirus 1 (Transmissible gastroenteritis virus), Torque teno virus 1, Brachyspira hyodysenteriae, and Brachyspira pilosicoli were not identified in PFTS-SICK pigs. Suid herpesvirus 2 (Porcine cytomegalovirus), Porcine enteric calicivirus, Torque teno virus 2, pathogenic Escherichia coli, and coccidia were detected in both PFTS-SICK and PFTS-HLTHY pigs. It was concluded that there is a lack of compelling evidence that PFTS is caused by any of these pathogens.

  11. Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions

    DEFF Research Database (Denmark)

    Morgani, Sophie M; Canham, Maurice A; Nichols, Jennifer

    2013-01-01

    Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought...... not directly support Nanog-positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants....

  12. Effects of sorbitol on porcine oocyte maturation and embryo development in vitro.

    Science.gov (United States)

    Lin, Tao; Zhang, Jin Yu; Diao, Yun Fei; Kang, Jung Won; Jin, Dong-Il

    2015-04-01

    In the present study, a porcine system was supplemented with sorbitol during in vitro maturation (IVM) or in vitro culture (IVC), and the effects of sorbitol on oocyte maturation and embryonic development following parthenogenetic activation were assessed. Porcine immature oocytes were treated with different concentrations of sorbitol during IVM, and the resultant metaphase II stage oocytes were activated and cultured in porcine zygote medium-3 (PZM-3) for 7 days. No significant difference was observed in cumulus expansion and the nuclear maturation between the control and sorbitol-treated groups, with the exception of the 100 mM group, which showed significantly decreased nuclear maturation and cumulus expansion. There was no significant difference in the intracellular reactive oxygen species (ROS) levels between oocytes matured with 10 or 20 mM sorbitol and control groups, but 50 and 100 mM groups had significantly higher ROS levels than other groups. The 20 mM group showed significant increases in intracellular glutathione and subsequent blastocyst formation rates following parthenogenetic activation compared with the other groups. During IVC, supplementation with sorbitol significantly reduced blastocyst formation and increased the apoptotic index compared with the control. The apoptotic index of blastocysts from the sorbitol-treated group for entire culture period was significantly higher than those of the partially sorbitol-exposed groups. Based on these findings, it can be concluded that the addition of a low concentration of sorbitol (20 mM) during IVM of porcine oocytes benefits subsequent blastocyst development and improves embryo quality, whereas sorbitol supplement during IVC has a negative effect on blastocyst formation.

  13. How the embryonic chick brain twists.

    Science.gov (United States)

    Chen, Zi; Guo, Qiaohang; Dai, Eric; Forsch, Nickolas; Taber, Larry A

    2016-11-01

    During early development, the tubular embryonic chick brain undergoes a combination of progressive ventral bending and rightward torsion, one of the earliest organ-level left-right asymmetry events in development. Existing evidence suggests that bending is caused by differential growth, but the mechanism for the predominantly rightward torsion of the embryonic brain tube remains poorly understood. Here, we show through a combination of in vitro experiments, a physical model of the embryonic morphology and mechanics analysis that the vitelline membrane (VM) exerts an external load on the brain that drives torsion. Our theoretical analysis showed that the force is of the order of 10 micronewtons. We also designed an experiment to use fluid surface tension to replace the mechanical role of the VM, and the estimated magnitude of the force owing to surface tension was shown to be consistent with the above theoretical analysis. We further discovered that the asymmetry of the looping heart determines the chirality of the twisted brain via physical mechanisms, demonstrating the mechanical transfer of left-right asymmetry between organs. Our experiments also implied that brain flexure is a necessary condition for torsion. Our work clarifies the mechanical origin of torsion and the development of left-right asymmetry in the early embryonic brain. © 2016 The Author(s).

  14. The 'ventral organs' of Pycnogonida (Arthropoda) are neurogenic niches of late embryonic and post-embryonic nervous system development.

    Science.gov (United States)

    Brenneis, Georg; Scholtz, Gerhard

    2014-01-01

    Early neurogenesis in arthropods has been in the focus of numerous studies, its cellular basis, spatio-temporal dynamics and underlying genetic network being by now comparably well characterized for representatives of chelicerates, myriapods, hexapods and crustaceans. By contrast, neurogenesis during late embryonic and/or post-embryonic development has received less attention, especially in myriapods and chelicerates. Here, we apply (i) immunolabeling, (ii) histology and (iii) scanning electron microscopy to study post-embryonic ventral nerve cord development in Pseudopallene sp., a representative of the sea spiders (Pycnogonida), the presumable sister group of the remaining chelicerates. During early post-embryonic development, large neural stem cells give rise to additional ganglion cell material in segmentally paired invaginations in the ventral ectoderm. These ectodermal cell regions - traditionally designated as 'ventral organs' - detach from the surface into the interior and persist as apical cell clusters on the ventral ganglion side. Each cluster is a post-embryonic neurogenic niche that features a tiny central cavity and initially still houses larger neural stem cells. The cluster stays connected to the underlying ganglionic somata cortex via an anterior and a posterior cell stream. Cell proliferation remains restricted to the cluster and streams, and migration of newly produced cells along the streams seems to account for increasing ganglion cell numbers in the cortex. The pycnogonid cluster-stream-systems show striking similarities to the life-long neurogenic system of decapod crustaceans, and due to their close vicinity to glomerulus-like neuropils, we consider their possible involvement in post-embryonic (perhaps even adult) replenishment of olfactory neurons - as in decapods. An instance of a potentially similar post-embryonic/adult neurogenic system in the arthropod outgroup Onychophora is discussed. Additionally, we document two transient posterior

  15. Interaction between SCO-spondin and low density lipoproteins from embryonic cerebrospinal fluid modulates their roles in early neurogenesis

    Directory of Open Access Journals (Sweden)

    América eVera

    2015-05-01

    Full Text Available During early stages of development, encephalic vesicles are composed by a layer of neuroepithelial cells surrounding a central cavity filled with embryonic cerebrospinal fluid (eCSF. This fluid contains several morphogens that regulate proliferation and differentiation of neuroepithelial cells. One of these neurogenic factors is SCO-spondin, a giant protein secreted to the eCSF from early stages of development. Inhibition of this protein in vivo or in vitro drastically decreases the neurodifferentiation process. Other important neurogenic factors of the eCSF are low density lipoproteins (LDL, the depletion of which generates a 60% decrease in mesencephalic explant neurodifferentiation. The presence of several LDL receptor class A (LDLrA domains (responsible for LDL binding in other proteins in the SCO-spondin sequence suggests a possible interaction between both molecules. This possibility was analyzed using three different experimental approaches: 1 Bioinformatics analyses of the SCO-spondin region, that contains eight LDLrA domains in tandem, and of comparisons with the LDL receptor consensus sequence; 2 Analysis of the physical interactions of both molecules through immunohistochemical colocalization in embryonic chick brains and through the immunoprecipitation of LDL with anti-SCO-spondin antibodies; and 3 Analysis of functional interactions during the neurodifferentiation process when these molecules were added to a culture medium of mesencephalic explants. The results revealed that LDL and SCO-spondin interact to form a complex that diminishes the neurogenic capacities that both molecules have separately. Our work suggests that the embryonic cerebrospinal fluid is an active signaling center with a complex regulation system that allows for correct brain development.

  16. The zinc finger transcription factor 191 is required for early embryonic development and cell proliferation

    International Nuclear Information System (INIS)

    Li Jianzhong; Chen Xia; Yang Hua; Wang Shuiliang; Guo Baoyu; Yu Long; Wang Zhugang; Fu Jiliang

    2006-01-01

    Human zinc finger protein 191 (ZNF191/ZNF24) was cloned and characterized as a SCAN family member, which shows 94% identity to its mouse homologue zinc finger protein 191 (Zfp191). ZNF191 can specifically interact with an intronic polymorphic TCAT repeat (HUMTH01) in the tyrosine hydroxylase (TH) gene. Allelic variations of HUMTH01 have been stated to have a quantitative silencing effect on TH gene expression and to correlate with quantitative and qualitative changes in the binding by ZNF191. Zfp191 is widely expressed during embryonic development and in multiple tissues and organs in adult. To investigate the functions of Zfp191 in vivo, we have used homologous recombination to generate mice that are deficient in Zfp191. Heterozygous Zfp191 +/- mice are normal and fertile. Homozygous Zfp191 -/- embryos are severely retarded in development and die at approximately 7.5 days post-fertilization. Unexpectedly, in Zfp191 -/- and Zfp191 +/- embryos, TH gene expression is not affected. Blastocyst outgrowth experiments and the RNA interference-mediated knockdown of ZNF191 in cultured cells revealed an essential role for Zfp191 in cell proliferation. In further agreement with this function, no viable Zfp191 -/- cell lines were obtained by derivation of embryonic stem (ES) cells from blastocysts of Zfp191 +/- intercrosses or by forced homogenotization of heterozygous ES cells at high concentrations of G418. These data show that Zfp191 is indispensable for early embryonic development and cell proliferation

  17. Glycogen and glucose metabolism are essential for early embryonic development of the red flour beetle Tribolium castaneum.

    Directory of Open Access Journals (Sweden)

    Amanda Fraga

    Full Text Available Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3 and hexokinase (HexA genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.

  18. Glycogen and Glucose Metabolism Are Essential for Early Embryonic Development of the Red Flour Beetle Tribolium castaneum

    Science.gov (United States)

    Fraga, Amanda; Ribeiro, Lupis; Lobato, Mariana; Santos, Vitória; Silva, José Roberto; Gomes, Helga; da Cunha Moraes, Jorge Luiz; de Souza Menezes, Jackson

    2013-01-01

    Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen. PMID:23750237

  19. Reproductive Toxicity of Zishen Yutai Pill in Rats: The Fertility and Early Embryonic Development Study (Segment I

    Directory of Open Access Journals (Sweden)

    Li Zhou

    2016-01-01

    Full Text Available Purpose. This study was aimed to investigate the reproductive toxicity of Zishen Yutai Pill (ZYP on fertility and early embryonic development in rats. Methods. SD rats were randomly divided into 5 groups: vehicle control group (distilled water, i.g., positive control group (80 mg/kg of cyclophosphamide, i.p., and three ZYP-treated groups (3, 6, and 12 g/kg/d, i.e., 12x, 24x, and 48x clinical doses, i.g.. The high dose was set as the maximum gavage dosage. Results. Cyclophosphamide showed diverse hazards, such as decreased weight of male reproductive organs and sperm density (P<0.05. However, there were no obvious effects of ZYP on physical signs, animal behavior, and survival rate, as well as on weight and food intake during the premating and gestation periods. Importantly, there were no significant adverse effects of ZYP on indexes of copulation, fecundity and fertility indexes, weights and coefficients of male reproductive organs, epididymal sperm number and motility, estrous cycle, preimplantation loss rate, and implantation rate. Besides, the numbers of live and resorbed fetuses per litter were not significantly altered. Conclusions. ZYP had no reproductive toxicities on fertility and early embryonic development in rats at 48x equivalent clinical doses.

  20. The CMV early enhancer/chicken beta actin (CAG) promoter can be used to drive transgene expression during the differentiation of murine embryonic stem cells into vascular progenitors

    DEFF Research Database (Denmark)

    Alexopoulou, Annika N; Couchman, John R; Whiteford, James

    2008-01-01

    BACKGROUND: Mouse embryonic stem cells cultured in vitro have the ability to differentiate into cells of the three germ layers as well as germ cells. The differentiation mimics early developmental events, including vasculogenesis and early angiogenesis and several differentiation systems are being...... used to identify factors that are important during the formation of the vascular system. Embryonic stem cells are difficult to transfect, while downregulation of promoter activity upon selection of stable transfectants has been reported, rendering the study of proteins by overexpression difficult....... RESULTS: CCE mouse embryonic stem cells were differentiated on collagen type IV for 4-5 days, Flk1+ mesodermal cells were sorted and replated either on collagen type IV in the presence of VEGFA to give rise to endothelial cells and smooth muscle cells or in collagen type I gels for the formation...

  1. Functional characterization of the ER stress induced X-box-binding protein-1 (Xbp-1 in the porcine system

    Directory of Open Access Journals (Sweden)

    Jin Dong-Il

    2011-05-01

    Full Text Available Abstract Background The unfolded protein response (UPR is an evolutionary conserved adaptive reaction for increasing cell survival under endoplasmic reticulum (ER stress conditions. X-box-binding protein-1 (Xbp1 is a key transcription factor of UPR that activates genes involved in protein folding, secretion, and degradation to restore ER function. The UPR induced by ER stress was extensively studied in diseases linked to protein misfolding and aggregations. However, in the porcine system, genes in the UPR pathway were not investigated. In this study, we isolated and characterized the porcine Xbp1 (pXbp1 gene in ER stress using porcine embryonic fibroblast (PEF cells and porcine organs. ER stress was induced by the treatment of tunicamycin and cell viability was investigated by the MTT assay. For cloning and analyzing the expression pattern of pXbp1, RT-PCR analysis and Western blot were used. Knock-down of pXbp1 was performed by the siRNA-mediated gene silencing. Results We found that the pXbp1 mRNA was the subject of the IRE1α-mediated unconventional splicing by ER stress. Knock-down of pXbp1 enhanced ER stress-mediated cell death in PEF cells. In adult organs, pXbp1 mRNA and protein were expressed and the spliced forms were detected. Conclusions It was first found that the UPR mechanisms and the function of pXbp1 in the porcine system. These results indicate that pXbp1 plays an important role during the ER stress response like other animal systems and open a new opportunity for examining the UPR pathway in the porcine model system.

  2. Early embryonic demise: no evidence of abnormal spiral artery transformation or trophoblast invasion.

    Science.gov (United States)

    Ball, E; Robson, S C; Ayis, S; Lyall, F; Bulmer, J N

    2006-03-01

    Invasion by extravillous trophoblast of uterine decidua and myometrium and the associated spiral artery 'transformation' are essential for the development of normal pregnancy. Small pilot studies of placental bed and basal plate tissues from miscarriages have suggested that impaired interstitial and endovascular trophoblast invasion may play a role in the pathogenesis of miscarriage. The hypothesis that early miscarriage is associated with reduced extravillous trophoblast invasion and spiral artery transformation was tested in a large series of placental bed biopsies containing decidua and myometrium and at least one spiral artery from early, karyotyped embryonic miscarriages (early miscarriage and also did not differ significantly from normal pregnancy. These findings suggest that failed trophoblast invasion and spiral artery transformation do not have a pivotal role in the pathogenesis of early miscarriage.

  3. The 'ventral organs' of Pycnogonida (Arthropoda are neurogenic niches of late embryonic and post-embryonic nervous system development.

    Directory of Open Access Journals (Sweden)

    Georg Brenneis

    Full Text Available Early neurogenesis in arthropods has been in the focus of numerous studies, its cellular basis, spatio-temporal dynamics and underlying genetic network being by now comparably well characterized for representatives of chelicerates, myriapods, hexapods and crustaceans. By contrast, neurogenesis during late embryonic and/or post-embryonic development has received less attention, especially in myriapods and chelicerates. Here, we apply (i immunolabeling, (ii histology and (iii scanning electron microscopy to study post-embryonic ventral nerve cord development in Pseudopallene sp., a representative of the sea spiders (Pycnogonida, the presumable sister group of the remaining chelicerates. During early post-embryonic development, large neural stem cells give rise to additional ganglion cell material in segmentally paired invaginations in the ventral ectoderm. These ectodermal cell regions - traditionally designated as 'ventral organs' - detach from the surface into the interior and persist as apical cell clusters on the ventral ganglion side. Each cluster is a post-embryonic neurogenic niche that features a tiny central cavity and initially still houses larger neural stem cells. The cluster stays connected to the underlying ganglionic somata cortex via an anterior and a posterior cell stream. Cell proliferation remains restricted to the cluster and streams, and migration of newly produced cells along the streams seems to account for increasing ganglion cell numbers in the cortex. The pycnogonid cluster-stream-systems show striking similarities to the life-long neurogenic system of decapod crustaceans, and due to their close vicinity to glomerulus-like neuropils, we consider their possible involvement in post-embryonic (perhaps even adult replenishment of olfactory neurons - as in decapods. An instance of a potentially similar post-embryonic/adult neurogenic system in the arthropod outgroup Onychophora is discussed. Additionally, we document two

  4. Inhibition of early AAA formation by aortic intraluminal pentagalloyl glucose (PGG) infusion in a novel porcine AAA model

    DEFF Research Database (Denmark)

    Kloster, Brian O; Lund, Lars; Lindholt, Jes S

    2016-01-01

    to prevent or delay their expansion. In this study, we investigated whether intraluminal delivered pentagalloyl glucose (PGG) can impair the early AAA development in a porcine model. METHODS: The infrarenal aorta was exposed in thirty pigs. Twenty underwent an elastase based AAA inducing procedure and ten...... of these received an additional intraluminal PGG infusion. The final 10 were sham operated and served as controls. RESULTS: All pigs who only had an elastase infusion developed macroscopically expanding AAAs. In pigs treated with an additional PGG infusion the growth rate of the AP-diameter rapidly returned...... and histology. CONCLUSION: In our model, intraluminal delivered PGG is able to penetrate the aortic wall from the inside and impair the early AAA development by stabilizing the elastic lamellae and preserving their integrity. The principle holds a high clinical potential if it can be translated to human...

  5. Ovulation rate and early embryonic survival rate in female rabbits of a synthetic line and a local Algerian population

    Directory of Open Access Journals (Sweden)

    R. Belabbas

    2016-12-01

    Full Text Available A higher litter size at birth has been reported in female rabbits from a Synthetic line than in those of the Local Algerian population. The aim of this work was to analyse whether this difference in litter size was due to a higher ovulation rate and/or embryonic survival rate in Synthetic line than in Local Algerian population. In total, 24 multiparous female rabbits from Synthetic line and 23 from Local population were used in this experiment. Litter size at birth was recorded up to the first 3 parities. Litter size was 20% higher in Synthetic line than Local population. At their 4th gestation, the females were euthanized at 72 h post coitum. Synthetic line females had 50% more ova and embryos than those of Local population (+4.42 ova and +3.92 embryos, respectively. Synthetic line displayed a lower percentage of normal embryos and a larger number of unfertilized oocytes than Local population (–2.81% and +0.64 oocytes, respectively, but differences were not relevant. Synthetic line showed a lesser embryonic stage of development at 72 h post coitum, showing a higher percentage of early morulae (31.50 vs. 8.50% and a lower percentage of compact morulae (51.45 vs. 78.65% than Local population. No relevant difference was found for early embryonic survival rate between Synthetic line and Local population. In conclusion, the difference in litter size was mainly due to a higher ovulation rate in the Synthetic line, allowing more embryos to develop in this line.

  6. Egg formation and the early embryonic development of Aspidogaster limacoides Diesing, 1835 (Aspidogastrea: Aspidogastridae), with comments on their phylogenetic significance

    Czech Academy of Sciences Publication Activity Database

    Świderski, Z.; Poddubnaya, L. G.; Gibson, D. I.; Levron, Céline; Młocicki, D.

    2011-01-01

    Roč. 60, č. 4 (2011), 371-380 ISSN 1383-5769 R&D Projects: GA ČR GAP506/10/1994 Institutional research plan: CEZ:AV0Z60220518 Keywords : Aspidogaster limacoides * Aspidogastrea * Eggshell * Early embryo * Embryonic envelope * Intrauterine eggs * Ultrastructure Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 2.132, year: 2011

  7. Vertebrate Embryonic Cleavage Pattern Determination.

    Science.gov (United States)

    Hasley, Andrew; Chavez, Shawn; Danilchik, Michael; Wühr, Martin; Pelegri, Francisco

    2017-01-01

    The pattern of the earliest cell divisions in a vertebrate embryo lays the groundwork for later developmental events such as gastrulation, organogenesis, and overall body plan establishment. Understanding these early cleavage patterns and the mechanisms that create them is thus crucial for the study of vertebrate development. This chapter describes the early cleavage stages for species representing ray-finned fish, amphibians, birds, reptiles, mammals, and proto-vertebrate ascidians and summarizes current understanding of the mechanisms that govern these patterns. The nearly universal influence of cell shape on orientation and positioning of spindles and cleavage furrows and the mechanisms that mediate this influence are discussed. We discuss in particular models of aster and spindle centering and orientation in large embryonic blastomeres that rely on asymmetric internal pulling forces generated by the cleavage furrow for the previous cell cycle. Also explored are mechanisms that integrate cell division given the limited supply of cellular building blocks in the egg and several-fold changes of cell size during early development, as well as cytoskeletal specializations specific to early blastomeres including processes leading to blastomere cohesion. Finally, we discuss evolutionary conclusions beginning to emerge from the contemporary analysis of the phylogenetic distributions of cleavage patterns. In sum, this chapter seeks to summarize our current understanding of vertebrate early embryonic cleavage patterns and their control and evolution.

  8. BLM has early and late functions in homologous recombination repair in mouse embryonic stem cells

    DEFF Research Database (Denmark)

    Chu, W K; Hanada, K; Kanaar, R

    2010-01-01

    function of BLM remains unclear. Multiple roles have been proposed for BLM in the homologous recombination (HR) repair pathway, including 'early' functions, such as the stimulation of resection of DNA double-strand break ends or displacement of the invading strand of DNA displacement loops, and 'late......' roles, such as dissolution of double Holliday junctions. However, most of the evidence for these putative roles comes from in vitro biochemical data. In this study, we report the characterization of mouse embryonic stem cells with disruption of Blm and/or Rad54 genes. We show that Blm has roles both...

  9. The ‘Ventral Organs’ of Pycnogonida (Arthropoda) Are Neurogenic Niches of Late Embryonic and Post-Embryonic Nervous System Development

    Science.gov (United States)

    Brenneis, Georg; Scholtz, Gerhard

    2014-01-01

    Early neurogenesis in arthropods has been in the focus of numerous studies, its cellular basis, spatio-temporal dynamics and underlying genetic network being by now comparably well characterized for representatives of chelicerates, myriapods, hexapods and crustaceans. By contrast, neurogenesis during late embryonic and/or post-embryonic development has received less attention, especially in myriapods and chelicerates. Here, we apply (i) immunolabeling, (ii) histology and (iii) scanning electron microscopy to study post-embryonic ventral nerve cord development in Pseudopallene sp., a representative of the sea spiders (Pycnogonida), the presumable sister group of the remaining chelicerates. During early post-embryonic development, large neural stem cells give rise to additional ganglion cell material in segmentally paired invaginations in the ventral ectoderm. These ectodermal cell regions – traditionally designated as ‘ventral organs’ – detach from the surface into the interior and persist as apical cell clusters on the ventral ganglion side. Each cluster is a post-embryonic neurogenic niche that features a tiny central cavity and initially still houses larger neural stem cells. The cluster stays connected to the underlying ganglionic somata cortex via an anterior and a posterior cell stream. Cell proliferation remains restricted to the cluster and streams, and migration of newly produced cells along the streams seems to account for increasing ganglion cell numbers in the cortex. The pycnogonid cluster-stream-systems show striking similarities to the life-long neurogenic system of decapod crustaceans, and due to their close vicinity to glomerulus-like neuropils, we consider their possible involvement in post-embryonic (perhaps even adult) replenishment of olfactory neurons – as in decapods. An instance of a potentially similar post-embryonic/adult neurogenic system in the arthropod outgroup Onychophora is discussed. Additionally, we document two transient

  10. Derivation of porcine pluripotent stem cells for biomedical research.

    Science.gov (United States)

    Shiue, Yow-Ling; Yang, Jenn-Rong; Liao, Yu-Jing; Kuo, Ting-Yung; Liao, Chia-Hsin; Kang, Ching-Hsun; Tai, Chein; Anderson, Gary B; Chen, Lih-Ren

    2016-07-01

    Pluripotent stem cells including embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced pluripotent stem cells (iPSCs) are capable of self-renew and limitlessly proliferating in vitro with undifferentiated characteristics. They are able to differentiate in vitro, spontaneously or responding to suitable signals, into cells of all three primary germ layers. Consequently, these pluripotent stem cells will be valuable sources for cell replacement therapy in numerous disorders. However, the promise of human ESCs and EGCs is cramped by the ethical argument about destroying embryos and fetuses for cell line creation. Moreover, there are still carcinogenic risks existing toward the goal of clinical application for human ESCs, EGCs, and iPSCs. Therefore, a suitable animal model for stem cell research will benefit the further development of human stem cell technology. The pigs, on the basis of their similarity in anatomy, immunology, physiology, and biochemical properties, have been wide used as model animals in the study of various human diseases. The development of porcine pluripotent stem cell lines will hold the opportunity to provide an excellent material for human counterpart to the transplantation in biomedical research and further development of cell-based therapeutic strategy. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Knockdown of Fanconi anemia genes in human embryonic stem cells reveals early developmental defects in the hematopoietic lineage.

    Science.gov (United States)

    Tulpule, Asmin; Lensch, M William; Miller, Justine D; Austin, Karyn; D'Andrea, Alan; Schlaeger, Thorsten M; Shimamura, Akiko; Daley, George Q

    2010-04-29

    Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by pediatric bone marrow failure and congenital anomalies. The effect of FA gene deficiency on hematopoietic development in utero remains poorly described as mouse models of FA do not develop hematopoietic failure and such studies cannot be performed on patients. We have created a human-specific in vitro system to study early hematopoietic development in FA using a lentiviral RNA interference (RNAi) strategy in human embryonic stem cells (hESCs). We show that knockdown of FANCA and FANCD2 in hESCs leads to a reduction in hematopoietic fates and progenitor numbers that can be rescued by FA gene complementation. Our data indicate that hematopoiesis is impaired in FA from the earliest stages of development, suggesting that deficiencies in embryonic hematopoiesis may underlie the progression to bone marrow failure in FA. This work illustrates how hESCs can provide unique insights into human development and further our understanding of genetic disease.

  12. Function of FEZF1 during early neural differentiation of human embryonic stem cells.

    Science.gov (United States)

    Liu, Xin; Su, Pei; Lu, Lisha; Feng, Zicen; Wang, Hongtao; Zhou, Jiaxi

    2018-01-01

    The understanding of the mechanism underlying human neural development has been hampered due to lack of a cellular system and complicated ethical issues. Human embryonic stem cells (hESCs) provide an invaluable model for dissecting human development because of unlimited self-renewal and the capacity to differentiate into nearly all cell types in the human body. In this study, using a chemical defined neural induction protocol and molecular profiling, we identified Fez family zinc finger 1 (FEZF1) as a potential regulator of early human neural development. FEZF1 is rapidly up-regulated during neural differentiation in hESCs and expressed before PAX6, a well-established marker of early human neural induction. We generated FEZF1-knockout H1 hESC lines using CRISPR-CAS9 technology and found that depletion of FEZF1 abrogates neural differentiation of hESCs. Moreover, loss of FEZF1 impairs the pluripotency exit of hESCs during neural specification, which partially explains the neural induction defect caused by FEZF1 deletion. However, enforced expression of FEZF1 itself fails to drive neural differentiation in hESCs, suggesting that FEZF1 is necessary but not sufficient for neural differentiation from hESCs. Taken together, our findings identify one of the earliest regulators expressed upon neural induction and provide insight into early neural development in human.

  13. The effect of dietary protein on reproduction in the mare. VII. Embryonic development, early embryonic death, foetal losses and their relationship with serum progestagen

    Directory of Open Access Journals (Sweden)

    F.E. Van Niekerk

    1998-07-01

    Full Text Available Sixty-four Thoroughbred and Anglo-Arab mares aged 6-12 years were randomly allocated to 4 dietary groups and fed diets that differed in the total protein content and quality (essential amino-acids. Forty mares were non-lactating and 24 lactating. Eight mares were withdrawn from the investigation owing to injuries or gynaecological pathology. An overall conception rate of 94.6%and a foaling rate of 80%was achieved. Five of 14 (35.7 % mares (Group 1 fed a low-quality protein diet suffered from early embryonic loss before 90 days of pregnancy compared to 3 of 41 (7.3 % mares in the remaining groups that received the higher-quality protein in their diets. Serum progestagen concentrations of mares in Group 1 that suffered foetal loss were indicative of luteal function insufficiency during the 1st 40 days post-ovulation. Non-lactating mares in all 4 groups gained on average approximately 30 kg in mass during the 90 days before the breeding period. Lactating mares in Group 1 (low-quality protein lost on average 25 kg in mass during lactation, with no weight loss observed among the lactating mares in the other 3 groups. No difference in the diameter of the embryonic vesicle was found between dietary groups until Day 35 of pregnancy.

  14. Light impacts embryonic and early larval development of the European eel, Anguilla anguilla

    DEFF Research Database (Denmark)

    Politis, Sebastian Nikitas; Butts, Ian; Tomkiewicz, Jonna

    2014-01-01

    Little is known about the natural ecology of European eel during early life history. Weextend our understandings on the ecology of this species by studying howearly life stages perform under various light regimes.We assessed the effects of intensity, photoperiod (12:12 and 24:0 h light/dark) and ......Little is known about the natural ecology of European eel during early life history. Weextend our understandings on the ecology of this species by studying howearly life stages perform under various light regimes.We assessed the effects of intensity, photoperiod (12:12 and 24:0 h light...... stages. In particular, for the 12:12 h photoperiod, embryonic survival, until 26 h post-fertilization was significantly higher when reared under low (62 ± 13%) than those reared under high intensity light (42 ± 13%). Furthermore, embryos reared in low light had a higher hatch success (16 ± 7%) than those...... in high intensity light (12 ± 7%). Larval yolk-sac area was significantly affected by photoperiod and body area was significantly affected by the interaction between intensity × photoperiod. The highest incidence of deformities (75%) occurred when embryos were reared in high intensity white light under...

  15. Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

    Science.gov (United States)

    Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

    2015-01-01

    We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

  16. Proteomic Profiling in Early Venous Stenosis Formation in a Porcine Model of Hemodialysis Graft

    Science.gov (United States)

    Misra, Sanjay; Fu, Alex A.; Puggioni, Alessandra; Glockner, James F.; McKusick, Michael A.; Bjarnason, Haraldur; Mukhopadhyay, Debabrata

    2009-01-01

    PURPOSE To use proteomic analysis to identify up- and downregulated proteins in early venous stenosis formation in a porcine model of hemodialysis graft failure. MATERIALS AND METHODS Pigs had chronic renal insufficiency created by subtotal renal infarction caused by renal artery embolization. Arteriovenous polytetrafluoroethylene grafts were placed 28 days later and the animals were killed after a further 3 days (n = 4), 7 days (n = 4), or 14 days (n = 4). Proteomic analysis with isotope-coded affinity tags and multidimensional liquid chromatography followed by tandem mass spectrometry was performed on the venous stenosis and control vessels. Expression of proteins was further confirmed by Western blot analysis. The blood urea nitrogen (BUN) and creatinine levels were determined before renal artery embolization and at the time of graft placement. RESULTS At graft placement, mean BUN and creatinine levels were significantly higher than before embolization (P < .05). Six proteins were identified that were common to all four animals at the same time point. Five proteins (α-fetoprotein, fetuin A, macrophage migration inhibitory factor, pyruvate dehydrogenase E1 component, and lactoferrin) were upregulated and one protein (decorin) was downregulated. Expression of macrophage migration inhibitory factor, α-fetoprotein, and lactoferrin was further validated with Western blotting. By day 14, lactoferrin and fetuin-A expression were increased significantly in early venous stenosis formation. CONCLUSIONS Significantly increased expression of lactoferrin and fetuin-A were observed in early venous stenosis by day 14. Understanding the role of lactoferrin and fetuin-A in hemodialysis vascular access failure could help in improving outcomes in patients undergoing hemodialysis. PMID:19028119

  17. Porcine cholecyst–derived scaffold promotes full-thickness wound healing in rabbit

    Directory of Open Access Journals (Sweden)

    Deepa Revi

    2013-12-01

    Full Text Available Graft-assisted healing is an important strategy for treating full-thickness skin wounds. This study evaluated the properties of porcine cholecyst–derived scaffold and its use for treating full-thickness skin wound in rabbit. The physical properties of cholecyst-derived scaffold were congenial for skin-graft application. Compared to a commercially available skin-graft substitute made of porcine small intestinal submucosa, the cholecyst-derived scaffold was rich in natural biomolecules like elastin and glycosaminoglycans. When used as a xenograft, it promoted healing with excess cell proliferation at early phases and acceptable collagen deposition in the later remodelling phases.

  18. The effect of oxygen tension on porcine embryonic development is dependent on embryo type

    DEFF Research Database (Denmark)

    Booth, Paul; Holm, Peter; Callesen, Henrik

    2005-01-01

    of the embryos prior to culture--a study was performed to examine the effect of O2 tension during culture on three different types of porcine embryos, namely: in vivo flushed embryos, and in vitro matured oocytes either fertilized in vitro or parthenogenetically activated. In vivo embryos (n=208) were flushed...... supplemented with 10% calf serum until day 7. The gas environment for IVM/IVF was 5% CO2 in air, while that for IVC was either 5% CO2 in air or 5% O2, 5% CO2 and 90% N2. Low O2 tension increased both day 7 blastocyst rates (high versus low O2, respectively; 9.3+/-2.9%: 26/280; 23.9+/-4.2%: 71/293; P...

  19. Differentiation of embryonic stem cells towards hematopoietic cells: progress and pitfalls.

    Science.gov (United States)

    Tian, Xinghui; Kaufman, Dan S

    2008-07-01

    Hematopoietic development from embryonic stem cells has been one of the most productive areas of stem cell biology. Recent studies have progressed from work with mouse to human embryonic stem cells. Strategies to produce defined blood cell populations can be used to better understand normal and abnormal hematopoiesis, as well as potentially improve the generation of hematopoietic cells with therapeutic potential. Molecular profiling, phenotypic and functional analyses have all been utilized to demonstrate that hematopoietic cells derived from embryonic stem cells most closely represent a stage of hematopoiesis that occurs at embryonic/fetal developmental stages. Generation of hematopoietic stem/progenitor cells comparable to hematopoietic stem cells found in the adult sources, such as bone marrow and cord blood, still remains challenging. However, genetic manipulation of intrinsic factors during hematopoietic differentiation has proven a suitable approach to induce adult definitive hematopoiesis from embryonic stem cells. Concrete evidence has shown that embryonic stem cells provide a powerful approach to study the early stage of hematopoiesis. Multiple hematopoietic lineages can be generated from embryonic stem cells, although most of the evidence suggests that hematopoietic development from embryonic stem cells mimics an embryonic/fetal stage of hematopoiesis.

  20. Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation

    International Nuclear Information System (INIS)

    Han, Jun; Wang, Qiao-Chu; Zhu, Cheng-Cheng; Liu, Jun; Zhang, Yu; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2016-01-01

    Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates agricultural staples and elicits a complex spectrum of toxic effects on humans and animals. It has been shown that DON impairs oocyte maturation, reproductive function and causes abnormal fetal development in mammals; however, the mechanisms remain unclear. In the present study, we investigate the possible reasons of the toxic effects of DON on porcine oocytes. Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level, which caused retardation of cell cycle progression. In addition, up-regulated LC3 protein expression and aberrant Lamp2, LC3 and mTOR mRNA levels were observed with DON exposure, together with Annexin V-FITC staining assay analysis, these results indicated that DON treatment induced autophagy/apoptosis in porcine oocytes. We also showed that DON exposure increased DNA methylation level in porcine oocytes through altering DNMT3A mRNA levels. Histone methylation levels were also changed showing with increased H3K27me3 and H3K4me2 protein levels, and mRNA levels of their relative methyltransferase genes, indicating that epigenetic modifications were affected. Taken together, our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics, cell cycle, autophagy/apoptosis and epigenetic modifications. - Highlights: • DON exposure disrupted meiotic spindle by reducing p-MAPK expression. • DON exposure caused retardation of cell cycle progression in porcine oocytes. • DON triggered autophagy and early-apoptosis in porcine oocytes. • DON exposure led to aberrant epigenetic modifications in porcine oocytes.

  1. Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation

    Energy Technology Data Exchange (ETDEWEB)

    Han, Jun; Wang, Qiao-Chu; Zhu, Cheng-Cheng; Liu, Jun; Zhang, Yu [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Cui, Xiang-Shun; Kim, Nam-Hyung [Department of Animal Science, Chungbuk National University, Cheongju 361-763 (Korea, Republic of); Sun, Shao-Chen, E-mail: sunsc@njau.edu.cn [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China)

    2016-06-01

    Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates agricultural staples and elicits a complex spectrum of toxic effects on humans and animals. It has been shown that DON impairs oocyte maturation, reproductive function and causes abnormal fetal development in mammals; however, the mechanisms remain unclear. In the present study, we investigate the possible reasons of the toxic effects of DON on porcine oocytes. Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level, which caused retardation of cell cycle progression. In addition, up-regulated LC3 protein expression and aberrant Lamp2, LC3 and mTOR mRNA levels were observed with DON exposure, together with Annexin V-FITC staining assay analysis, these results indicated that DON treatment induced autophagy/apoptosis in porcine oocytes. We also showed that DON exposure increased DNA methylation level in porcine oocytes through altering DNMT3A mRNA levels. Histone methylation levels were also changed showing with increased H3K27me3 and H3K4me2 protein levels, and mRNA levels of their relative methyltransferase genes, indicating that epigenetic modifications were affected. Taken together, our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics, cell cycle, autophagy/apoptosis and epigenetic modifications. - Highlights: • DON exposure disrupted meiotic spindle by reducing p-MAPK expression. • DON exposure caused retardation of cell cycle progression in porcine oocytes. • DON triggered autophagy and early-apoptosis in porcine oocytes. • DON exposure led to aberrant epigenetic modifications in porcine oocytes.

  2. Impaired embryonic development in mice overexpressing the RNA-binding protein TIAR.

    Directory of Open Access Journals (Sweden)

    Yacine Kharraz

    Full Text Available BACKGROUND: TIA-1-related (TIAR protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs. Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translational arrest observed in cells exposed to environmental stress. However, the in vivo role of this protein has not been studied so far mainly due to severe embryonic lethality upon tiar invalidation. METHODOLOGY/PRINCIPAL FINDINGS: To examine potential TIAR tissue-specificity in various cellular contexts, either embryonic or adult, we constructed a TIAR transgenic allele (loxPGFPloxPTIAR allowing the conditional expression of TIAR protein upon Cre recombinase activity. Here, we report the role of TIAR during mouse embryogenesis. We observed that early TIAR overexpression led to low transgene transmission associated with embryonic lethality starting at early post-implantation stages. Interestingly, while pre-implantation steps evolved correctly in utero, in vitro cultured embryos were very sensitive to culture medium. Control and transgenic embryos developed equally well in the G2 medium, whereas culture in M16 medium led to the phosphorylation of eIF2alpha that accumulated in cytoplasmic granules precluding transgenic blastocyst hatching. Our results thus reveal a differential TIAR-mediated embryonic response following artificial or natural growth environment. CONCLUSIONS/SIGNIFICANCE: This study reports the importance of the tightly balanced expression of the RNA-binding protein TIAR for normal embryonic development, thereby emphasizing the role of post-transcriptional regulations in early embryonic programming.

  3. Dual effect of fetal bovine serum on early development depends on stage-specific reactive oxygen species demands in pigs.

    Directory of Open Access Journals (Sweden)

    Seong-Eun Mun

    Full Text Available Despite the application of numerous supplements to improve in vitro culture (IVC conditions of mammalian cells, studies regarding the effect of fetal bovine serum (FBS on mammalian early embryogenesis, particularly in relation to redox homeostasis, are lacking. Herein, we demonstrated that early development of in vitro-produced (IVP porcine embryos highly depends on the combination of FBS supplementation timing and embryonic reactive oxygen species (ROS requirements. Interestingly, FBS significantly reduced intracellular ROS levels in parthenogenetically activated (PA embryos regardless of the developmental stage. However, the beneficial effect of FBS on early embryogenesis was found only during the late phase (IVC 4-6 days treatment group. In particular, developmental competence parameters, such as blastocyst formation rate, cellular survival, total cell number and trophectoderm proportion, were markedly increased by FBS supplementation during the late IVC phase. In addition, treatment with FBS elevated antioxidant transcript levels during the late IVC phase. In contrast, supplementation with FBS during the entire period (1-6 days or during the early IVC phase (1-2 days greatly impaired the developmental parameters. Consistent with the results from PA embryos, the developmental competence of in vitro fertilization (IVF or somatic cell nuclear transfer (SCNT embryos were markedly improved by treatment with FBS during the late IVC phase. Moreover, the embryonic stage-specific effects of FBS were reversed by the addition of an oxidant and were mimicked by treatment with an antioxidant. These findings may increase our understanding of redox-dependent early embryogenesis and contribute to the large-scale production of high-quality IVP embryos.

  4. Global and stage specific patterns of Krüppel-associated-box zinc finger protein gene expression in murine early embryonic cells.

    Directory of Open Access Journals (Sweden)

    Andrea Corsinotti

    Full Text Available Highly coordinated transcription networks orchestrate the self-renewal of pluripotent stem cell and the earliest steps of mammalian development. KRAB-containing zinc finger proteins represent the largest group of transcription factors encoded by the genomes of higher vertebrates including mice and humans. Together with their putatively universal cofactor KAP1, they have been implicated in events as diverse as the silencing of endogenous retroelements, the maintenance of imprinting and the pluripotent self-renewal of embryonic stem cells, although the genomic targets and specific functions of individual members of this gene family remain largely undefined. Here, we first generated a list of Ensembl-annotated KRAB-containing genes encoding the mouse and human genomes. We then defined the transcription levels of these genes in murine early embryonic cells. We found that the majority of KRAB-ZFP genes are expressed in mouse pluripotent stem cells and other early progenitors. However, we also identified distinctively cell- or stage-specific patterns of expression, some of which are pluripotency-restricted. Finally, we determined that individual KRAB-ZFP genes exhibit highly distinctive modes of expression, even when grouped in genomic clusters, and that these cannot be correlated with the presence of prototypic repressive or activating chromatin marks. These results pave the way to delineating the role of specific KRAB-ZFPs in early embryogenesis.

  5. Fucoidan promotes early step of cardiac differentiation from human embryonic stem cells and long-term maintenance of beating areas.

    Science.gov (United States)

    Hamidi, Sofiane; Letourneur, Didier; Aid-Launais, Rachida; Di Stefano, Antonio; Vainchenker, William; Norol, Françoise; Le Visage, Catherine

    2014-04-01

    Somatic stem cells require specific niches and three-dimensional scaffolds provide ways to mimic this microenvironment. Here, we studied a scaffold based on Fucoidan, a sulfated polysaccharide known to influence morphogen gradients during embryonic development, to support human embryonic stem cells (hESCs) differentiation toward the cardiac lineage. A macroporous (pore 200 μm) Fucoidan scaffold was selected to support hESCs attachment and proliferation. Using a protocol based on the cardiogenic morphogen bone morphogenic protein 2 (BMP2) and transforming growth factor (TGFβ) followed by tumor necrosis factor (TNFα), an effector of cardiopoietic priming, we examined the cardiac differentiation in the scaffold compared to culture dishes and embryoid bodies (EBs). At day 8, Fucoidan scaffolds supported a significantly higher expression of the 3 genes encoding for transcription factors marking the early step of embryonic cardiac differentiation NKX2.5 (prelease TGFβ and TNFα was confirmed by Luminex technology. We also found that Fucoidan scaffolds supported the late stage of embryonic cardiac differentiation marked by a significantly higher atrial natriuretic factor (ANF) expression (pstress in the soft hydrogel impaired sarcomere formation, as confirmed by molecular analysis of the cardiac muscle myosin MYH6 and immunohistological staining of sarcomeric α-actinin. Nevertheless, Fucoidan scaffolds contributed to the development of thin filaments connecting beating areas through promotion of smooth muscle cells, thus enabling maintenance of beating areas for up to 6 months. In conclusion, Fucoidan scaffolds appear as a very promising biomaterial to control cardiac differentiation from hESCs that could be further combined with mechanical stress to promote sarcomere formation at terminal stages of differentiation.

  6. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    NARCIS (Netherlands)

    van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during

  7. Regulation of cap-dependent translation initiation in the early stage porcine parthenotes

    Czech Academy of Sciences Publication Activity Database

    Šušor, Andrej; Jelínková, Lucie; Karabínová, Pavla; Torner, H.; Tomek, W.; Kovářová, Hana; Kubelka, Michal

    2008-01-01

    Roč. 75, č. 12 (2008), s. 1716-1725 ISSN 1040-452X R&D Projects: GA ČR GA524/04/0104; GA ČR GA524/07/1087 Institutional research plan: CEZ:AV0Z50450515 Keywords : meiosis * translation initiation * porcine parthenotes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.287, year: 2008

  8. The role of RNA-polymerase II transcription in embryonic nucleologenesis by bovine embryos

    DEFF Research Database (Denmark)

    Kovalská, Mária; Petrovicová, Ida; Strejcek, Frantisek

    2010-01-01

    The early stages of embryonic development are maternally driven. As development proceeds, maternally inherited informational molecules decay, and embryogenesis becomes dependent on de novo synthesized RNAs of embryonic genome. The aim of the present study is to investigate the role of de novo tra...

  9. Porcine endogenous retroviral nucleic acid in peripheral tissues is associated with migration of porcine cells post islet transplant.

    Science.gov (United States)

    Binette, Tanya M; Seeberger, Karen L; Lyon, James G; Rajotte, Ray V; Korbutt, Gregory S

    2004-07-01

    Porcine islets represent an alternative source of insulin-producing tissue, however, porcine endogenous retrovirus (PERV) remains a concern. In this study, SCID mice were transplanted with nonencapsulated (non-EC), microencapsulated (EC) or macroencapsulated (in a TheraCyte trade mark device) neonatal porcine islets (NPIs), and peripheral tissues were screened for presence of viral DNA and mRNA. To understand the role of an intact immune system in PERV incidence, mice with established NPI grafts were reconstituted with splenocytes. Peripheral tissues were screened for PERV and porcine DNA using PCR. Tissues with positive DNA were analyzed for PERV mRNA using RT-PCR. No significant difference was observed between non-EC and EC transplants regarding presence of PERV or porcine-specific DNA or mRNA. In reconstituted animals, little PERV or porcine DNA, and no PERV mRNA was detected. No PERV or porcine-specific DNA was observed in animals implanted with a TheraCyte trade mark device. In conclusion, an intact immune system significantly lowered the presence of PERV. Microencapsulation of islets did not alter PERV presence, however, macroencapsulation in the TheraCyte device did. Lower PERV incidence coincided with lower porcine DNA in peripheral tissues, linking the presence of PERV to migration of porcine cells.

  10. Probing Embryonic Stem Cell Autocrine and Paracrine Signaling Using Microfluidics

    Science.gov (United States)

    Przybyla, Laralynne; Voldman, Joel

    2012-07-01

    Although stem cell fate is traditionally manipulated by exogenously altering the cells' extracellular signaling environment, the endogenous autocrine and paracrine signals produced by the cells also contribute to their two essential processes: self-renewal and differentiation. Autocrine and/or paracrine signals are fundamental to both embryonic stem cell self-renewal and early embryonic development, but the nature and contributions of these signals are often difficult to fully define using conventional methods. Microfluidic techniques have been used to explore the effects of cell-secreted signals by controlling cell organization or by providing precise control over the spatial and temporal cellular microenvironment. Here we review how such techniques have begun to be adapted for use with embryonic stem cells, and we illustrate how many remaining questions in embryonic stem cell biology could be addressed using microfluidic technologies.

  11. Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency.

    Science.gov (United States)

    Pang, Yun-Wei; An, Lei; Wang, Peng; Yu, Yong; Yin, Qiu-Dan; Wang, Xiao-Hong; Xin-Zhang; Qian-Zhang; Yang, Mei-Ling; Min-Guo; Wu, Zhong-Hong; Tian, Jian-Hui

    2013-05-01

    This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10(-6 )M or 10(-8) M, melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10(-10) M melatonin-treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10(-9) M or 10(-12) M melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis-related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7-fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7-fold and 23.2-fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53-mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor-independent, possibly through melatonin's ability to directly scavenge free radicals. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

  12. Delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx.

    Science.gov (United States)

    Meenakumari, Karukayil J; Krishna, Amitabh

    2005-01-01

    The unusual feature of the breeding cycle of Cynopterus sphinx at Varanasi is the significant variation in gestation length of the two successive pregnancies of the year. The aim of this study was to investigate whether the prolongation of the first pregnancy in C. sphinx is due to delayed embryonic development. The first (winter) pregnancy commences in late October and lasts until late March and has a gestation period of about 150 days. The second (summer) pregnancy commences in April and lasts until the end of July or early August with a gestation period of about 125 days. Changes in the size and weight of uterine cornua during the two successive pregnancies suggest retarded embryonic growth during November and December. Histological analysis during the period of retarded embryonic development in November and December showed a slow gastrulation process. The process of amniogenesis was particularly slow. When the embryos attained the early primitive streak stage, their developmental rate suddenly increased considerably. During the summer pregnancy, on the other hand, the process of gastrulation was much faster and proceeded quickly. A comparison of the pattern of embryonic development for 4 consecutive years consistently showed retarded or delayed embryonic development during November and December. The time of parturition and post-partum oestrus showed only a limited variation from 1 year to another. This suggests that delayed embryonic development in C. sphinx may function to synchronize parturition among females. The period of delayed embryonic development in this species clearly coincides with the period of fat deposition. The significance of this correlation warrants further investigation.

  13. Cigarette smoking during early pregnancy reduces the number of embryonic germ and somatic cells

    DEFF Research Database (Denmark)

    Mamsen, Linn; Lutterodt, M C; Andersen, Elisabeth Anne Wreford

    2010-01-01

    BACKGROUND: Cigarette smoking during pregnancy is associated with negative reproductive consequences for male fetuses in adult life such as reduced testicular volume and sperm concentration. The present study evaluates the number of germ and somatic cells present in human embryonic first-trimeste......BACKGROUND: Cigarette smoking during pregnancy is associated with negative reproductive consequences for male fetuses in adult life such as reduced testicular volume and sperm concentration. The present study evaluates the number of germ and somatic cells present in human embryonic first......-trimester gonads in relation to maternal smoking. METHODS: The study includes 24 human first-trimester testes, aged 37-68 days post-conception, obtained from women undergoing legal termination of pregnancy. A questionnaire was used to obtain information about smoking and drinking habits during pregnancy. Validated...... confounders such as alcohol and coffee consumption (P = 0.002). The number of germ cells in embryonic gonads, irrespective of gender, was also significantly reduced by 41% (95% CI 58-19%, P = 0.001) in exposed versus non-exposed embryonic gonads. CONCLUSIONS: Prenatal exposure to maternal cigarette smoke...

  14. Toxic effects and possible mechanisms of hydrogen sulfide and/or ammonia on porcine oocyte maturation in vitro.

    Science.gov (United States)

    Yang, Lei-Lei; Zhao, Yong; Luo, Shi-Ming; Ma, Jun-Yu; Ge, Zhao-Jia; Shen, Wei; Yin, Shen

    2018-03-15

    Previous studies suggest that hydrogen sulfide (H 2 S) and ammonia (NH 3 ) are two major air pollutants which can cause damage to porcine health. However, the mechanisms underlying toxic effects of these compounds on porcine oocyte maturation are not clear. To clarify the mechanism, we evaluated the oocyte quality by detecting some events during oocytes maturation. In our study, porcine oocytes were cultured with different concentrations of Na 2 S and/or NH 4 Cl in vitro and the rate of the first polar body extrusion decreased significantly. Also, actin filament was seriously disrupted to damage the cytoskeleton which resulted in reduced rate of oocyte maturation. We explored the reactive oxygen species (ROS) generation and found that the ROS level was increased significantly after Na 2 S treatment but not after NH 4 Cl treatment. Moreover, early stage apoptosis rate was significantly increased and autophagy protein LC3 B expression level was higher in oocytes treated with Na 2 S and/or NH 4 Cl, which might be caused by ROS elevation. Additionally, exposure to Na 2 S and/or NH 4 Cl also caused ROS generation and early apoptosis in cumulus cells, which might further affect oocyte maturation in vitro. In summary, our data suggested that exposure to H 2 S and/or NH 3 decreased porcine oocyte maturation in vitro, which might be caused by actin disruption, ROS generation, early apoptosis and autophagy. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Molecular characterization and analysis of the porcine NURR1 gene

    Directory of Open Access Journals (Sweden)

    Knud Larsen

    2016-12-01

    Here we report the isolation and characterization of porcine NURR1 cDNA. The NURR1 cDNA was RT-PCR cloned using NURR1-specific oligonucleotide primers derived from in silico sequences. The porcine NURR1 cDNA encodes a polypeptide of 598 amino acids, displaying a very high similarity with bovine, human and mouse (99% NURR1 protein. Expression analysis revealed a differential NURR1 mRNA expression in various organs and tissues. NURR1 transcripts could be detected as early as at 60 days of embryo development in different brain tissues. A significant increase in NURR1 transcript in the cerebellum and a decrease in NURR1 transcript in the basal ganglia was observed during embryo development. The porcine NURR1 gene was mapped to chromosome 15. Two missense mutations were found in exon 3, the first coding exon of NURR1. Methylation analysis of the porcine NURR1 gene body revealed a high methylation degree in brain tissue, whereas methylation of the promoter was very low. A decrease in DNA methylation in a discrete region of the NURR1 promoter was observed in pig frontal cortex during pig embryo development. This observation correlated with an increase in NURR1 transcripts. Therefore, methylation might be a determinant of NURR1 expression at certain time points in embryo development.

  16. Carcino-Embryonic Antigen

    International Nuclear Information System (INIS)

    Akute, O.

    1999-02-01

    Tumour marker analysis has increased our understanding of the presence of tumours in the body. Carcino-embryonic antigen, CEA, is one of the best studied tumour markers and has proved an ideal diagnostic adjuvant. It has helped in quantifying the amount of disease present in a patient and thence to make accurate prognosis on the various diagnosed ailments. At UCH, it is observed that there is an increase in cancer related ailments and therefore the need for early diagnosis is more compelling in our environment to mitigate future cost of managing advanced manifestation

  17. Chronology of early embryonic development and embryo uterine migration in alpacas.

    Science.gov (United States)

    Picha, Y; Tibary, A; Memon, M; Kasimanickam, R; Sumar, J

    2013-03-01

    The objectives were to: (1) describe the chronology of early embryonic development from ovulation to entry into the uterus; and (2) to determine the timing of embryo migration to the left uterine horn when ovulation occurred from the right ovary. The experiment was conducted in Peru. Females (n = 132) were randomly assigned to 15 experimental groups. All females were mated to an intact male, given 50 μg GnRH im (Cystorelin) and ovulation time determined by transrectal ultrasonography, conducted every 6 hours, starting 24 hours postmating. Animals were slaughtered at a specific intervals postovulation and reproductive tracts were recovered and subjected to oviductal and uterine flushing for females slaughtered between 1 and 6 days postovulation (dpo; Day 0 = ovulation) and uterine flushing for females slaughtered from 7 to 15 dpo for recovery of oocytes/embryos. Season of mating did not influence the interval from mating to ovulation (winter: 29 ± 6 hours vs. summer: 30 ± 6 hours; P = 0.49). Ovulation rates for females mated during winter and summer were 92% versus 100%, respectively (P = 0.05). Fertilization rates for winter and summer mated females were 72% and 82% (P = 0.29). Unfertilized ova were not retained in the uterine tube. All embryos collected were in the uterine tube ipsilateral to the side of ovulation between 1 and 5 dpo. Embryos reached the uterus on 6 dpo. Embryos began to elongate on 9 dpo; at this time, 83% of embryos derived from right-ovary ovulations were collected from the left uterine horn. Embryos occupied the entire uterine cavity by 10 dpo. In conclusion, we characterized early embryo development and location of embryo during its early developmental stages in alpaca. This was apparently the first report regarding chronology of embryo development and migration to the left horn in alpaca which merits further investigation regarding its role in maternal recognition of pregnancy. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Dynamic expression of calretinin in embryonic and early fetal human cortex

    Directory of Open Access Journals (Sweden)

    Miriam eGonzalez-Gomez

    2014-06-01

    Full Text Available Calretinin (CR is one of the earliest neurochemical markers in human corticogenesis. In embryos from Carnegie stages (CS 17 to 23, calbindin (CB and CR stain opposite poles of the incipient cortex suggesting early regionalization: CB marks the neuroepithelium of the medial boundary of the cortex with the choroid plexus (cortical hem. By contrast, CR is confined to the subventricular zone (SVZ of the lateral and caudal ganglionic eminences at the pallial-subpallial boundary (PSB, or antihem, from where CR+/Tbr1- neurons migrate toward piriform cortex and amygdala as a component of the lateral cortical stream. At CS 19, columns of CR+ cells arise in the rostral cortex, and contribute at CS 20 to the monolayer of horizontal Tbr1+/CR+ and GAD+ cells in the preplate. At CS 21, the pioneer cortical plate appears as a radial aggregation of CR+/Tbr1+ neurons, which cover the entire future neocortex and extend the first corticofugal axons. CR expression in early human corticogenesis is thus not restricted to interneurons, but is also present in the first excitatory projection neurons of the cortex. At CS 21/22, the cortical plate is established following a lateral to medial gradient, when Tbr1+/CR- neurons settle within the pioneer cortical plate, and thus separate superficial and deep pioneer neurons. CR+ pioneer neurons disappear shortly after the formation of the cortical plate. Reelin+ Cajal-Retzius cells begin to express CR around CS21 (7/8 PCW. At CS 21-23, the CR+ SVZ at the PSB is the source of CR+ interneurons migrating into the cortical SVZ. In turn, CB+ interneurons migrate from the subpallium into the intermediate zone following the fibers of the internal capsule. Early CR+ and CB+ interneurons thus have different origins and migratory routes. CR+ cell populations in the embryonic telencephalon take part in a complex sequence of events not analyzed so far in other mammalian species, which may represent a distinctive trait of the initial steps

  19. Somatic donor cell type correlates with embryonic, but not extra-embryonic, gene expression in postimplantation cloned embryos.

    Directory of Open Access Journals (Sweden)

    Ryutaro Hirasawa

    Full Text Available The great majority of embryos generated by somatic cell nuclear transfer (SCNT display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts. The embryos retrieved from the uteri were separated into embryonic (epiblast and extraembryonic (extraembryonic ectoderm and ectoplacental cone tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs (>2-fold vs. controls than did the extraembryonic tissues (P<1.0 × 10(-26. In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1-5% per embryos transferred in our laboratory, because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.

  20. 7 CFR 1230.611 - Porcine animal.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Porcine animal. 1230.611 Section 1230.611 Agriculture... CONSUMER INFORMATION Procedures for the Conduct of Referendum Definitions § 1230.611 Porcine animal. The term Porcine animal means a swine, that is raised: (a) As a feeder pig, that is, a young pig sold to...

  1. Temperature profiles of different cooling methods in porcine pancreas procurement.

    Science.gov (United States)

    Weegman, Bradley P; Suszynski, Thomas M; Scott, William E; Ferrer Fábrega, Joana; Avgoustiniatos, Efstathios S; Anazawa, Takayuki; O'Brien, Timothy D; Rizzari, Michael D; Karatzas, Theodore; Jie, Tun; Sutherland, David E R; Hering, Bernhard J; Papas, Klearchos K

    2014-01-01

    Porcine islet xenotransplantation is a promising alternative to human islet allotransplantation. Porcine pancreas cooling needs to be optimized to reduce the warm ischemia time (WIT) following donation after cardiac death, which is associated with poorer islet isolation outcomes. This study examines the effect of four different cooling Methods on core porcine pancreas temperature (n = 24) and histopathology (n = 16). All Methods involved surface cooling with crushed ice and chilled irrigation. Method A, which is the standard for porcine pancreas procurement, used only surface cooling. Method B involved an intravascular flush with cold solution through the pancreas arterial system. Method C involved an intraductal infusion with cold solution through the major pancreatic duct, and Method D combined all three cooling Methods. Surface cooling alone (Method A) gradually decreased core pancreas temperature to <10 °C after 30 min. Using an intravascular flush (Method B) improved cooling during the entire duration of procurement, but incorporating an intraductal infusion (Method C) rapidly reduced core temperature 15-20 °C within the first 2 min of cooling. Combining all methods (Method D) was the most effective at rapidly reducing temperature and providing sustained cooling throughout the duration of procurement, although the recorded WIT was not different between Methods (P = 0.36). Histological scores were different between the cooling Methods (P = 0.02) and the worst with Method A. There were differences in histological scores between Methods A and C (P = 0.02) and Methods A and D (P = 0.02), but not between Methods C and D (P = 0.95), which may highlight the importance of early cooling using an intraductal infusion. In conclusion, surface cooling alone cannot rapidly cool large (porcine or human) pancreata. Additional cooling with an intravascular flush and intraductal infusion results in improved core porcine pancreas temperature profiles during procurement and

  2. An in vivo characterization of colostrum protein uptake in porcine gut during early lactation

    DEFF Research Database (Denmark)

    Danielsen, Marianne; Pedersen, Lene Juul; Bendixen, Emøke

    2011-01-01

    Understanding the bioactive roles of colostrum proteins has gained much attention, and in particular, their potential use in human and veterinary medicine has been extensively studied. However, studies of bioactivity have mainly been conducted in vitro, but it has not yet been well characterized...... at the individual protein level which colostrum components are internalized by the intestinal tissue of the neonate. The aim of this study was to characterize the in vivo processing of porcine colostrum in the gastrointestinal tract, and describe which of the potential bioactive proteins can be observed...... in the small intestinal tissue, and therefore may be functionally important. Using 2D-LC-MS/MS analysis we mapped the proteins in porcine colostrum. The colostrum proteins were then traced in the stomach content, as well as in the small intestinal tissue of 5 piglets suckled for 24 h. For comparison, we also...

  3. Embryonic chirality and the evolution of spiralian left–right asymmetries

    Science.gov (United States)

    2016-01-01

    The group Spiralia includes species with one of the most significant cases of left–right asymmetries in animals: the coiling of the shell of gastropod molluscs (snails). In this animal group, an early event of embryonic chirality controlled by cytoskeleton dynamics and the subsequent differential activation of the genes nodal and Pitx determine the left–right axis of snails, and thus the direction of coiling of the shell. Despite progressive advances in our understanding of left–right axis specification in molluscs, little is known about left–right development in other spiralian taxa. Here, we identify and characterize the expression of nodal and Pitx orthologues in three different spiralian animals—the brachiopod Novocrania anomala, the annelid Owenia fusiformis and the nemertean Lineus ruber—and demonstrate embryonic chirality in the biradial-cleaving spiralian embryo of the bryozoan Membranipora membranacea. We show asymmetric expression of nodal and Pitx in the brachiopod and annelid, respectively, and symmetric expression of Pitx in the nemertean. Our findings indicate that early embryonic chirality is widespread and independent of the cleavage programme in the Spiralia. Additionally, our study illuminates the evolution of nodal and Pitx signalling by demonstrating embryonic asymmetric expression in lineages without obvious adult left–right asymmetries. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’. PMID:27821523

  4. High efficient differentiation of functional hepatocytes from porcine induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Ying Ao

    Full Text Available Hepatocyte transplantation is considered to be a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs provide an unlimited source for the generation of functional hepatocytes. In this study, we generated iPSCs from porcine ear fibroblasts (PEFs by overexpressing Sox2, Klf4, Oct4, and c-Myc (SKOM, and developed a novel strategy for the efficient differentiation of hepatocyte-like cells from porcine iPSCs by following the processes of early liver development. The differentiated cells displayed the phenotypes of hepatocytes, exhibited classic hepatocyte-associated bio-functions, such as LDL uptake, glycogen storage and urea secretion, as well as possessed the metabolic activities of cytochrome P-450 (CYP 3A and 2C. Furthermore, we compared the hepatocyte differentiation efficacy of our protocol with another published method, and the results demonstrated that our differentiation strategy could significantly improve the generation of morphological and functional hepatocyte-like cells from porcine iPSCs. In conclusion, this study establishes an efficient method for in vitro generation of functional hepatocytes from porcine iPSCs, which could represent a promising cell source for preclinical testing of cell-based therapeutics for liver failure and for pharmacological applications.

  5. Low oxygen levels slow embryonic development of Limulus polyphemus

    DEFF Research Database (Denmark)

    Funch, Peter; Wang, Tobias; Pertoldi, Cino

    2016-01-01

    The American horseshoe crab Limulus polyphemus typically spawns in the upper intertidal zone, where the developing embryos are exposed to large variations in abiotic factors such as temperature, humidity, salinity, and oxygen, which affect the rate of development. It has been shown that embryonic...... pronounced hypoxia in later embryonic developmental stages, but also in earlier, previously unexplored, developmental stages....... development is slowed at both high and low salinities and temperatures, and that late embryos close to hatching tolerate periodic hypoxia. In this study we investigated the influence of hypoxia on both early and late embryonic development in L. polyphemus under controlled laboratory conditions. Embryos were...

  6. Quantitative phosphoproteomic analysis of porcine muscle within 24 h postmortem

    DEFF Research Database (Denmark)

    Huang, Honggang; Larsen, Martin Røssel; Palmisano, Giuseppe

    2014-01-01

    in meat quality development, a quantitative mass spectrometry-based phosphoproteomic study was performed to analyze the porcine muscle within 24h PM using dimethyl labeling combined with the TiSH phosphopeptide enrichment strategy. In total 305 unique proteins were identified, including 160...... phosphorylation levels in muscle within 24 h PM. The high phosphorylation level of heat shock proteins (HSPs) in early PM may be an adaptive response to slaughter stress and protect muscle cell from apoptosis, as observed in the serine 84 of HSP27. This work indicated that PM muscle proteins underwent significant...... and rigor mortis development in PM muscle. BIOLOGICAL SIGNIFICANCE: The manuscript describes the characterization of postmortem (PM) porcine muscle within 24 h postmortem from the perspective of protein phosphorylation using advanced phosphoproteomic techniques. In the study, the authors employed...

  7. Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Joseph Landry

    2008-10-01

    Full Text Available We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor, the largest subunit of NURF (Nucleosome Remodeling Factor in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf(-/- embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf(-/- embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

  8. Monoclonal antibodies specific to heat-treated porcine blood.

    Science.gov (United States)

    Raja Nhari, Raja Mohd Hafidz; Hamid, Muhajir; Rasli, Nurmunirah Mohamad; Omar, Abdul Rahman; El Sheikha, Aly Farag; Mustafa, Shuhaimi

    2016-05-01

    Porcine blood is potentially being utilized in food as a binder, gelling agent, emulsifier or colorant. However, for certain communities, the usage of animal blood in food is strictly prohibited owing to religious concerns and health reasons. This study reports the development of monoclonal antibodies (MAbs) against heat-treated soluble proteins (HSPs) of autoclaved porcine blood; characterization of MAbs against blood, non-blood and plasma from different animal species using qualitative indirect non-competitive enzyme-linked immunosorbent assay (ELISA); and immunoblotting of antigenic components in HSPs of porcine blood. Fifteen MAbs are specific to heat-treated and raw porcine blood and not cross-reacted with other animal blood and non-blood proteins (meat and non-meat). Twelve MAbs are specific to porcine plasma, while three MAbs specific to porcine plasma are cross-reacted with chicken plasma. Immunoblotting revealed antigenic protein bands (∼60, ∼85-100 and ∼250 kDa) in porcine blood and plasma recognized by the MAbs. Selection of MAbs that recognized 60 kDa HSPs of porcine blood and plasma as novel monoclonal antibodies would be useful for detection of porcine plasma in processed food using the immunoassay method. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  9. Are there factors preventing cancer development during embryonic life

    International Nuclear Information System (INIS)

    Einhorn, L.

    1983-01-01

    On the basis of the following literature observations, a hypothesis is advanced that the development of cancer is actively inhibited during embryonic life. Although the processes of cell differentiation and proliferation are - without comparison - most pronounced during embryonic life, cancer is rarely found in the newborn and is seldom a cause of neonatal death or spontaneous abortion. Attempts to induce cancer in early-stage animal embryos by irradiation or by transplacental chemical carcinogenesis have been unsuccessful, even when exposed animals have been observed throughout their lifetime. After the period of major organogenesis, however, the embryos become susceptible to carcinogenesis. In humans, the most common embryonic tumors arise in tissues which have an unusually late ongoing development and are still partly immature at or shortly before birth. For many human embryonic tumors the survival rates are higher, and spontaneous regression more frequent, in younger children, i.e. prognosis is age-dependent. Thus, although cancer generally appears in tissues capable of proliferation and differentiation, induction of malignancy in the developmentally most active tissues seems to be beset with difficulty. One possible explanation for this paradox could be that cancer is controlled by the regulators influencing development, regulators that are most active during embryonic life. (Auth.)

  10. Endolymphatic potassium of the chicken vestibule during embryonic development.

    Science.gov (United States)

    Masetto, Sergio; Zucca, Giampiero; Bottà, Luisa; Valli, Paolo

    2005-08-01

    The endolymph fills the lumen of the inner ear membranous labyrinth. Its ionic composition is unique in vertebrates as an extracellular fluid for its high-K(+)/low-Na(+) concentration. The endolymph is actively secreted by specialized cells located in the vestibular and cochlear epithelia. We have investigated the early phases of endolymph secretion by measuring the endolymphatic K(+) concentration in the chicken vestibular system during pre-hatching development. Measurements were done by inserting K(+)-selective microelectrodes in chicken embryo ampullae dissected at different developmental stages from embryonic day 9 up to embryonic day 21 (day of hatching). We found that the K(+) concentration is low (<10mM/L) up to embryonic day 11, afterward it increases steeply to reach a plateau level of about 140 mM/L at embryonic day 19--21. We have developed a short-term in vitro model of endolymph secretion by culturing vestibular ampullae dissected from embryonic day 11 chicken embryos for a few days. The preparation reproduced a double compartment system where the luminal K(+) concentration increased along with the days of culturing. This model could be important for (1) investigating the development of cellular mechanisms contributing to endolymph homeostasis and (2) testing compounds that influence those mechanisms.

  11. Porcine respiratory disease complex: Interaction of vaccination and porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, and Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Chae, Chanhee

    2016-06-01

    Porcine respiratory disease is a multifactorial and complex disease caused by a combination of infectious pathogens, environmental stressors, differences in production systems, and various management practices; hence the name porcine respiratory disease complex (PRDC) is used. Porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and Mycoplasma hyopneumoniae are considered to be the most important pathogens that cause PRDC. Although interactions among the three major respiratory pathogens are well documented, it is also necessary to understand the interaction between vaccines and the three major respiratory pathogens. PRRSV and M. hyopneumoniae are well known to potentiate PCV2-associated lesions; however, PRRSV and mycoplasmal vaccines can both enhance PCV2 viraemia regardless of the effects of the actual PRRSV or M. hyopneumoniae infection. On the other hand, M. hyopneumoniae potentiates the severity of pneumonia induced by PRRSV, and vaccination against M. hyopneumoniae alone is also able to decrease PRRSV viraemia and PRRSV-induced lung lesions in dually infected pigs. This review focuses on (1) interactions between PCV2, PRRSV, and M. hyopneumoniae; and (2) interactions between vaccines and the three major respiratory pathogens. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Pang, Daxin, E-mail: pdx@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Ouyang, Hongsheng, E-mail: ouyh@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China)

    2011-07-29

    Highlights: {yields} Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. {yields} The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. {yields} A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 {mu}g/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  13. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    International Nuclear Information System (INIS)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue; Pang, Daxin; Ouyang, Hongsheng

    2011-01-01

    Highlights: → Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. → The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. → A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 μg/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  14. Germline transgenic pigs by Sleeping Beauty transposition in porcine zygotes and targeted integration in the pig genome.

    Directory of Open Access Journals (Sweden)

    Wiebke Garrels

    Full Text Available Genetic engineering can expand the utility of pigs for modeling human diseases, and for developing advanced therapeutic approaches. However, the inefficient production of transgenic pigs represents a technological bottleneck. Here, we assessed the hyperactive Sleeping Beauty (SB100X transposon system for enzyme-catalyzed transgene integration into the embryonic porcine genome. The components of the transposon vector system were microinjected as circular plasmids into the cytoplasm of porcine zygotes, resulting in high frequencies of transgenic fetuses and piglets. The transgenic animals showed normal development and persistent reporter gene expression for >12 months. Molecular hallmarks of transposition were confirmed by analysis of 25 genomic insertion sites. We demonstrate germ-line transmission, segregation of individual transposons, and continued, copy number-dependent transgene expression in F1-offspring. In addition, we demonstrate target-selected gene insertion into transposon-tagged genomic loci by Cre-loxP-based cassette exchange in somatic cells followed by nuclear transfer. Transposase-catalyzed transgenesis in a large mammalian species expands the arsenal of transgenic technologies for use in domestic animals and will facilitate the development of large animal models for human diseases.

  15. Nitric oxide synthase during early embryonic development in silkworm Bombyx mori: Gene expression, enzyme activity, and tissue distribution.

    Science.gov (United States)

    Kitta, Ryo; Kuwamoto, Marina; Yamahama, Yumi; Mase, Keisuke; Sawada, Hiroshi

    2016-12-01

    To elucidate the mechanism for embryonic diapause or the breakdown of diapause in Bombyx mori, we biochemically analyzed nitric oxide synthase (NOS) during the embryogenesis of B. mori. The gene expression and enzyme activity of B. mori NOS (BmNOS) were examined in diapause, non-diapause, and HCl-treated diapause eggs. In the case of HCl-treated diapause eggs, the gene expression and enzyme activity of BmNOS were induced by HCl treatment. However, in the case of diapause and non-diapause eggs during embryogenesis, changes in the BmNOS activity and gene expressions did not coincide except 48-60 h after oviposition in diapause eggs. The results imply that changes in BmNOS activity during the embryogenesis of diapause and non-diapause eggs are regulated not only at the level of transcription but also post-transcription. The distribution and localization of BmNOS were also investigated with an immunohistochemical technique using antibodies against the universal NOS; the localization of BmNOS was observed mainly in the cytoplasm of yolk cells in diapause eggs and HCl-treated diapause eggs. These data suggest that BmNOS has an important role in the early embryonic development of the B. mori. © 2016 Japanese Society of Developmental Biologists.

  16. Relationship between delayed embryonic development and metabolic factors and fat deposition in fruit bat Cynopterus sphinx.

    Science.gov (United States)

    Banerjee, Arnab; Meenakumari, K J; Krishna, Amitabh

    2007-01-01

    The present study was undertaken in the fruit bat Cynopterus sphinx, which breeds twice in quick succession at Varanasi, India. Its gestation period varies significantly in the two successive pregnancies of the year owing to delayed embryonic development during the first (winter) pregnancy. The primary aim of the present study was to determine the role of metabolic factors in delayed embryonic development in the fruit bat C. sphinx. Variation in bodyweight, fat deposition, oxygen (O(2)) consumption rate, basal metabolic rate (BMR), body temperature (Tb) and hepatic succinate dehydrogenase (SDH) activity, along with circulating levels of thyroid hormones (tri-iodothyronine and thyroxine), were examined as metabolic factors during the two successive pregnancies in C. sphinx. The increase in bodyweight observed in November was due to accumulation of white adipose tissue in the posterior abdominal region. A significant decline in O(2) consumption rate, BMR, Tb and SDH activity was found in early winter in November-December, which coincides closely with the period of fat accumulation and with the period of delayed embryonic development in C. sphinx. A significantly higher O(2) consumption rate, BMR, Tb and SDH activity was noted during the second pregnancy in, when embryonic development was relatively faster. Thyroid hormone levels were high during the period of embryonic delay compared with levels during the remaining months. The results of the present study suggest that the delayed embryonic development in C. sphinx during early winter may be due to a low O(2) consumption rate, BMR, Tb and SDH activity in November-December. The energy saved by suppressing embryonic development in this species may be advantageous for fat accumulation. Increased thyroid hormone levels during the early winter period might facilitate fat accumulation in C. sphinx.

  17. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Gao, Xiugong; Sprando, Robert L.; Yourick, Jeffrey J.

    2015-01-01

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds

  18. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Xiugong, E-mail: xiugong.gao@fda.hhs.gov; Sprando, Robert L.; Yourick, Jeffrey J.

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds.

  19. Interlaboratory testing of porcine sera for antibodies to porcine circovirus type 2

    DEFF Research Database (Denmark)

    McNair, I.; Marshall, M.; McNeilly, F.

    2004-01-01

    A panel of 20 porcine sera was distributed to 5 laboratories across Europe and Canada. Each center was requested to test the sera for the presence of porcine circovirus type 2 antibodies using the routine assays, indirect immunofluorescence assay (IFA) and indirect immunoperoxidase monolayer assa...... than did IFA, and paraformaldehyde gave higher titers than did acetone or ethyl alcohol. This report highlights the need for standardized procedures and biologicals for this virus....

  20. The effect of dietary fatty acids on post-operative inflammatory response in a porcine model

    DEFF Research Database (Denmark)

    Langerhuus, Sine Nygaard; Jensen, Karin Hjelholt; Tønnesen, Else Kirstine

    2012-01-01

    ), sunflower oil (SO, n 28), or animal fat (AF, n 28) was evaluated with respect to post-operative responses in inflammatory markers in a porcine model on aortic vascular prosthetic graft infection. In the early post-operative period (0 necrosis factor...

  1. GLUT3 gene expression is critical for embryonic growth, brain development and survival.

    Science.gov (United States)

    Carayannopoulos, Mary O; Xiong, Fuxia; Jensen, Penny; Rios-Galdamez, Yesenia; Huang, Haigen; Lin, Shuo; Devaskar, Sherin U

    2014-04-01

    Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dose-dependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Effects of Exogenous Oxytocin on Embryonic Survival in Cows

    Directory of Open Access Journals (Sweden)

    A. Yildiz

    2006-01-01

    Full Text Available The aim of this study was to evaluate the effect of oxytocin on embryonic survival in dairy cows. Pregnancy was verified using the early pregnancy factor (EPF activity on Day 4 after artificial insemination (AI. Pregnant cows were randomly allotted to two groups: treated (n = 8 and control (n = 8. Oxytocin (100 IU, 5 ml, DIF Turkey was administered twice daily by intravenous injections to treated cows and sterile saline (5 ml to control cows immediately before milking on days 4 to 7 after AI. Blood samples were taken via jugular vein every day from day 4 to 8 and every other day until Day 20 following insemination to evaluate the effect of oxytocin on embryonic survival. The embryonic loss was diagnosed in 3 of the 8 cows treated with oxytocin, and embryonic survival rate was 62.5% in this group versus 87.5% in controls. Short cycles occurred in 37.5% of oxytocin-treated cows. At the same time their serum progesterone concentrations rose more slowly than in controls. It was concluded that cows administered oxytocin on days 4 to 7 after insemination are at a higher risk of pregnancy loss.

  3. Co-expression network analysis to identify pluripotency biomarkers in bovine and porcine embryos

    DEFF Research Database (Denmark)

    Mazzoni, Gianluca; Freude, Karla Kristine; Hall, Vanessa Jane

    Differentiated somatic cells can be reprogrammed in induced pluripotent stem cells (iPSCs); a cell type with great potentials in regenerative medicine and in vitro disease modeling. In the pig, we have developed iPSCs, but proper culture conditions for maintaining pluripotency over time are still...... lacking. Hence, there is a need for a more fundamental dissection of the pluripotency apparatus in the pig as well as in cattle. The aim of this study is to analyze RNA-seq data to increase the knowledge about biological pathways in porcine and bovine embryonic pluripotent cell populations exploiting...... the mouse data as proof of principle. In particular we studied cell populations from three different stages of pluripotency after fertilization: the inner cell mass, the epithelial epiblast and the gastrulating epiblast. Reads quality was checked with FASTQC, then the reads were pre-processed using Prinseq...

  4. Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Yao, Chaogang; Zhou, Yang; Zhu, Jianguo; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

    2011-12-01

    Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

  5. Mechanical signaling coordinates the embryonic heartbeat

    Science.gov (United States)

    Chiou, Kevin K.; Rocks, Jason W.; Chen, Christina Yingxian; Cho, Sangkyun; Merkus, Koen E.; Rajaratnam, Anjali; Robison, Patrick; Tewari, Manorama; Vogel, Kenneth; Majkut, Stephanie F.; Prosser, Benjamin L.; Discher, Dennis E.; Liu, Andrea J.

    2016-01-01

    In the beating heart, cardiac myocytes (CMs) contract in a coordinated fashion, generating contractile wave fronts that propagate through the heart with each beat. Coordinating this wave front requires fast and robust signaling mechanisms between CMs. The primary signaling mechanism has long been identified as electrical: gap junctions conduct ions between CMs, triggering membrane depolarization, intracellular calcium release, and actomyosin contraction. In contrast, we propose here that, in the early embryonic heart tube, the signaling mechanism coordinating beats is mechanical rather than electrical. We present a simple biophysical model in which CMs are mechanically excitable inclusions embedded within the extracellular matrix (ECM), modeled as an elastic-fluid biphasic material. Our model predicts strong stiffness dependence in both the heartbeat velocity and strain in isolated hearts, as well as the strain for a hydrogel-cultured CM, in quantitative agreement with recent experiments. We challenge our model with experiments disrupting electrical conduction by perfusing intact adult and embryonic hearts with a gap junction blocker, β-glycyrrhetinic acid (BGA). We find this treatment causes rapid failure in adult hearts but not embryonic hearts—consistent with our hypothesis. Last, our model predicts a minimum matrix stiffness necessary to propagate a mechanically coordinated wave front. The predicted value is in accord with our stiffness measurements at the onset of beating, suggesting that mechanical signaling may initiate the very first heartbeats. PMID:27457951

  6. Embryonic exposure of medaka (Oryzias latipes) to propylparaben: Effects on early development and post-hatching growth

    International Nuclear Information System (INIS)

    González-Doncel, Miguel; García-Mauriño, José Enrique; San Segundo, Laura; Beltrán, Eulalia M.; Sastre, Salvador; Fernández Torija, Carlos

    2014-01-01

    Here we proposed a battery of non-invasive biomarkers and a histological survey to examine physiological/anatomical features in embryos, eleutheroembryos (13 days post-fertilization, dpf), and larvae (28–42 dpf) of medaka to investigate the effects of embryonic exposure to propylparaben (PrP). Concentrations <1000 μg PrP/L didn't exert early or late toxic effects. However, survivorship was affected at 4000 μg/L in eleutheroembryos and at ≥1000 μg/L in larvae. Histological alterations were found in 37.5% of eleutheroembryos exposed to 4000 μg PrP/L. Morphometric analysis of the gallbladder revealed significant dilation at ≥400 μg/L throughout embryo development. Ethoxyresorufin-O-deethylase (EROD), as indicator of cytochrome P4501A activity, didn't reveal induction/inhibition although its combination with a P4501A agonist (i.e. β-naphthoflavone) resulted in a synergic EROD response. Results suggest a low toxicity of PrP for fish and support the use of fish embryos and eleutheroembryos as alternatives of in vivo biomarkers indicative of exposure/toxicity. -- Highlights: • Addressing pre- and post-hatch effects from medaka embryo exposure to propylparaben. • Macroscopical effects (length, mortality) seen primarily after hatch at ≥400 μg/L. • Synergic EROD embryonic response when propylparaben combined with a CYP1A agonist. • Significant gallbladder dilation seen at ≥400 μg PrP/L and as soon as discernible. • Histological harm to eleutheroembryos in peritoneal cavity, liver, kidney and brain. -- PrP resulted in low toxicity based on non-invasive biomarkers and histological tools to analyze pre- and post-hatch effects after medaka embryo exposure

  7. Investigating the Flow and Biomechanics of the Embryonic Zebrafish Heart

    Science.gov (United States)

    Johnson, Brennan; Garrity, Deborah; Dasi, Lakshmi

    2010-11-01

    Understanding flow and kinematic characteristics of the embryonic heart is a prerequisite to devise early intervention or detection methods in the context of congenital heart defects. In this study, the kinematics and fluid dynamics of the embryonic zebrafish heart were analyzed through the early stages of cardiac development (24-48 hours post-fertilization) in vivo using optical microscopy and high-speed video. Endocardial walls and individual blood cells were segmented from raw images and were tracked through the cardiac cycle. Particle tracking velocimetry analysis yielded quantitative blood cell velocity field, chamber volume, and flow rate information. It was seen that the pumping mechanism starts as a combined peristaltic and suction pump while the heart is in the tube configuration and transforms into a positive displacement pump after cardiac looping. Strong two-phase nature of the fluid is evident. This work provides us new understanding of the spatio-temporal characteristics of kinematics and blood cell velocity field inside the developing heart.

  8. 7 CFR 1230.18 - Porcine animal.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Porcine animal. 1230.18 Section 1230.18 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... animal. Porcine animal means a swine, that is raised as (a) a feeder pig, that is, a young pig sold to...

  9. Embryonic health: new insights, mHealth and personalised patient care.

    Science.gov (United States)

    Steegers-Theunissen, Régine P M; Steegers, Eric A P

    2015-05-01

    The worldwide epidemic of non-communicable diseases (NCD), including obesity, is a burden to which poor lifestyles contribute significantly. Events in early life may enhance susceptibility to NCD, with transmission into succeeding generations. This may also explain, in part, why interventions in adulthood are less effective to reduce NCD risk. New insights reveal that the early embryo, in particular, is extremely sensitive to signals from gametes, trophoblastic tissue and periconception maternal lifestyles. Embryonic size and growth as determinants of embryonic health seem to impact future health. A relatively small embryo for gestational age is associated with pregnancy complications, as well as with the risk of early features of NCD in childhood. Although personal lifestyles are modifiable, they are extremely difficult to change. Therefore, adopting a life course approach from the periconception period onwards and integrated into patient care with short-term reproductive health benefits may have important implications for future prevention of NCD. The current reproductive population is used to Internet and social media. Therefore, they can be reached via mobile phone (mHealth) platforms that provide personalised lifestyle (pre)pregnancy programs. This will offer opportunities and possibly great benefits for the health of current and succeeding generations.

  10. PTBP1 is required for embryonic development before gastrulation.

    Science.gov (United States)

    Suckale, Jakob; Wendling, Olivia; Masjkur, Jimmy; Jäger, Melanie; Münster, Carla; Anastassiadis, Konstantinos; Stewart, A Francis; Solimena, Michele

    2011-02-17

    Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

  11. Mouse embryonic retina delivers information controlling cortical neurogenesis.

    Directory of Open Access Journals (Sweden)

    Ciro Bonetti

    2010-12-01

    Full Text Available The relative contribution of extrinsic and intrinsic mechanisms to cortical development is an intensely debated issue and an outstanding question in neurobiology. Currently, the emerging view is that interplay between intrinsic genetic mechanisms and extrinsic information shape different stages of cortical development. Yet, whereas the intrinsic program of early neocortical developmental events has been at least in part decoded, the exact nature and impact of extrinsic signaling are still elusive and controversial. We found that in the mouse developing visual system, acute pharmacological inhibition of spontaneous retinal activity (retinal waves-RWs during embryonic stages increase the rate of corticogenesis (cell cycle withdrawal. Furthermore, early perturbation of retinal spontaneous activity leads to changes of cortical layer structure at a later time point. These data suggest that mouse embryonic retina delivers long-distance information capable of modulating cell genesis in the developing visual cortex and that spontaneous activity is the candidate long-distance acting extrinsic cue mediating this process. In addition, these data may support spontaneous activity to be a general signal coordinating neurogenesis in other developing sensory pathways or areas of the central nervous system.

  12. Comparison of neurotrophin and repellent sensitivities of early embryonic geniculate and trigeminal axons

    NARCIS (Netherlands)

    Rochlin, M William; O'Connor, R.; Giger, Roman J; Verhaagen, J; Farbman, A I

    2000-01-01

    Geniculate (gustatory) and trigeminal (somatosensory) afferents take different routes to the tongue during rat embryonic development. To learn more about the mechanisms controlling neurite outgrowth and axon guidance, we are studying the roles of diffusible factors. We previously profiled the in

  13. Gastrin-releasing peptide in the porcine pancreas

    DEFF Research Database (Denmark)

    Holst, J J; Poulsen, Steen Seier

    1987-01-01

    to consist of one main form, namely the 27-amino acid peptide originally extracted from porcine stomach, and small amounts of a C-terminal fragment identical with the C-terminal 10-amino acid peptide. Gastrin-releasing peptide-like immunoreactivity released from the isolated perfused porcine pancreas during...... electrical vagal stimulation was shown by gel filtration to consist of the same two forms. By use of immunocytochemical techniques employing an antiserum directed against its N terminus, GRP was localized to varicose nerve fibers in close association with the exocrine tissue of the porcine pancreas...... in particular. Some fibers were found penetrating into pancreatic islets also. Immunoreactive nerve cell bodies as well as fibers were found within intrapancreatic ganglia. The potency of GRP in stimulating exocrine as well as endocrine secretion from the porcine pancreas, its presence in close contact...

  14. Influence of high- and low-LET radiation on the cardiac differentiation of mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Helm, Alexander

    2013-07-19

    The in utero exposure to ionising radiation poses a risk for the radiosensitive developing embryo. Effects of low-LET radiation on different developmental stages of the embryo are relatively well known due to experimental studies and epidemiological data. Data for effects on the very early stage of the embryonic development, particularly the effects of high-LET radiation instead are rather limited. However, unanticipated exposures of the early embryo to ionising radiation may occur through diagnostic or therapeutic applications or through radiation accidents. Additionally, protons and carbon ions are increasingly used in radiotherapy. Thus, a risk estimation of high-LET exposure especially to the early embryo is of a certain importance. To address this topic, pluripotent mouse embryonic stem cells resembling the blastocyst stage were irradiated with high-LET carbon ions or low-LET X-rays and subsequently differentiated to mimic the early embryonic development. The occurrence of spontaneously contracting cardiomyocytes was used as a marker to asses the radiation effects on the differentiation. Among others, cell inactivation, cell death and gene expression were analysed. A delay in the cardiac differentiation after radiation exposure was found. The results point to radiation-induced cell killing as the main effector of the developmental delay. Carbon ions were found to be more effective than X-rays.

  15. Rapamycin Rescues the Poor Developmental Capacity of Aged Porcine Oocytes

    Directory of Open Access Journals (Sweden)

    Seung Eun Lee

    2014-05-01

    Full Text Available Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; 44 h+10 μM rapamycin/24 h, 47.52±5.68 or control oocytes (44 h IVM; 42.14±4.40 significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.04±5.68 (p<0.05. Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK, and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1 compared with untreated, 24 h-aged IVM oocytes (p<0.05. Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS activity and DNA fragmentation (p<0.05, and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05 and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1, anti-apoptosis (BCL2L1 and BIRC5; p<0.05, and development (NANOG and SOX2; p<0.05 genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3 compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.

  16. Aminopeptidase-N-independent entry of porcine epidemic diarrhea virus into Vero or porcine small intestine epithelial cells.

    Science.gov (United States)

    Ji, Chun-Miao; Wang, Bin; Zhou, Jiyong; Huang, Yao-Wei

    2018-04-01

    A monkey cell line Vero (ATCC CCL-81) is commonly used for porcine epidemic diarrhea virus (PEDV) propagation in vitro. However, it is still controversial whether the porcine aminopeptidase N (pAPN) counterpart on Vero cells (Vero-APN) confers PEDV entry. We found that endogenous expression of Vero-APN was undetectable in the mRNA and the protein levels in Vero cells. We cloned the partial Vero-APN gene (3340-bp) containing exons 1 to 9 from cellular DNA and subsequently generated two APN-knockout Vero cell lines by CRISPR/Cas9 approach. PEDV infection of two APN-knockout Vero cells had the same efficiency as the Vero cells with or without neuraminidase treatment. A Vero cells stably expressing pAPN did not increase PEDV production. SiRNA-knockdown of pAPN in porcine jejunum epithelial cells had no effects on PEDV infection. The results suggest that there exists an additional cellular receptor on Vero or porcine jejunal cells independent of APN for PEDV entry. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. How does blastomere removal affect embryonic development? : A time-lapse analysis

    DEFF Research Database (Denmark)

    Kirkegaard, Kirstine; Hindkjær, Johnny Juhl; Ingerslev, Hans Jakob

    of the 6-10 cell embryo. It has been argued that blastomere removal does not affect embryonic development, but few studies have focussed on safety of the procedure. Recently, time-lapse studies on mice have suggested that blastomere removal affects embryonic development. The present study was conducted...... to evaluate the effect of blastomere biopsy on early human embryonic development using time-lapse analysis. Materials and methods: Couples undergoing IVF treatment or PGD were requested permission to include embryos in the project. The diagnosis healthy/diseased was made by analysis of a single blastomere....... For PGD 56 human embryos were biopsied 68 hours after fertilisation, the majority at the eight cell stage. As controls 43 non-biopsied embryos at the 6-8 cell stage were selected. All embryos were cultured until 5 days after fertilisation in a time-lapse incubator (EmbryoScope™). Key events such as time...

  18. Quantification of Porcine Vocal Fold Geometry.

    Science.gov (United States)

    Stevens, Kimberly A; Thomson, Scott L; Jetté, Marie E; Thibeault, Susan L

    2016-07-01

    The aim of this study was to quantify porcine vocal fold medial surface geometry and three-dimensional geometric distortion induced by freezing the larynx, especially in the region of the vocal folds. The medial surface geometries of five excised porcine larynges were quantified and reported. Five porcine larynges were imaged in a micro-CT scanner, frozen, and rescanned. Segmentations and three-dimensional reconstructions were used to quantify and characterize geometric features. Comparisons were made with geometry data previously obtained using canine and human vocal folds as well as geometries of selected synthetic vocal fold models. Freezing induced an overall expansion of approximately 5% in the transverse plane and comparable levels of nonuniform distortion in sagittal and coronal planes. The medial surface of the porcine vocal folds was found to compare reasonably well with other geometries, although the compared geometries exhibited a notable discrepancy with one set of published human female vocal fold geometry. Porcine vocal folds are qualitatively geometrically similar to data available for canine and human vocal folds, as well as commonly used models. Freezing of tissue in the larynx causes distortion of around 5%. The data can provide direction in estimating uncertainty due to bulk distortion of tissue caused by freezing, as well as quantitative geometric data that can be directly used in developing vocal fold models. Copyright © 2016 The Voice Foundation. Published by Elsevier Inc. All rights reserved.

  19. Time--temperature relation of embryonic development in the northwestern salamander, Ambystoma gracile

    Energy Technology Data Exchange (ETDEWEB)

    Brown, H A

    1976-04-01

    A field and laboratory study on temperature-related embryonic development of Ambystoma gracile was made on a population from northwestern Washington. Natural spawning began in the beaver pond during early March, and the duration of embryonic development (stages 1 to 46) was about 62 days. Average water temperature in the pond during embryonic development was 8.5/sup 0/C (range, 4.4 to 14.3/sup 0/C). The laboratory data of embryonic development at constant temperatures show that the limits of temperature tolerance are about 5 to 22.5/sup 0/C. Rate of development was measured by determining time required to develop from first cleavage (stage 2) to gill circulation (stage 37); representative rates are 12.7 days at 20/sup 0/C, 27 days at 12/sup 0/C, and 89 days at 7/sup 0/C. Embryos of A. gracile have the slowest rate of development when compared with embryos of four other species of Ambystoma (maculatum, mexicanum, tigrinum, and jeffersonianum) and with embryos of three Pacific Northwest frogs (Ascaphus truei, Rana aurora, and Hyla regilla).

  20. Do embryonic polar bodies commit suicide?

    Science.gov (United States)

    Fabian, Dušan; Čikoš, Štefan; Rehák, Pavol; Koppel, Juraj

    2014-02-01

    The extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.

  1. RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells.

    Science.gov (United States)

    Pandolfini, Luca; Luzi, Ettore; Bressan, Dario; Ucciferri, Nadia; Bertacchi, Michele; Brandi, Rossella; Rocchiccioli, Silvia; D'Onofrio, Mara; Cremisi, Federico

    2016-05-06

    Embryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role. We performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells. We propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.

  2. Comparison of the biomechanical tensile and compressive properties of decellularised and natural porcine meniscus.

    Science.gov (United States)

    Abdelgaied, A; Stanley, M; Galfe, M; Berry, H; Ingham, E; Fisher, J

    2015-06-01

    Meniscal repair is widely used as a treatment for meniscus injury. However, where meniscal damage has progressed such that repair is not possible, approaches for partial meniscus replacement are now being developed which have the potential to restore the functional role of the meniscus, in stabilising the knee joint, absorbing and distributing stress during loading, and prevent early degenerative joint disease. One attractive potential solution to the current lack of meniscal replacements is the use of decellularised natural biological scaffolds, derived from xenogeneic tissues, which are produced by treating the native tissue to remove the immunogenic cells. The current study investigated the effect of decellularisation on the biomechanical tensile and compressive (indentation and unconfined) properties of the porcine medial meniscus through an experimental-computational approach. The results showed that decellularised medial porcine meniscus maintained the tensile biomechanical properties of the native meniscus, but had lower tensile initial elastic modulus. In compression, decellularised medial porcine meniscus generally showed lower elastic modulus and higher permeability compared to that of the native meniscus. These changes in the biomechanical properties, which ranged from less than 1% to 40%, may be due to the reduction of glycosaminoglycans (GAG) content during the decellularisation process. The predicted biomechanical properties for the decellularised medial porcine meniscus were within the reported range for the human meniscus, making it an appropriate biological scaffold for consideration as a partial meniscus replacement. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. Embryonic demise caused by targeted disruption of a cysteine protease Dub-2.

    Science.gov (United States)

    Baek, Kwang-Hyun; Lee, Heyjin; Yang, Sunmee; Lim, Soo-Bin; Lee, Wonwoo; Lee, Jeoung Eun; Lim, Jung-Jin; Jun, Kisun; Lee, Dong-Ryul; Chung, Young

    2012-01-01

    A plethora of biological metabolisms are regulated by the mechanisms of ubiquitination, wherein this process is balanced with the action of deubiquitination system. Dub-2 is an IL-2-inducible, immediate-early gene that encodes a deubiquitinating enzyme with growth regulatory activity. DUB-2 presumably removes ubiquitin from ubiquitin-conjugated target proteins regulating ubiquitin-mediated proteolysis, but its specific target proteins are unknown yet. To elucidate the functional role of Dub-2, we generated genetically modified mice by introducing neo cassette into the second exon of Dub-2 and then homologous recombination was done to completely abrogate the activity of DUB-2 proteins. We generated Dub-2+/- heterozygous mice showing a normal phenotype and are fertile, whereas new born mouse of Dub-2-/- homozygous alleles could not survive. In addition, Dub-2-/- embryo could not be seen between E6.5 and E12.5 stages. Furthermore, the number of embryos showing normal embryonic development for further stages is decreased in heterozygotes. Even embryonic stem cells from inner cell mass of Dub-2-/- embryos could not be established. Our study suggests that the targeted disruption of Dub-2 may cause embryonic lethality during early gestation, possibly due to the failure of cell proliferation during hatching process.

  4. Embryonic demise caused by targeted disruption of a cysteine protease Dub-2.

    Directory of Open Access Journals (Sweden)

    Kwang-Hyun Baek

    Full Text Available BACKGROUND: A plethora of biological metabolisms are regulated by the mechanisms of ubiquitination, wherein this process is balanced with the action of deubiquitination system. Dub-2 is an IL-2-inducible, immediate-early gene that encodes a deubiquitinating enzyme with growth regulatory activity. DUB-2 presumably removes ubiquitin from ubiquitin-conjugated target proteins regulating ubiquitin-mediated proteolysis, but its specific target proteins are unknown yet. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the functional role of Dub-2, we generated genetically modified mice by introducing neo cassette into the second exon of Dub-2 and then homologous recombination was done to completely abrogate the activity of DUB-2 proteins. We generated Dub-2+/- heterozygous mice showing a normal phenotype and are fertile, whereas new born mouse of Dub-2-/- homozygous alleles could not survive. In addition, Dub-2-/- embryo could not be seen between E6.5 and E12.5 stages. Furthermore, the number of embryos showing normal embryonic development for further stages is decreased in heterozygotes. Even embryonic stem cells from inner cell mass of Dub-2-/- embryos could not be established. CONCLUSIONS: Our study suggests that the targeted disruption of Dub-2 may cause embryonic lethality during early gestation, possibly due to the failure of cell proliferation during hatching process.

  5. Inhibition of early AAA formation by aortic intraluminal pentagalloyl glucose (PGG) infusion in a novel porcine AAA model.

    Science.gov (United States)

    Kloster, Brian O; Lund, Lars; Lindholt, Jes S

    2016-05-01

    The vast majority of abdominal aortic aneurysms found in screening programs are small, and as no effective treatment exits, many will expand until surgery is indicated. Therefore, it remains intriguing to develop a safe and low cost treatment of these small aneurysms, that is able to prevent or delay their expansion. In this study, we investigated whether intraluminal delivered pentagalloyl glucose (PGG) can impair the early AAA development in a porcine model. The infrarenal aorta was exposed in thirty pigs. Twenty underwent an elastase based AAA inducing procedure and ten of these received an additional intraluminal PGG infusion. The final 10 were sham operated and served as controls. All pigs who only had an elastase infusion developed macroscopically expanding AAAs. In pigs treated with an additional PGG infusion the growth rate of the AP-diameter rapidly returned to physiological values as seen in the control group. In the elastase group, histology revealed more or less complete resolution of the elastic lamellae in the media while they were more abundant, coherent and structurally organized in the PGG group. The control group displayed normal physiological growth and histology. In our model, intraluminal delivered PGG is able to penetrate the aortic wall from the inside and impair the early AAA development by stabilizing the elastic lamellae and preserving their integrity. The principle holds a high clinical potential if it can be translated to human conditions, since it, if so, potentially could represent a new drug for stabilizing small abdominal aneurysms.

  6. Cell Cycle Control in the Early Embryonic Development of Aquatic Animal Species

    Science.gov (United States)

    Siefert, Joseph C.; Clowdus, Emily A.; Sansam, Christopher L.

    2016-01-01

    The cell cycle is integrated with many aspects of embryonic development. Not only is proper control over the pace of cell proliferation important, but also the timing of cell cycle progression is coordinated with transcription, cell migration, and cell differentiation. Due to the ease with which the embryos of aquatic organisms can be observed and manipulated, they have been a popular choice for embryologists throughout history. In the cell cycle field, aquatic organisms have been extremely important because they have played a major role in the discovery and analysis of key regulators of the cell cycle. In particular, the frog Xenopus laevis has been instrumental for understanding how the basic embryonic cell cycle is regulated. More recently, the zebrafish has been used to understand how the cell cycle is remodeled during vertebrate development and how it is regulated during morphogenesis. This review describes how some of the unique strengths of aquatic species have been leveraged for cell cycle research and suggests how species such as Xenopus and zebrafish will continue to reveal the roles of the cell cycle in human biology and disease. PMID:26475527

  7. Maternal Diabetes Leads to Adaptation in Embryonic Amino Acid Metabolism during Early Pregnancy.

    Science.gov (United States)

    Gürke, Jacqueline; Hirche, Frank; Thieme, René; Haucke, Elisa; Schindler, Maria; Stangl, Gabriele I; Fischer, Bernd; Navarrete Santos, Anne

    2015-01-01

    During pregnancy an adequate amino acid supply is essential for embryo development and fetal growth. We have studied amino acid composition and branched chain amino acid (BCAA) metabolism at day 6 p.c. in diabetic rabbits and blastocysts. In the plasma of diabetic rabbits the concentrations of 12 amino acids were altered in comparison to the controls. Notably, the concentrations of the BCAA leucine, isoleucine and valine were approximately three-fold higher in diabetic rabbits than in the control. In the cavity fluid of blastocysts from diabetic rabbits BCAA concentrations were twice as high as those from controls, indicating a close link between maternal diabetes and embryonic BCAA metabolism. The expression of BCAA oxidizing enzymes and BCAA transporter was analysed in maternal tissues and in blastocysts. The RNA amounts of three oxidizing enzymes, i.e. branched chain aminotransferase 2 (Bcat2), branched chain ketoacid dehydrogenase (Bckdha) and dehydrolipoyl dehydrogenase (Dld), were markedly increased in maternal adipose tissue and decreased in liver and skeletal muscle of diabetic rabbits than in those of controls. Blastocysts of diabetic rabbits revealed a higher Bcat2 mRNA and protein abundance in comparison to control blastocysts. The expression of BCAA transporter LAT1 and LAT2 were unaltered in endometrium of diabetic and healthy rabbits, whereas LAT2 transcripts were increased in blastocysts of diabetic rabbits. In correlation to high embryonic BCAA levels the phosphorylation amount of the nutrient sensor mammalian target of rapamycin (mTOR) was enhanced in blastocysts caused by maternal diabetes. These results demonstrate a direct impact of maternal diabetes on BCAA concentrations and degradation in mammalian blastocysts with influence on embryonic mTOR signalling.

  8. Maternal Diabetes Leads to Adaptation in Embryonic Amino Acid Metabolism during Early Pregnancy.

    Directory of Open Access Journals (Sweden)

    Jacqueline Gürke

    Full Text Available During pregnancy an adequate amino acid supply is essential for embryo development and fetal growth. We have studied amino acid composition and branched chain amino acid (BCAA metabolism at day 6 p.c. in diabetic rabbits and blastocysts. In the plasma of diabetic rabbits the concentrations of 12 amino acids were altered in comparison to the controls. Notably, the concentrations of the BCAA leucine, isoleucine and valine were approximately three-fold higher in diabetic rabbits than in the control. In the cavity fluid of blastocysts from diabetic rabbits BCAA concentrations were twice as high as those from controls, indicating a close link between maternal diabetes and embryonic BCAA metabolism. The expression of BCAA oxidizing enzymes and BCAA transporter was analysed in maternal tissues and in blastocysts. The RNA amounts of three oxidizing enzymes, i.e. branched chain aminotransferase 2 (Bcat2, branched chain ketoacid dehydrogenase (Bckdha and dehydrolipoyl dehydrogenase (Dld, were markedly increased in maternal adipose tissue and decreased in liver and skeletal muscle of diabetic rabbits than in those of controls. Blastocysts of diabetic rabbits revealed a higher Bcat2 mRNA and protein abundance in comparison to control blastocysts. The expression of BCAA transporter LAT1 and LAT2 were unaltered in endometrium of diabetic and healthy rabbits, whereas LAT2 transcripts were increased in blastocysts of diabetic rabbits. In correlation to high embryonic BCAA levels the phosphorylation amount of the nutrient sensor mammalian target of rapamycin (mTOR was enhanced in blastocysts caused by maternal diabetes. These results demonstrate a direct impact of maternal diabetes on BCAA concentrations and degradation in mammalian blastocysts with influence on embryonic mTOR signalling.

  9. Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

    International Nuclear Information System (INIS)

    Ahn, Kwang Sung; Won, Ji Young; Park, Jin-Ki; Sorrell, Alice M.; Heo, Soon Young; Kang, Jee Hyun; Woo, Jae-Seok; Choi, Bong-Hwan; Chang, Won-Kyong; Shim, Hosup

    2010-01-01

    Research highlights: → Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. → hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. → hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

  10. Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Kwang Sung; Won, Ji Young [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Park, Jin-Ki [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Sorrell, Alice M. [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Heo, Soon Young; Kang, Jee Hyun [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Woo, Jae-Seok [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Choi, Bong-Hwan [Genomics and Bioinformatics Division, National Institute of Animal Science, Suwon (Korea, Republic of); Chang, Won-Kyong [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of)

    2010-10-01

    Research highlights: {yields} Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. {yields} hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. {yields} hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

  11. Tyrosine pathway regulation is host-mediated in the pea aphid symbiosis during late embryonic and early larval development.

    Science.gov (United States)

    Rabatel, Andréane; Febvay, Gérard; Gaget, Karen; Duport, Gabrielle; Baa-Puyoulet, Patrice; Sapountzis, Panagiotis; Bendridi, Nadia; Rey, Marjolaine; Rahbé, Yvan; Charles, Hubert; Calevro, Federica; Colella, Stefano

    2013-04-10

    Nutritional symbioses play a central role in insects' adaptation to specialized diets and in their evolutionary success. The obligatory symbiosis between the pea aphid, Acyrthosiphon pisum, and the bacterium, Buchnera aphidicola, is no exception as it enables this important agricultural pest insect to develop on a diet exclusively based on plant phloem sap. The symbiotic bacteria provide the host with essential amino acids lacking in its diet but necessary for the rapid embryonic growth seen in the parthenogenetic viviparous reproduction of aphids. The aphid furnishes, in exchange, non-essential amino acids and other important metabolites. Understanding the regulations acting on this integrated metabolic system during the development of this insect is essential in elucidating aphid biology. We used a microarray-based approach to analyse gene expression in the late embryonic and the early larval stages of the pea aphid, characterizing, for the first time, the transcriptional profiles in these developmental phases. Our analyses allowed us to identify key genes in the phenylalanine, tyrosine and dopamine pathways and we identified ACYPI004243, one of the four genes encoding for the aspartate transaminase (E.C. 2.6.1.1), as specifically regulated during development. Indeed, the tyrosine biosynthetic pathway is crucial for the symbiotic metabolism as it is shared between the two partners, all the precursors being produced by B. aphidicola. Our microarray data are supported by HPLC amino acid analyses demonstrating an accumulation of tyrosine at the same developmental stages, with an up-regulation of the tyrosine biosynthetic genes. Tyrosine is also essential for the synthesis of cuticular proteins and it is an important precursor for cuticle maturation: together with the up-regulation of tyrosine biosynthesis, we observed an up-regulation of cuticular genes expression. We were also able to identify some amino acid transporter genes which are essential for the switch

  12. Characterizing early embryonic development of Brown Tsaiya Ducks (Anas platyrhynchos in comparison with Taiwan Country Chicken (Gallus gallus domestics.

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    Chompunut Lumsangkul

    Full Text Available Avian embryos are among the most convenient and the primary representatives for the study of classical embryology. It is well-known that the hatching time of duck embryos is approximately one week longer than that of chicken embryos. However, the key features associated with the slower embryonic development in ducks have not been adequately described. This study aimed to characterize the pattern and the speed of early embryogenesis in Brown Tsaiya Ducks (BTD compared with those in Taiwan Country Chicken (TCC by using growth parameters including embryonic crown-tail length (ECTL, primitive streak formation, somitogenesis, and other development-related parameters, during the first 72 h of incubation. Three hundred and sixty eggs from BTD and TCC, respectively, were incubated at 37.2°C, and were then dissected hourly to evaluate their developmental stages. We found that morphological changes of TCC embryos shared a major similarity with that of the Hamburger and Hamilton staging system during early chick embryogenesis. The initial primitive streak in TCC emerged between 6 and 7 h post-incubation, but its emergence was delayed until 10 to 13 h post-incubation in BTD. Similarly, the limb primordia (wing and limb buds were observed at 51 h post-incubation in TCC embryos compared to 64 h post-incubation in BTD embryos. The allantois first appeared around 65 to 68 h in TCC embryos, but it was not observed in BTD embryos. At the 72 h post-incubation, 40 somites were clearly formed in TCC embryos while only 32 somites in BTD embryos. Overall, the BTD embryos developed approximately 16 h slower than the chicken embryo during the first 72 h of development. To our best knowledge, this is the first study to describe two distinct developmental time courses between TCC and BTD, which would facilitate future embryogenesis-related studies of the two important avian species in Taiwan.

  13. Characterizing early embryonic development of Brown Tsaiya Ducks (Anas platyrhynchos) in comparison with Taiwan Country Chicken (Gallus gallus domestics)

    Science.gov (United States)

    Lumsangkul, Chompunut; Fan, Yang-Kwang; Chang, Shen-Chang; Ju, Jyh-Cherng

    2018-01-01

    Avian embryos are among the most convenient and the primary representatives for the study of classical embryology. It is well-known that the hatching time of duck embryos is approximately one week longer than that of chicken embryos. However, the key features associated with the slower embryonic development in ducks have not been adequately described. This study aimed to characterize the pattern and the speed of early embryogenesis in Brown Tsaiya Ducks (BTD) compared with those in Taiwan Country Chicken (TCC) by using growth parameters including embryonic crown-tail length (ECTL), primitive streak formation, somitogenesis, and other development-related parameters, during the first 72 h of incubation. Three hundred and sixty eggs from BTD and TCC, respectively, were incubated at 37.2°C, and were then dissected hourly to evaluate their developmental stages. We found that morphological changes of TCC embryos shared a major similarity with that of the Hamburger and Hamilton staging system during early chick embryogenesis. The initial primitive streak in TCC emerged between 6 and 7 h post-incubation, but its emergence was delayed until 10 to 13 h post-incubation in BTD. Similarly, the limb primordia (wing and limb buds) were observed at 51 h post-incubation in TCC embryos compared to 64 h post-incubation in BTD embryos. The allantois first appeared around 65 to 68 h in TCC embryos, but it was not observed in BTD embryos. At the 72 h post-incubation, 40 somites were clearly formed in TCC embryos while only 32 somites in BTD embryos. Overall, the BTD embryos developed approximately 16 h slower than the chicken embryo during the first 72 h of development. To our best knowledge, this is the first study to describe two distinct developmental time courses between TCC and BTD, which would facilitate future embryogenesis-related studies of the two important avian species in Taiwan. PMID:29742160

  14. Tachykinins in the porcine pancreas

    DEFF Research Database (Denmark)

    Schmidt, P T; Tornøe, K; Poulsen, Steen Seier

    2000-01-01

    The localization, release, and effects of substance P and neurokinin A were studied in the porcine pancreas and the localization of substance P immunoreactive nerve fibers was examined by immunohistochemistry. The effects of electrical vagus stimulation and capsaicin infusion on tachykinin release...... and the effects of substance P and neurokinin A infusion on insulin, glucagon, somatostatin, and exocrine secretion were studied using the isolated perfused porcine pancreas with intact vagal innervation. NK-1 and NK-2 receptor antagonists were used to investigate receptor involvement. Substance P immunoreactive...

  15. Methods for the detection and serum depletion of porcine galectin-3.

    Science.gov (United States)

    Eliaz, Isaac; Patil, Aarti; Navarro-Alvarez, Nalu; Wang, Zhirui; Eliaz, Amity; Weil, Elaine; Wilk, Barry; Sachs, David H; Huang, Christene A

    2017-10-01

    Circulating galectin-3 (Gal-3) is elevated in systemic inflammatory disorders, fibrotic diseases, and in cancers. Gal-3 is a promising cancer target where it promotes tumorigenesis and metastasis, as well as in renal, pulmonary, hepatic, and cardiovascular diseases, because of its role as a driver of fibrotic remodeling. This reports goal was to establish methods for the detection and removal of porcine Gal-3 that will enable further studies of the therapeutic potential of Gal-3 depletion by apheresis in porcine disease models. The long-term aim is to develop a safe, effective method of removing Gal-3 via apheresis as a standalone therapeutic tool and as an adjuvant to other therapies. Purified recombinant porcine Gal-3 was prepared and used as the standard for development of a porcine Gal-3 enzyme-linked immunosorbent assay (ELISA). Different affinity column matrices that incorporated either a rat IgG2a anti-Gal-3 monoclonal antibody or carbohydrate ligand were assessed for depletion of Gal-3 from porcine serum. A porcine Gal-3 ELISA with a linear range from 0.3 to 20 ng/mL was able to detect native porcine Gal-3 in both fetal (∼150-200 ng/mL) and juvenile (∼5-15 ng/mL) porcine serum samples. Use of an anti-Gal-3 monoclonal antibody affinity column depleted Gal-3 from porcine serum to at least 313 pg/mL, the limit of ELISA detection. Methods have been developed for the detection and depletion of porcine Gal-3. These methods will be used to study the specific effects of Gal-3 depletion via apheresis in porcine models of disease. © 2017 Wiley Periodicals, Inc.

  16. Huntingtin Protein is Essential for Mitochondrial Metabolism, Bioenergetics and Structure in Murine Embryonic Stem Cells

    Science.gov (United States)

    Ismailoglu, Ismail; Chen, Qiuying; Popowski, Melissa; Yang, Lili; Gross, Steven S.; Brivanlou, Ali H.

    2014-01-01

    Mutations in the Huntington locus (htt) have devastating consequences. Gain-of-poly-Q repeats in Htt protein causes Huntington's disease (HD), while htt-/- mutants display early embryonic lethality. Despite its importance, the function of Htt remains elusive. To address this, we compared more than 3,700 compounds in three syngeneic mouse embryonic stem cell (mESC) lines: htt-/-, extended poly-Q (Htt-Q140/7), and wildtype mESCs (Htt-Q7/7) using untargeted metabolite profiling. While Htt-Q140/7 cells, did not show major differences in cellular bioenergetics, we find extensive metabolic aberrations in htt-/- mESCs, including: (i) complete failure of ATP production despite preservation of the mitochondrial membrane potential; (ii) near-maximal glycolysis, with little or no glycolytic reserve; (iii) marked ketogenesis; (iv) depletion of intracellular NTPs; (v) accelerated purine biosynthesis and salvage; and (vi) loss of mitochondrial structural integrity. Together, our findings reveal that Htt is necessary for mitochondrial structure and function from the earliest stages of embryogenesis, providing a molecular explanation for htt-/- early embryonic lethality. PMID:24780625

  17. EMBRYONIC VASCULAR DISRUPTION ADVERSE OUTCOMES: LINKING HIGH THROUGHPUT SIGNALING SIGNATURES WITH FUNCTIONAL CONSEQUENCES

    Science.gov (United States)

    Embryonic vascular disruption is an important adverse outcome pathway (AOP) given the knowledge that chemical disruption of early cardiovascular system development leads to broad prenatal defects. High throughput screening (HTS) assays provide potential building blocks for AOP d...

  18. Lentiviral Vector Gene Transfer to Porcine Airways

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    Patrick L Sinn

    2012-01-01

    Full Text Available In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE. Interestingly, feline immunodeficiency virus (FIV-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1–based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF.

  19. EFFECTS OF SILVER NANOPARTICLES IN SOLUTION AND LIPOSOMAL FORM ON SOME BLOOD PARAMETERS IN FEMALE RABBITS DURING FERTILIZATION AND EARLY EMBRYONIC DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    Vasyl Syrvatka

    2014-02-01

    Full Text Available Silver nanoparticles are the most rapidly growing classes of nanoproducts. In this study, we investigated the influence of subcutaneous injections of silver nanoparticles in solution and in liposomal form on hematological and biochemical parameters of blood of New Zealand White rabbits during hormonal treatment, fertilization and early embryonic development. The females treated by free silver nanoparticles and silver nanoparticles in liposomal form received silver at a dose of 10 µg/kg/day in 5 % glucose solution during 28 days. Blood sampling was done four times: the day before the compounds administration; on day 7 after the compounds administration; in the period after hormonal induction and fertilization and on the 14th day of pregnancy. Our results showed changes in some biochemical (lactate dehydrogenase activities, progesterone and estradiol concentration, malondialdehyde level, etc. and hematological (hematocrit, mean cell volume, mean corpuscular hemoglobin concentration, etc. parameters under the influence of hormonal treatment and pregnancy. The concentration of progesterone showed significantly higher values (P˂0.05 on GDs 1 in S group than in C group. The percentage of neutrophils was significantly higher in SG rabbits after 7 days of silver nanoparticles administration than that in the CG. There were no significant changes in red blood cells parameters, platelets, and activity of some ferments (ALP, AST, ALT, LDH, GGT between control and silver groups during the entire period of experiment. In conclusion, the hematological and biochemical values of blood obtained in the given study showed that free silver nanoparticles and silver nanoparticles in liposomal form in the investigated concentrations had no toxic effect on hormonal treatment, fertilization and early embryonic development in New Zealand White rabbits.

  20. Porcine circovirus diseases

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    Ristoski Trpe

    2009-05-01

    Full Text Available Porcine circovirus type 2 belongs on the family Circoviridae. This virus family includes small, non-enveloped viruses, with a circular, single-standed DNA genome.This virus causes mainly subclinical infections, but a number of diseases have been linked to it (porcine circovirus diseases, PCVD. The most economically important PCVD is postweaning multisystemic wasting syndrome (PMWS, which mainly affects pigs of 2 to 5 months of age, with progressive wasting, diarrhea and respiratory disorders. Main PMWS lesions are found in lymphoid tissues, which are characterized by lymphocyte depletion with granulomatous (histiocytic and multinucleate giant cell infiltration. PMWS is considered as multifactorial disease, with a number of infectious and non-infectious factors able to act as disease triggering in PCV2 infected pigs. PCVDs are worldwide distributed, and PMWS was diagnosed in Macedonia in 2007.

  1. The fine structure of human germ layers in vivo: clues to the early differentiation of embryonic stem cells in vitro.

    Science.gov (United States)

    Sathananthan, Henry; Selvaraj, Kamala; Clark, Joan

    2011-08-01

    The fine structure of the three germ layers in human ectopic embryos (stage 7) have been documented by digital light and electron microscopy. The formation of ectoderm, endoderm and mesoderm and notochordal cells, and also the extraembryonic membranes, amnion and yolk sac, are imaged. The germ layers give rise to all the cells and tissues of the human body. Possible clues to the early differentiation of embryonic stem cells (ESC) in vitro were obtained, since these events are more or less mimicked in cultures of ESC derived from the inner cell mass of human blastocysts. The findings are discussed with reference to previous studies on the fine structure of ESC using the same technique. Copyright © 2011. Published by Elsevier Ltd.

  2. Parental and embryonic experiences with predation risk affect prey offspring behaviour and performance.

    Science.gov (United States)

    Donelan, Sarah C; Trussell, Geoffrey C

    2018-03-14

    Because phenotypic plasticity can operate both within and between generations, phenotypic outcomes are often shaped by a complex history of environmental signals. For example, parental and embryonic experiences with predation risk can both independently and interactively influence prey offspring traits early in their life. Parental and embryonic risk experiences can also independently shape offspring phenotypes throughout an offspring's ontogeny, but the persistence of their interactive effects throughout offspring ontogeny is unknown. We examined the effects of parental and embryonic experiences with predation risk on the response of 1-year-old prey (the carnivorous snail, Nucella lapillus ) offspring to current predation risk. We found that parental and embryonic risk experiences had largely independent effects on offspring performance and that these effects were context dependent. Parental experience with risk had strong impacts on multiple offspring traits in the presence of current risk that generally improved offspring performance under risk, but embryonic risk experience had relatively weaker effects and only operated in the absence of current risk to reduce offspring growth. These results illustrate that past environmental experiences can dynamically shape organism phenotypes across ontogeny and that attention to these effects is key to a better understanding of predator/prey dynamics in natural systems. © 2018 The Author(s).

  3. Systemic thioridazine in combination with dicloxacillin against early aortic graft infections caused by Staphylococcus aureus in a porcine model: In vivo results do not reproduce the in vitro synergistic activity.

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    Michael Stenger

    Full Text Available Conservative treatment solutions against aortic prosthetic vascular graft infection (APVGI for inoperable patients are limited. The combination of antibiotics with antibacterial helper compounds, such as the neuroleptic drug thioridazine (TDZ, should be explored.To investigate the efficacy of conservative systemic treatment with dicloxacillin (DCX in combination with TDZ (DCX+TDZ, compared to DCX alone, against early APVGI caused by methicillin-sensitive Staphylococcus aureus (MSSA in a porcine model.The synergism of DCX+TDZ against MSSA was initially assessed in vitro by viability assay. Thereafter, thirty-two pigs had polyester grafts implanted in the infrarenal aorta, followed by inoculation with 106 CFU of MSSA, and were randomly administered oral systemic treatment with either 1 DCX or 2 DCX+TDZ. Treatment was initiated one week postoperatively and continued for a further 21 days. Weight, temperature, and blood samples were collected at predefined intervals. By termination, bacterial quantities from the graft surface, graft material, and perigraft tissue were obtained.Despite in vitro synergism, the porcine experiment revealed no statistical differences for bacteriological endpoints between the two treatment groups, and none of the treatments eradicated the APVGI. Accordingly, the mixed model analyses of weight, temperature, and blood samples revealed no statistical differences.Conservative systemic treatment with DCX+TDZ did not reproduce in vitro results against APVGI caused by MSSA in this porcine model. However, unexpected severe adverse effects related to the planned dose of TDZ required a considerable reduction to the administered dose of TDZ, which may have compromised the results.

  4. Deciphering the porcine intestinal microRNA transcriptome

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    Keller Andreas

    2010-04-01

    Full Text Available Abstract Background While more than 700 microRNAs (miRNAs are known in human, a comparably low number has been identified in swine. Because of the close phylogenetic distance to humans, pigs serve as a suitable model for studying e.g. intestinal development or disease. Recent studies indicate that miRNAs are key regulators of intestinal development and their aberrant expression leads to intestinal malignancy. Results Here, we present the identification of hundreds of apparently novel miRNAs in the porcine intestine. MiRNAs were first identified by means of deep sequencing followed by miRNA precursor prediction using the miRDeep algorithm as well as searching for conserved miRNAs. Second, the porcine miRNAome along the entire intestine (duodenum, proximal and distal jejunum, ileum, ascending and transverse colon was unraveled using customized miRNA microarrays based on the identified sequences as well as known porcine and human ones. In total, the expression of 332 intestinal miRNAs was discovered, of which 201 represented assumed novel porcine miRNAs. The identified hairpin forming precursors were in part organized in genomic clusters, and most of the precursors were located on chromosomes 3 and 1, respectively. Hierarchical clustering of the expression data revealed subsets of miRNAs that are specific to distinct parts of the intestine pointing to their impact on cellular signaling networks. Conclusions In this study, we have applied a straight forward approach to decipher the porcine intestinal miRNAome for the first time in mammals using a piglet model. The high number of identified novel miRNAs in the porcine intestine points out their crucial role in intestinal function as shown by pathway analysis. On the other hand, the reported miRNAs may share orthologs in other mammals such as human still to be discovered.

  5. Growth hormone-specific induction of the nuclear localization of porcine growth hormone receptor in porcine hepatocytes.

    Science.gov (United States)

    Lan, H N; Hong, P; Li, R N; Shan, A S; Zheng, X

    2017-10-01

    The phenomenon of nuclear translocation of growth hormone receptor (GHR) in human, rat, and fish has been reported. To date, this phenomenon has not been described in a domestic animal (such as pig). In addition, the molecular mechanisms of GHR nuclear translocation have not been thoroughly elucidated. To this end, porcine hepatocytes were isolated and used as a cell model. We observed that porcine growth hormone (pGH) can induce porcine GHR's nuclear localization in porcine hepatocytes. Subsequently, the dynamics of pGH-induced pGHR's nuclear localization were analyzed and demonstrated that pGHR's nuclear localization occurs in a time-dependent manner. Next, we explored the mechanism of pGHR nuclear localization using different pGHR ligands, and we demonstrated that pGHR's nuclear translocation is GH(s)-dependent. We also observed that pGHR translocates into cell nuclei in a pGH dimerization-dependent fashion, whereas further experiments indicated that IMPα/β is involved in the nuclear translocation of the pGH-pGHR dimer. The pGH-pGHR dimer may form a pGH-GHR-JAK2 multiple complex in cell nuclei, which would suggest that similar to its function in the cell membrane, the nuclear-localized pGH-pGHR dimer might still have the ability to signal. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Critical early roles for col27a1a and col27a1b in zebrafish notochord morphogenesis, vertebral mineralization and post-embryonic axial growth.

    Science.gov (United States)

    Christiansen, Helena E; Lang, Michael R; Pace, James M; Parichy, David M

    2009-12-29

    Fibrillar collagens are well known for their links to human diseases, with which all have been associated except for the two most recently identified fibrillar collagens, type XXIV collagen and type XXVII collagen. To assess functions and potential disease phenotypes of type XXVII collagen, we examined its roles in zebrafish embryonic and post-embryonic development. We identified two type XXVII collagen genes in zebrafish, col27a1a and col27a1b. Both col27a1a and col27a1b were expressed in notochord and cartilage in the embryo and early larva. To determine sites of type XXVII collagen function, col27a1a and col27a1b were knocked down using morpholino antisense oligonucleotides. Knockdown of col27a1a singly or in conjunction with col27a1b resulted in curvature of the notochord at early stages and formation of scoliotic curves as well as dysmorphic vertebrae at later stages. These defects were accompanied by abnormal distributions of cells and protein localization in the notochord, as visualized by transmission electron microscopy, as well as delayed vertebral mineralization as detected histologically. Together, our findings indicate a key role for type XXVII collagen in notochord morphogenesis and axial skeletogenesis and suggest a possible human disease phenotype.

  7. Comparative Analysis of the Regulatory T Cells Dynamics in Peripheral Blood in Human and Porcine Polytrauma

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    Rafael Serve

    2018-03-01

    intracellular FACS stain.ConclusionDespite minor percental differences in the recovery of Treg populations after trauma, our findings show a comparable decrease of Treg early after polytrauma, and strengthen the immunological significance of the porcine polytrauma model. Furthermore, the Treg subpopulation CD4+CD25+CD127− was characterized in porcine samples.

  8. Porcine sialoadhesin (CD169/Siglec-1 is an endocytic receptor that allows targeted delivery of toxins and antigens to macrophages.

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    Peter L Delputte

    Full Text Available Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that sialoadhesin is an endocytic receptor. In this report, we show that porcine sialoadhesin-specific antibodies and F(ab'₂ fragments trigger sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the sialoadhesin endocytosis mechanism, two sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine sialoadhesin-specific immunotoxins efficiently kill sialoadhesin-expressing macrophages. Furthermore, porcine sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to sialoadhesin-expressing macrophages.

  9. Sequence conservation between porcine and human LRRK2

    DEFF Research Database (Denmark)

    Larsen, Knud; Madsen, Lone Bruhn

    2009-01-01

     Leucine-rich repeat kinase 2 (LRRK2) is a member of the ROCO protein superfamily (Ras of complex proteins (Roc) with a C-terminal Roc domain). Mutations in the LRRK2 gene lead to autosomal dominant Parkinsonism. We have cloned the porcine LRRK2 cDNA in an attempt to characterize conserved...... and expression patterns are conserved across species. The porcine LRRK2 gene was mapped to chromosome 5q25. The results obtained suggest that the LRRK2 gene might be of particular interest in our attempt to generate a transgenic porcine model for Parkinson's disease...

  10. Combined sequencing of mRNA and DNA from human embryonic stem cells

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    Florian Mertes

    2016-06-01

    Full Text Available Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO database under accession number GSE69471.

  11. First identification of porcine parvovirus 6 in North America by viral metagenomic sequencing of serum from pigs infected with porcine reproductive and respiratory syndrome virus.

    Science.gov (United States)

    Schirtzinger, Erin E; Suddith, Andrew W; Hause, Benjamin M; Hesse, Richard A

    2015-10-16

    Currently, eight species in four genera of parvovirus have been described that infect swine. These include ungulate protoparvovirus 1 (classical porcine parvovirus, PPV), ungulate tetraparvovirus 2 (PPV3), ungulate tetraparvovirus 3 (which includes PPV2, porcine hokovirus, porcine partetravirus and porcine PARV4), ungulate copiparvovirus 2 (which includes PPV4 and PPV5), ungulate bocaparvovirus 2 (which includes porcine bocavirus 1, 2 and 6), ungulate bocaparvovirus 3 (porcine bocavirus 5), ungulate bocaparvovirus 4 (porcine bocavirus 7) and ungulate bocaparvovirus 5 (porcine bocavirus 3, 4-1 and 4-2). PPV6, the most recently described porcine parvovirus, was first identified in China in late 2014 in aborted pig fetuses. Prevalence of PPV6 in China was found to be similar in finishing age pigs from farms with and without evidence of swine reproductive failure. Porcine parvovirus 6 (PPV6) was detected by sequence-independent single primer amplification (SISPA) and confirmed by overlapping and real-time PCR in the serum of porcine reproductive and respiratory virus (PRRSv) positive samples. Seven nearly complete genomes of PPV6 were identified in PRRSv genotype 2 positive serum samples submitted to state veterinary diagnostic laboratories in 2014. Further testing using overlapping and real-time PCR determined PPV6 to be present in 13.2 % of the serums tested. Additionally, PPV6 was present in samples from all of the geographic locations sampled encompassing nine states in the United States and one state in Mexico. The presence of PPV6 in serum indicates that the PPV6 infection is disseminated and not localized to a specific tissue type. Alignments of the near full length genomes, NS1, and capsid genes identified one of the five PPV6 isolates from China (98.6-99.5 % identity with the North American strains) to be the North American strains nearest relative. These results are the first to report the presence of PPV6 in North America and demonstrate that the virus is

  12. Occurance of apoptosis during ischemia in porcine pancreas islet cells.

    Science.gov (United States)

    Stadlbauer, V; Schaffellner, S; Iberer, F; Lackner, C; Liegl, B; Zink, B; Kniepeiss, D; Tscheliessnigg, K H

    2003-03-01

    Pancreas islet transplantation is a potential treatment of diabetes mellitus and porcine organs provide an easily available source of cells. Unfortunately quality and quantity of isolated islets are still not satisfactory. Apoptosis occurs in freshly isolated islets and plays a significant role in early graft loss. We evaluated the influence of four storage solutions on porcine pancreas islets. After warm ischemia of 15-20 minutes 12 organs were stored in 4 cold preservation solutions: Histidine-Tryptophan-Ketoglutarate solution (HTK), Hank's buffered saline solution (HBSS), University of Wisconsin (UW) solution and Ringer-Lactate (R). After cold ischemia for 100 minutes, organs were fixed in 3% formalin. Apoptotic cells were counted on hematocylin-eosin stainings. Most apoptotic cells were found in organs stored in R. Low numbers were found in the other groups. The difference between organs stored in R and organs stored in UW, HTK, or HBSS was highly significant. No significant difference could be found between UW, HTK and HBSS. Cold and warm ischemia of the pancreas seems to induce apoptosis in islet cells. Preservation solutions cause less apoptosis than electrolyte solution. No significant differences could be found among the preservation solutions.

  13. The association between autism and errors in early embryogenesis: what is the causal mechanism?

    NARCIS (Netherlands)

    Ploeger, A.; Raijmakers, M.E.J.; van der Maas, H.L.J.; Galis, F.

    2010-01-01

    The association between embryonic errors and the development of autism has been recognized in the literature, but the mechanism underlying this association remains unknown. We propose that pleiotropic effects during a very early and specific stage of embryonic development—early organogenesis—can

  14. Spatial clustering of porcine cysticercosis in Mbulu district, northern Tanzania.

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    Helena A Ngowi

    Full Text Available BACKGROUND: Porcine cysticercosis is caused by a zoonotic tapeworm, Taenia solium, which causes serious disease syndromes in human. Effective control of the parasite requires knowledge on the burden and pattern of the infections in order to properly direct limited resources. The objective of this study was to establish the spatial distribution of porcine cysticercosis in Mbulu district, northern Tanzania, to guide control strategies. METHODOLOGY/PRINCIPAL FINDINGS: This study is a secondary analysis of data collected during the baseline and follow-up periods of a randomized community trial aiming at reducing the incidence rate of porcine cysticercosis through an educational program. At baseline, 784 randomly selected pig-keeping households located in 42 villages in 14 wards were included. Lingual examination of indigenous pigs aged 2-12 (median 8 months, one randomly selected from each household, were conducted. Data from the control group of the randomized trial that included 21 of the 42 villages were used for the incidence study. A total of 295 pig-keeping households were provided with sentinel pigs (one each and reassessed for cysticercosis incidence once or twice for 2-9 (median 4 months using lingual examination and antigen ELISA. Prevalence of porcine cysticercosis was computed in Epi Info 3.5. The prevalence and incidence of porcine cysticercosis were mapped at household level using ArcView 3.2. K functions were computed in R software to assess general clustering of porcine cysticercosis. Spatial scan statistics were computed in SatScan to identify local clusters of the infection. The overall prevalence of porcine cysticercosis was 7.3% (95% CI: 5.6, 9.4; n = 784. The K functions revealed a significant overall clustering of porcine cysticercosis incidence for all distances between 600 m and 5 km from a randomly chosen case household based on Ag-ELISA. Lingual examination revealed clustering from 650 m to 6 km and between 7.5 and 10 km

  15. Porcine SLITRK1

    DEFF Research Database (Denmark)

    Larsen, Knud Erik; Momeni, Jamal; Farajzadeh, Leila

    2014-01-01

    The membrane protein SLITRK1 functions as a developmentally regulated stimulator of neurite outgrowth and variants in this gene have been implicated in Tourette syndrome. In the current study we have cloned and characterized the porcine SLITRK1 gene. The genomic organization of SLITRK1 lacks...

  16. Redeployment of germ layers related TFs shows regionalized expression during two non-embryonic developments.

    Science.gov (United States)

    Ricci, Lorenzo; Cabrera, Fabien; Lotito, Sonia; Tiozzo, Stefano

    2016-08-01

    In all non-vertebrate metazoan phyla, species that evolved non-embryonic developmental pathways as means of propagation or regeneration can be found. In this context, new bodies arise through asexual reproduction processes (such as budding) or whole body regeneration, that lack the familiar temporal and spatial cues classically associated with embryogenesis, like maternal determinants, or gastrulation. The molecular mechanisms underlying those non-embryonic developments (i.e., regeneration and asexual reproduction), and their relationship to those deployed during embryogenesis are poorly understood. We have addressed this question in the colonial ascidian Botryllus schlosseri, which undergoes an asexual reproductive process via palleal budding (PB), as well as a whole body regeneration by vascular budding (VB). We identified early regenerative structures during VB and then followed the fate of differentiating tissues during both non-embryonic developments (PB and VB) by monitoring the expression of genes known to play key functions in germ layer specification with well conserved expression patterns in solitary ascidian embryogenesis. The expression patterns of FoxA1, GATAa, GATAb, Otx, Bra, Gsc and Tbx2/3 were analysed during both PB and VB. We found that the majority of these transcription factors were expressed during both non-embryonic developmental processes, revealing a regionalization of the palleal and vascular buds. Knockdown of GATAa by siRNA in palleal buds confirmed that preventing the correct development of one of these regions blocks further tissue specification. Our results indicate that during both normal and injury-induced budding, a similar alternative developmental program operates via early commitment of epithelial regions. Copyright © 2016. Published by Elsevier Inc.

  17. FGF signaling via MAPK is required early and improves Activin A-induced definitive endoderm formation from human embryonic stem cells

    International Nuclear Information System (INIS)

    Sui, Lina; Mfopou, Josué K.; Geens, Mieke; Sermon, Karen; Bouwens, Luc

    2012-01-01

    Highlights: ► Deep study the FGF signaling role during DE specification in the context of hESCs. ► DE differentiation from hESCs has an early dependence on FGF signaling. ► A serum-free DE protocol is developed based on the findings. ► The DE cells showed potential to differentiate into pancreatic progenitor cells. -- Abstract: Considering their unlimited proliferation and pluripotency properties, human embryonic stem cells (hESCs) constitute a promising resource applicable for cell replacement therapy. To facilitate this clinical translation, it is critical to study and understand the early stage of hESCs differentiation wherein germ layers are defined. In this study, we examined the role of FGF signaling in Activin A-induced definitive endoderm (DE) differentiation in the absence of supplemented animal serum. We found that activated FGF/MAPK signaling is required at the early time point of Activin A-induced DE formation. In addition, FGF activation increased the number of DE cells compared to Activin A alone. These DE cells could further differentiate into PDX1 and NKX6.1 positive pancreatic progenitors in vitro. We conclude that Activin A combined with FGF/MAPK signaling efficiently induce DE cells in the absence of serum. These findings improve our understanding of human endoderm formation, and constitute a step forward in the generation of clinical grade hESCs progenies for cell therapy.

  18. Targeted Porcine Genome Engineering with TALENs

    DEFF Research Database (Denmark)

    Luo, Yonglun; Lin, Lin; Golas, Mariola Monika

    2015-01-01

    confers precisely editing (e.g., mutations or indels) or insertion of a functional transgenic cassette to user-designed loci. Techniques for targeted genome engineering are growing dramatically and include, e.g., zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs......, including construction of sequence-specific TALENs, delivery of TALENs into primary porcine fibroblasts, and detection of TALEN-mediated cleavage, is described. This chapter is useful for scientists who are inexperienced with TALEN engineering of porcine cells as well as of other large animals....

  19. Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells

    Energy Technology Data Exchange (ETDEWEB)

    Cong, Yingying; Li, Xiaoxue; Bai, Yunyun [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); Lv, Xiaonan [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); CAS Key Lab for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience & Technology of China, Beijing 100090 (China); Herrler, Georg [Institute for Virology, University of Veterinary Medicine, Hannover D-30559 (Germany); Enjuanes, Luis [Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid (Spain); Zhou, Xingdong [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); Qu, Bo [Faculty of Life Sciences, Northeast Agricultural University, Harbin 150030 (China); Meng, Fandan [Institute for Virology, University of Veterinary Medicine, Hannover D-30559 (Germany); Cong, Chengcheng [College Animal Husbandry and Veterinary Medicine, Shenyang Agricultural University, Shenyang 110161 (China); Ren, Xiaofeng; Li, Guangxing [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China)

    2015-04-15

    Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus released into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells. - Highlights: • PEDV infection of polarized intestinal epithelial cells (IECs) was characterized. • Porcine aminpeptidase N (pAPN) facilitated PEDV infection in IECs. • PEDV entry into and release from polarized cell via its apical membrane. • PEDV infection may proceed by lateral spread of virus in IECs.

  20. Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells

    International Nuclear Information System (INIS)

    Cong, Yingying; Li, Xiaoxue; Bai, Yunyun; Lv, Xiaonan; Herrler, Georg; Enjuanes, Luis; Zhou, Xingdong; Qu, Bo; Meng, Fandan; Cong, Chengcheng; Ren, Xiaofeng; Li, Guangxing

    2015-01-01

    Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus released into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells. - Highlights: • PEDV infection of polarized intestinal epithelial cells (IECs) was characterized. • Porcine aminpeptidase N (pAPN) facilitated PEDV infection in IECs. • PEDV entry into and release from polarized cell via its apical membrane. • PEDV infection may proceed by lateral spread of virus in IECs

  1. Comparison of commercial and experimental porcine circovirus type 2 (PCV2) vaccines using a triple challenge with PCV2, porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus (PPV).

    Science.gov (United States)

    Shen, H G; Beach, N M; Huang, Y W; Halbur, P G; Meng, X J; Opriessnig, T

    2010-08-23

    The efficacies of commercial porcine circovirus type 2 (PCV2) vaccines and a live PCV1-2a chimeric vaccine were compared in conventional, PCV2-positive piglets using a PCV2-porcine reproductive and respiratory syndrome virus (PRRSV)-porcine parvovirus (PPV) coinfection challenge model. Seventy-three, 2-week-old pigs were randomized into seven groups including five vaccinated and two control groups. Pigs in the vaccinated groups were vaccinated at 3 weeks (one dose) or at 3 and 6 weeks (two dose) of age. All vaccine regimens tested were effective in reducing naturally occurring PCV2 viremia at 16 weeks of age and after PCV2 challenge, demonstrating the capability of the products to induce a lasting protective immunity despite the presence of PCV2 viremia at the time of vaccination. Copyright 2010 Elsevier Ltd. All rights reserved.

  2. Circovirose suína Porcine circovirosis: a review

    Directory of Open Access Journals (Sweden)

    Ticiana do Nascimento França

    2005-06-01

    Full Text Available Por meio de revisão da literatura pertinente foram coligidos e são apresentados os principais dados relativos aos aspectos epidemiológicos, clínicos, anátomo e histopatológicos observados na infecção por Circovírus Porcino tipo 2 (PCV-2 em suínos. São abordados a Síndrome Definhante Multissistêmica dos Suínos Desmamados (SDMDS, o Tremor Congênito Suíno (TCS, a Síndrome da Nefropatia e Dermatite Porcina (SNDP, bem como outras enfermidades associadas ou correlatas, a Síndrome Respiratória e Reprodutiva Porcina (SRRP, a Pneumonia Necrotizante Proliferativa (PNP e as falhas reprodutivas. Uma vez que a SDMSD já foi registrada na Região Sul do Brasil e no Estado do Rio de Janeiro esse estudo objetiva chamar a atenção para o especial significado dessa virose para a suinocultura brasileira, em função dos prejuízos econômicos por ela determinados.The literature of Porcine Circovirosis, including the main data on epidemiology and clinical, macroscopic and microscopic alterations of the infection of swine by Porcine Circovirus type 2 (PCV-2, is reviewed. There are various forms of infection: the [Porcine] Postweaning Multisystemic Wasting Syndrome (PMWS, Porcine Congenital Tremor, Porcine Dermatitis and Nephropathy Syndrome, and other associated or correlated diseases as the Porcine Reproductive and Respiratory Syndrome, Proliferative Necrotizing Pneumonia, and reproductive disorders. As PMWS already has been reported from southern Brazil and from the state of Rio de Janeiro, the objective of this review is to draw attention to the implications of this virosis for swine production in Brazil and its economical importance.

  3. Mechanobiology of embryonic limb development.

    Science.gov (United States)

    Nowlan, Niamh C; Murphy, Paula; Prendergast, Patrick J

    2007-04-01

    Considerable evidence exists to support the hypothesis that mechanical forces have an essential role in healthy embryonic skeletal development. Clinical observations and experimental data indicate the importance of muscle contractions for limb development. However, the influence of these forces is seldom referred to in biological descriptions of bone development, and perhaps this is due to the fact that the hypothesis that mechanical forces are essential for normal embryonic skeletal development is difficult to test and elaborate experimentally in vivo, particularly in humans. Computational modeling has the potential to address this issue by simulating embryonic growth under a range of loading conditions but the potential of such models has yet to be fully exploited. In this article, we review the literature on mechanobiology of limb development in three main sections: (a) experimental alteration of the mechanical environment, (b) mechanical properties of embryonic tissues, and (c) the use of computational models. Then we analyze the main issues, and suggest how experimental and computational fields could work closer together to enhance our understanding of mechanobiology of the embryonic skeleton.

  4. Absence of Rybp Compromises Neural Differentiation of Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Gergo Kovacs

    2016-01-01

    Full Text Available Rybp (Ring1 and Yy1 Binding Protein is a transcriptional regulator and member of the noncanonical polycomb repressive complex 1 with essential role in early embryonic development. We have previously described that alteration of Rybp dosage in mouse models induced striking neural tube defects (NTDs, exencephaly, and disorganized neurocortex. In this study we further investigated the role of Rybp in neural differentiation by utilising wild type (rybp+/+ and rybp null mutant (rybp-/- embryonic stem cells (ESCs and tried to uncover underlying molecular events that are responsible for the observed phenotypic changes. We found that rybp null mutant ESCs formed less matured neurons, astrocytes, and oligodendrocytes from existing progenitors than wild type cells. Furthermore, lack of rybp coincided with altered gene expression of key neural markers including Pax6 and Plagl1 pinpointing a possible transcriptional circuit among these genes.

  5. Functional hierarchy of two L domains in porcine endogenous retrovirus (PERV) that influence release and infectivity

    International Nuclear Information System (INIS)

    Marcucci, Katherine T.; Martina, Yuri; Harrison, Frank; Wilson, Carolyn A.; Salomon, Daniel R.

    2008-01-01

    The porcine endogenous retrovirus (PERV) Gag protein contains two late (L) domain motifs, PPPY and P(F/S)AP. Using viral release assays we demonstrate that PPPY is the dominant L domain involved in PERV release. PFAP represents a novel retroviral L domain variant and is defined by abnormal viral assembly phenotypes visualized by electron microscopy and attenuation of early PERV release as measured by viral genomes. PSAP is functionally dominant over PFAP in early PERV release. PSAP virions are 3.5-fold more infectious in vitro by TCID 50 and in vivo results in more RNA positive tissues and higher levels of proviral DNA using our human PERV-A receptor (HuPAR-2) transgenic mouse model [Martina, Y., Marcucci, K.T., Cherqui, S., Szabo, A., Drysdale, T., Srinivisan, U., Wilson, C.A., Patience, C., Salomon, D.R., 2006. Mice transgenic for a human porcine endogenous retrovirus receptor are susceptible to productive viral infection. J. Virol. 80 (7), 3135-3146]. The functional hierarchies displayed by PERV L domains, demonstrates that L domain selection in viral evolution exists to promote efficient viral assembly, release and infectivity in the virus-host context

  6. Plasma membrane proteomics of human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Braam, S.R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2008-01-01

    Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired

  7. Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland.

    Science.gov (United States)

    Lilja, Anna M; Rodilla, Veronica; Huyghe, Mathilde; Hannezo, Edouard; Landragin, Camille; Renaud, Olivier; Leroy, Olivier; Rulands, Steffen; Simons, Benjamin D; Fre, Silvia

    2018-06-01

    Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.5 and no statistically discernable bipotency after E15.5. To gain insights into the mechanisms governing the switch from multipotency to unipotency, we used gain-of-function Notch1 mice and demonstrated that Notch activation cell autonomously dictates luminal cell fate specification to both embryonic and basally committed mammary cells. These functional studies have important implications for understanding the signals underlying cell plasticity and serve to clarify how reactivation of embryonic programs in adult cells can lead to cancer.

  8. Anterograde Tracing Method using DiI to Label Vagal Innervation of the Embryonic and Early Postnatal Mouse Gastrointestinal Tract

    Science.gov (United States)

    Murphy, Michelle C.; Fox, Edward A.

    2007-01-01

    The mouse is an extremely valuable model for studying vagal development in relation to strain differences, genetic variation, gene manipulations, or pharmacological manipulations. Therefore, a method using 1, 1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was developed for labeling vagal innervation of the gastrointestinal (GI) tract in embryonic and postnatal mice. DiI labeling was adapted and optimized for this purpose by varying several facets of the method. For example, insertion and crushing of DiI crystals into the nerve led to faster DiI diffusion along vagal axons and diffusion over longer distances as compared with piercing the nerve with a micropipette tip coated with dried DiI oil. Moreover, inclusion of EDTA in the fixative reduced leakage of DiI out of nerve fibers that occurred with long incubations. Also, mounting labeled tissue in PBS was superior to glycerol with n-propyl gallate, which resulted in reduced clarity of DiI labeling that may have been due to DiI leaking out of fibers. Optical sectioning of flattened wholemounts permitted examination of individual tissue layers of the GI tract wall. This procedure aided identification of nerve ending types because in most instances each type innervates a different tissue layer. Between embryonic day 12.5 and postnatal day 8, growth of axons into the GI tract, formation and patterning of fiber bundles in the myenteric plexus and early formation of putative afferent and efferent nerve terminals were observed. Thus, the DiI tracing method developed here has opened up a window for investigation during an important phase of vagal development. PMID:17418900

  9. The kinetics of interaction of porcine - alpha-, and porcine - beta -trypsin with intact and modified soybean trypsin inhibitor (kunitz)

    International Nuclear Information System (INIS)

    Hamid, M.A.

    1994-01-01

    The association of porcine trypsin with soybean trypsin inhibitor (Kunitz) resulted in characteristic changes in absorption spectrum, indicating an alteration of the micro environments of the enzyme chromophores as a consequence of the interaction. The rates of formation of the stable trypsin - inhibitor complexes from porcine - alpha - trypsin and soybean trypsin inhibitor and from porcine - beta - trypsin and either intact or modified soybean trypsin inhibitor were measured by mixing the equimolar concentration of the reactants in a Stopped - Flow apparatus at pH (4.5 to 10.0). The reaction of trypsin with soybean trypsin inhibitor was of first order with respect to the concentration of the reactants used. The rates of dissociation of the stable complexes, alpha - trypsin - soybean trypsin inhibitor, beta -trypsin - soybean trypsin inhibitor and beta -trypsin modified soybean trypsin inhibitor were also measured at pH (1.92 to 3.58). The values of first order rate constant, k/sub D/ obtained for the dissociation of all the three complexes were identical with one another. The kinetics results obtained for the porcine trypsin were compared with those of bovine trypsin system and it was suggested that the reaction mechanisms in both these systems were identical. (author)

  10. The porcine lymphotropic herpesvirus 1 encodes functional regulators of gene expression

    International Nuclear Information System (INIS)

    Lindner, I.; Ehlers, B.; Noack, S.; Dural, G.; Yasmum, N.; Bauer, C.; Goltz, M.

    2007-01-01

    The porcine lymphotropic herpesviruses (PLHV) are discussed as possible risk factors in xenotransplantation because of the high prevalence of PLHV-1, PLHV-2 and PLHV-3 in pig populations world-wide and the fact that PLHV-1 has been found to be associated with porcine post-transplant lymphoproliferative disease. To provide structural and functional knowledge on the PLHV immediate-early (IE) transactivator genes, the central regions of the PLHV genomes were characterized by genome walking, sequence and splicing analysis. Three spliced genes were identified (ORF50, ORFA6/BZLF1 h , ORF57) encoding putative IE transactivators, homologous to (i) ORF50 and BRLF1/Rta (ii) K8/K-bZIP and BZLF1/Zta and (iii) ORF57 and BMLF1 of HHV-8 and EBV, respectively. Expressed as myc-tag or HA-tag fusion proteins, they were located to the cellular nucleus. In reporter gene assays, several PLHV-promoters were mainly activated by PLHV-1 ORF50, to a lower level by PLHV-1 ORFA6/BZLF1 h and not by PLHV-1 ORF57. However, the ORF57-encoded protein acted synergistically on ORF50-mediated activation

  11. Paternal identity impacts embryonic development for two species of freshwater fish

    DEFF Research Database (Denmark)

    Siddique, Mohammad Abdul Momin; Linhart, Otomar; Krejszeff, Sławomir

    2017-01-01

    then partition variation in embryonic phenotypic performance to maternal, paternal, and parental interactions using the Restricted Maximum Likelihood (REML) model. Results showed that paternal, maternal, and the paternal. ×. maternal interaction terms were highly significant for both species; clearly......Paternal, compared to maternal, contributions were believed to have only a limited influence on embryonic development and larval fitness traits in fishes. Therefore, the perspective of male influence on early life history traits has come under scrutiny. This study was conducted to determine...... demonstrating that certain family combinations were more compatible than others. Paternal effects explained 20.24% of the total variance, which was 2-fold higher than the maternal effects (10.73%) in Ide, while paternal effects explained 18.9% of the total variance, which was 15-fold higher than the maternal...

  12. Establishing the Embryonic Axes: Prime Time for Teratogenic Insults

    Directory of Open Access Journals (Sweden)

    Thomas W. Sadler

    2017-09-01

    Full Text Available A long standing axiom in the field of teratology states that the teratogenic period, when most birth defects are produced, occurs during the third to eighth weeks of development post-fertilization. Any insults prior to this time are thought to result in a slowing of embryonic growth from which the conceptus recovers or death of the embryo followed by spontaneous abortion. However, new insights into embryonic development during the first two weeks, including formation of the anterior-posterior, dorsal-ventral, and left-right axes, suggests that signaling pathways regulating these processes are prime targets for genetic and toxic insults. Establishment of the left-right (laterality axis is particularly sensitive to disruption at very early stages of development and these perturbations result in a wide variety of congenital malformations, especially heart defects. Thus, the time for teratogenic insults resulting in birth defects should be reset to include the first two weeks of development.

  13. Bacteriospermia in extended porcine semen.

    Science.gov (United States)

    Althouse, Gary C; Lu, Kristina G

    2005-01-15

    Bacteriospermia is a frequent finding in freshly extended porcine semen and can result in detrimental effects on semen quality and longevity if left uncontrolled. The primary source of bacterial contamination is the boar. Other sources that have been identified include environment, personnel, and the water used for extender preparation. A 1-year retrospective study was performed on submissions of extended porcine semen for routine quality control bacteriological screening at the University of Pennsylvania. Out of 250 sample submissions, 78 (31.2%) tested positive for bacterial contamination. The most popular contaminants included Enterococcus spp. (20.5%), Stenotrophomonas maltophilia (15.4%), Alcaligenes xylosoxidans (10.3%), Serratia marcescens (10.3%), Acinetobacter lwoffi (7.7%), Escherichia coli (6.4%), Pseudomonas spp. (6.4%), and others (23.0%). Prudent individual hygiene, good overall sanitation, and regular monitoring can contribute greatly in controlling bacterial load. Strategies that incorporate temperature-dependent bacterial growth and hyperthermic augmentation of antimicrobial activity are valuable for effective control of susceptible bacterial loads. Aminoglycosides remain the most popular antimicrobial class used in porcine semen extenders, with beta-lactam and lincosamide use increasing. With the advent of more novel antimicrobial selection and semen extender compositions in swine, prudent application and understanding of in vitro pharmacodynamics are becoming paramount to industry success in the use of this breeding modality.

  14. The influence of IVF/ICSI treatment on human embryonic growth trajectories.

    Science.gov (United States)

    Eindhoven, S C; van Uitert, E M; Laven, J S E; Willemsen, S P; Koning, A H J; Eilers, P H C; Exalto, N; Steegers, E A P; Steegers-Theunissen, R P M

    2014-12-01

    groups (βIVF/ICSI = 6 g; P = 0.36 and βIVF/ICSI = 80 g; P = 0.24, respectively). Variations in embryonic growth trajectories of spontaneously conceived pregnancies with reliable pregnancy dating may partially be a result of less precise pregnancy dating and differences in endometrium receptivity compared with IVF/ICSI pregnancies. The absence of a significant difference in embryonic and fetal growth trajectories suggests safety of IVF/ICSI treatment with regard to early embryonic growth. However, further research is warranted to ascertain the influence of IVF/ICSI treatments in a larger study population, and to estimate the impact of the underlying causes of the subfertility and other periconceptional exposures on human embryonic and fetal growth trajectories. This study was supported by the Department of Obstetrics and Gynaecology of the Erasmus MC, University Medical Centre. No competing interests are declared. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine, and rabbit oocytes and nuclear donor cells of various species.

    Science.gov (United States)

    Lagutina, Irina; Zakhartchenko, Valeri; Fulka, Helena; Colleoni, Silvia; Wolf, Eckhard; Fulka, Josef; Lazzari, Giovanna; Galli, Cesare

    2011-04-01

    The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.

  16. Simultaneous cell death and desquamation of the embryonic diffusion barrier during epidermal development

    International Nuclear Information System (INIS)

    Saathoff, Manuela; Blum, Barbara; Quast, Thomas; Kirfel, Gregor; Herzog, Volker

    2004-01-01

    The periderm is an epithelial layer covering the emerging epidermis in early embryogenesis of vertebrates. In the chicken embryo, an additional cellular layer, the subperiderm, occurs at later embryonic stages underneath the periderm. The questions arose what is the function of both epithelial layers and, as they are transitory structures, by which mechanism are they removed. By immunocytochemistry, the tight junction (TJ) proteins occludin and claudin-1 were localized in the periderm and in the subperiderm, and sites of close contact between adjacent cells were detected by electron microscopy. Using horseradish peroxidase (HRP) as tracer, these contacts were identified as tight junctions involved in the formation of the embryonic diffusion barrier. This barrier was lost by desquamation at the end of the embryonic period, when the cornified envelope of the emerging epidermis was formed. By TUNEL and DNA ladder assays, we detected simultaneous cell death in the periderm and the subperiderm shortly before hatching. The absence of caspases-3, -6, and -7 activity, key enzymes of apoptosis, and the lack of typical morphological criteria of apoptosis such as cell fragmentation or membrane blebbing point to a special form of programmed cell death (PCD) leading to the desquamation of the embryonic diffusion barrier

  17. Observation of human embryonic behavior in vitro by high-resolution time-lapse cinematography.

    Science.gov (United States)

    Iwata, Kyoko; Mio, Yasuyuki

    2016-07-01

    Assisted reproductive technology (ART) has yielded vast amounts of information and knowledge on human embryonic development in vitro; however, still images provide limited data on dynamic changes in the developing embryos. Using our high-resolution time-lapse cinematography (hR-TLC) system, we were able to describe normal human embryonic development continuously from the fertilization process to the hatched blastocyst stage in detail. Our hR-TLC observation also showed the embryonic abnormality of a third polar body (PB)-like substance likely containing a small pronucleus being extruded and resulting in single-pronucleus (1PN) formation, while our molecular biological investigations suggested the possibility that some 1PN embryos could be diploid, carrying both maternal and paternal genomes. Furthermore, in some embryos the extruded third PB-like substance was eventually re-absorbed into the ooplasm resulting in the formation of an uneven-sized, two-PN zygote. In addition, other hR-TLC observations showed that cytokinetic failure was correlated with equal-sized, multi-nucleated blastomeres that were also observed in the embryo showing early initiation of compaction. Assessment combining our hR-TLC with molecular biological techniques enables a better understanding of embryonic development and potential improvements in ART outcomes.

  18. The embryonic origin of the ampullate silk glands of the spider Cupiennius salei.

    Science.gov (United States)

    Hilbrant, Maarten; Damen, Wim G M

    2015-05-01

    Silk production in spiders is considered a key innovation, and to have been vital for the diversification of the clade. The evolutionary origin of the organs involved in spider silk production, however, and in particular of the silk glands, is poorly understood. Homologies have been proposed between these and other glands found in arachnids, but lacking knowledge of the embryonic development of spider silk glands hampers an evaluation of hypotheses. This study focuses on the embryonic origin of the largest silk glands of the spider Cupiennius salei, the major and minor ampullate glands. We show how the ampullate glands originate from ectodermal invaginations on the embryonic spinneret limb buds, in relation to morphogenesis of these buds. Moreover, we visualize the subsequent growth of the ampullate glands in sections of the early postembryonic stages. The invaginations are shown to correlate with expression of the proneural gene CsASH2, which is remarkable since it has been proposed that spider silk glands and their nozzles originate from sensory bristles. Hence, by confirming the ectodermal origin of spider silk glands, and by describing the (post-)embryonic morphogenesis of the ampullate glands, this work provides a starting point for further investigating into the genetic program that underlies their development. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Paternal identity impacts embryonic development for two species of freshwater fish.

    Science.gov (United States)

    Siddique, Mohammad Abdul Momin; Linhart, Otomar; Krejszeff, Sławomir; Żarski, Daniel; Pitcher, Trevor E; Politis, Sebastian Nikitas; Butts, Ian Anthony Ernest

    2017-05-01

    Paternal, compared to maternal, contributions were believed to have only a limited influence on embryonic development and larval fitness traits in fishes. Therefore, the perspective of male influence on early life history traits has come under scrutiny. This study was conducted to determine parental effects on the rate of eyed embryos of Ide Leuciscus idus and Northern pike Esox lucius. Five sires and five dams from each species were crossed using a quantitative genetic breeding design and the resulting 25 sib groups of each species were reared to the embryonic eyed stage. We then partition variation in embryonic phenotypic performance to maternal, paternal, and parental interactions using the Restricted Maximum Likelihood (REML) model. Results showed that paternal, maternal, and the paternal×maternal interaction terms were highly significant for both species; clearly demonstrating that certain family combinations were more compatible than others. Paternal effects explained 20.24% of the total variance, which was 2-fold higher than the maternal effects (10.73%) in Ide, while paternal effects explained 18.9% of the total variance, which was 15-fold higher than the maternal effects (1.3%) in Northern pike. Together, these results indicate that male effects are of major importance during embryonic development for these species. Furthermore, this study demonstrates that genetic compatibility between sires and dams plays an important role and needs to be taken into consideration for reproduction of these and likely other economically important fish species. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Tissue-engineered bone with 3-dimensionally printed β-tricalcium phosphate and polycaprolactone scaffolds and early implantation: an in vivo pilot study in a porcine mandible model.

    Science.gov (United States)

    Konopnicki, Sandra; Sharaf, Basel; Resnick, Cory; Patenaude, Adam; Pogal-Sussman, Tracy; Hwang, Kyung-Gyun; Abukawa, Harutsugi; Troulis, Maria J

    2015-05-01

    Deep bone penetration into implanted scaffolds remains a challenge in tissue engineering. The purpose of this study was to evaluate bone penetration depth within 3-dimensionally (3D) printed β-tricalcium phosphate (β-TCP) and polycaprolactone (PCL) scaffolds, seeded with porcine bone marrow progenitor cells (pBMPCs), and implanted early in vivo. Scaffolds were 3D printed with 50% β-TCP and 50% PCL. The pBMPCs were harvested, isolated, expanded, and differentiated into osteoblasts. Cells were seeded into the scaffolds and constructs were incubated in a rotational oxygen-permeable bioreactor system for 14 days. Six 2- × 2-cm defects were created in each mandible (N = 2 minipigs). In total, 6 constructs were placed within defects and 6 defects were used as controls (unseeded scaffolds, n = 3; empty defects, n = 3). Eight weeks after surgery, specimens were harvested and analyzed by hematoxylin and eosin (H&E), 4',6-diamidino-2-phenylindole (DAPI), and CD31 staining. Analysis included cell counts, bone penetration, and angiogenesis at the center of the specimens. All specimens (N = 12) showed bone formation similar to native bone at the periphery. Of 6 constructs, 4 exhibited bone formation in the center. Histomorphometric analysis of the H&E-stained sections showed an average of 22.1% of bone in the center of the constructs group compared with 1.87% in the unseeded scaffolds (P bone in the center, showed massive cell penetration depth by DAPI staining, with an average of 2,109 cells/0.57 mm(2) in the center compared with 1,114 cells/0.57 mm(2) in the controls (P printed β-TCP and PCL scaffolds seeded with pBMPCs and implanted early into porcine mandibular defects display good bone penetration depth. Further study with a larger sample and larger bone defects should be performed before human applications. Copyright © 2015 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

  1. FGF signaling via MAPK is required early and improves Activin A-induced definitive endoderm formation from human embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Sui, Lina, E-mail: linasui@vub.ac.be [Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels (Belgium); Mfopou, Josue K. [Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels (Belgium); Geens, Mieke; Sermon, Karen [Department of Embryology and Genetics, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels (Belgium); Bouwens, Luc [Cell Differentiation Unit, Diabetes Research Center, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels (Belgium)

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Deep study the FGF signaling role during DE specification in the context of hESCs. Black-Right-Pointing-Pointer DE differentiation from hESCs has an early dependence on FGF signaling. Black-Right-Pointing-Pointer A serum-free DE protocol is developed based on the findings. Black-Right-Pointing-Pointer The DE cells showed potential to differentiate into pancreatic progenitor cells. -- Abstract: Considering their unlimited proliferation and pluripotency properties, human embryonic stem cells (hESCs) constitute a promising resource applicable for cell replacement therapy. To facilitate this clinical translation, it is critical to study and understand the early stage of hESCs differentiation wherein germ layers are defined. In this study, we examined the role of FGF signaling in Activin A-induced definitive endoderm (DE) differentiation in the absence of supplemented animal serum. We found that activated FGF/MAPK signaling is required at the early time point of Activin A-induced DE formation. In addition, FGF activation increased the number of DE cells compared to Activin A alone. These DE cells could further differentiate into PDX1 and NKX6.1 positive pancreatic progenitors in vitro. We conclude that Activin A combined with FGF/MAPK signaling efficiently induce DE cells in the absence of serum. These findings improve our understanding of human endoderm formation, and constitute a step forward in the generation of clinical grade hESCs progenies for cell therapy.

  2. New stepwise cooling system for short-term porcine islet preservation.

    Science.gov (United States)

    Ikemoto, Tetsuya; Noguchi, Hirofumi; Fujita, Yasutaka; Takita, Morihito; Shimoda, Masayuki; Sugimoto, Koji; Jackson, Andrew; Naziruddin, Bashoo; Shimada, Mitsuo; Levy, Marlon F; Matsumoto, Shinichi

    2010-10-01

    Porcine islets are the most suitable for xeno-islet transplantation. However, it is necessary to establish an effective preservation method against its fragility. Recently, we developed a new cooling and preservation (Keep and Fresh [KFC]; FUJIYA Co, Tokushima, Japan) system, which can maintain viability of hepatocyte. In this study, we examined the KFC for porcine islet preservation. Isolated porcine islets were preserved in CMRL 1066 culture media with bovine serum at 37°C, 22°C, and 4°C and KFC for 24, 48, and 72 hours. Islet recovery rate, purity, and viability were evaluated. After 24-hour preservation, the recovery rate was the highest in the KFC, but no significant difference was found. After 48-hour preservation, the recovery rate by the KFC was 73.9% ± 17.3%, which was significantly higher than the other groups (48.7% ± 28.6% at 37°C, P KFC group, purities and viabilities were the highest among the groups after 24-, 48-, and 72-hour preservation. The KFC system significantly improved porcine islet preservation; therefore, the KFC might be useful for porcine islet preservation.

  3. Growth trajectories of the human embryonic head and periconceptional maternal conditions.

    Science.gov (United States)

    Koning, I V; Baken, L; Groenenberg, I A L; Husen, S C; Dudink, J; Willemsen, S P; Gijtenbeek, M; Koning, A H J; Reiss, I K M; Steegers, E A P; Steegers-Theunissen, R P M

    2016-05-01

    Can growth trajectories of the human embryonic head be created using 3D ultrasound (3D-US) and virtual reality (VR) technology, and be associated with second trimester fetal head size and periconceptional maternal conditions? Serial first trimester head circumference (HC) and head volume (HV) measurements were used to create reliable growth trajectories of the embryonic head, which were significantly associated with fetal head size and periconceptional maternal smoking, age and ITALIC! in vitro fertilization (IVF)/intra-cytoplasmic sperm injection (ICSI) treatment. Fetal growth is influenced by periconceptional maternal conditions. We selected 149 singleton pregnancies with a live born non-malformed fetus from the Rotterdam periconception cohort. Bi-parietal diameter and occipital frontal diameter to calculate HC, HV and crown-rump length (CRL) were measured weekly between 9 + 0 and 12 + 6 weeks gestational age (GA) using 3D-US and VR. Fetal HC was obtained from second trimester structural anomaly scans. Growth trajectories of the embryonic head were created with general additive models and linear mixed models were used to estimate associations with maternal periconceptional conditions as a function of GA and CRL, respectively. A total of 303 3D-US images of 149 pregnancies were eligible for embryonic head measurements (intra-class correlation coefficients >0.99). Associations were found between embryonic HC and fetal HC ( ITALIC! ρ = 0.617, ITALIC! P head measured by HC and HV (All ITALIC! P head may be of benefit in future early antenatal care. This study was funded by the Department of Obstetrics and Gynaecology, Erasmus MC University Medical Centre and Sophia Foundation for Medical Research, Rotterdam, The Netherlands (SSWO grant number 644). No competing interests are declared. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email

  4. Embryonic Stem Cell Proteins and MicroRNAs in the Etiology of Germ Cell Cancer

    NARCIS (Netherlands)

    R. Eini (Ronak)

    2013-01-01

    textabstractIn the early 1980s, a population of unique cells was isolated from the inner cell mass (ICM) of the mouse pre-implantation embryo named embryonic stem (ES) cells. These cells were generated by removing the ICM from pre-implantation blastocysts. The resulting cells were found to be

  5. Extraction Socket Preservation Using Porcine-Derived Collagen Membrane Alone or Associated with Porcine-Derived Bone. Clinical Results of Randomized Controlled Study

    Directory of Open Access Journals (Sweden)

    Renzo Guarnieri

    2017-03-01

    Full Text Available Objectives: The aim of present randomized controlled clinical trial was to clinically evaluate hard tissue changes after extraction socket preservation procedures compared to natural spontaneous healing. Material and Methods: Thirty patients were enrolled in the present study and underwent single-tooth extraction in the premolar/molar areas. Ten sites were grafted with porcine-derived bone covered by collagen membrane, 10 covered by porcine-derived collagen membrane alone, and 10 underwent natural spontaneous healing. Vertical and horizontal bone changes after 3-month were evaluated at implant placement. Results: The vertical and horizontal bone changes at the extraction sockets treated with collagen membrane alone (vertical: -0.55 [SD 0.11] mm, and horizontal: -1.21 [SD 0.69] mm and collagen membrane plus porcine-derived bone (vertical: -0.37 [SD 0.7] mm, and horizontal: -0.91 [SD 0.53] mm were found significantly lower (P < 0.001, when compared to non-grafted sockets (vertical: -2.09 [SD 0.19] mm, and horizontal: -3.96 [SD 0.87] mm. In type 1 extraction sockets, in premolar sites, and in presence of vestibular bone thicknesses ≥ 1.5 mm, the use of collagen membrane alone revealed similar outcomes to those with additional graft material. Conclusions: At the re-entry surgery, extraction sockets grafted with porcine-derived bone and covered by collagen membrane, and extraction sockets covered by porcine-derived collagen membrane alone, showed significantly lower vertical and horizontal bone changes, compared to extraction sockets sites underwent natural spontaneous healing. However, a complete prevention of remodelling is not achievable, irrespective of the technique used.

  6. X-linked gene transcription patterns in female and male in vivo, in vitro and cloned porcine individual blastocysts.

    Directory of Open Access Journals (Sweden)

    Chi-Hun Park

    Full Text Available To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF, and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process

  7. Localization of DNA methyltransferase-1 during oocyte differentiation, in vitro maturation and early embryonic development in cow

    Directory of Open Access Journals (Sweden)

    A. M. Luciano

    2009-12-01

    Full Text Available DNA methyltransferase-1 (Dnmt1 is involved in the maintenance of DNA methylation patterns and is crucial for normal mammalian development. The aim of the present study was to assess the localization of Dnmt1 in cow, during the latest phases of oocyte differentiation and during the early stages of segmentation. Dnmt1 expression and localization were assessed in oocytes according to the chromatin configuration, which in turn provides an important epigenetic mechanism for the control of global gene expression and represents a morphological marker of oocyte differentiation.We found that the initial chromatin condensation was accompanied by a slight increase in the level of global DNA methylation, as assessed by 5-methyl-cytosine immunostaining followed by laser scanning confocal microscopy analysis (LSCM. RT-PCR confirmed the presence of Dnmt1 transcripts throughout this phase of oocyte differentiation. Analogously, Dnmt1 immunodetection and LSCM indicated that the protein was always present and localized in the cytoplasm, regardless the chromatin configuration and the level of global DNA methylation. Moreover, our data indicate that while Dnmt1 is retained in the cytoplasm in metaphase II stage oocytes and zygotes, it enters the nuclei of 8-16 cell stage embryos. As suggested in mouse, the functional meaning of the presence of Dnmt1 in the bovine embryo nuclei could be the maintainement of the methylation pattern of imprinted genes. In conclusion, the present work provides useful elements for the study of Dnmt1 function during the late stage of oocyte differentiation, maturation and early embryonic development in mammals.

  8. Curcumin is a promising inhibitor of genotype 2 porcine reproductive and respiratory syndrome virus infection.

    Science.gov (United States)

    Du, Taofeng; Shi, Yunpeng; Xiao, Shuqi; Li, Na; Zhao, Qin; Zhang, Angke; Nan, Yuchen; Mu, Yang; Sun, Yani; Wu, Chunyan; Zhang, Hongtao; Zhou, En-Min

    2017-10-10

    Porcine reproductive and respiratory syndrome virus (PRRSV) could lead to pandemic diseases and huge financial losses to the swine industry worldwide. Curcumin, a natural compound, has been reported to serve as an entry inhibitor of hepatitis C virus, chikungunya virus and vesicular stomatitis virus. In this study, we investigated the potential effect of curcumin on early stages of PRRSV infection. Curcumin inhibited infection of Marc-145 cells and porcine alveolar macrophages (PAMs) by four different genotype 2 PRRSV strains, but had no effect on the levels of major PRRSV receptor proteins on Marc-145 cells and PAMs or on PRRSV binding to Marc-145 cells. However, curcumin did block two steps of the PRRSV infection process: virus internalization and virus-mediated cell fusion. Our results suggested that an inhibition of genotype 2 PRRSV infection by curcumin is virus strain-independent, and mainly inhibited by virus internalization and cell fusion mediated by virus. Collectively, these results demonstrate that curcumin holds promise as a new anti-PRRSV drug.

  9. Expression and proteasomal degradation of the major vault protein (MVP) in mammalian oocytes and zygotes.

    Science.gov (United States)

    Sutovsky, Peter; Manandhar, Gaurishankar; Laurincik, Jozef; Letko, Juraj; Caamaño, Jose Nestor; Day, Billy N; Lai, Liangxue; Prather, Randall S; Sharpe-Timms, Kathy L; Zimmer, Randall; Sutovsky, Miriam

    2005-03-01

    Major vault protein (MVP), also called lung resistance-related protein is a ribonucleoprotein comprising a major part (>70%) of the vault particle. The function of vault particle is not known, although it appears to be involved in multi-drug resistance and cellular signaling. Here we show that MVP is expressed in mammalian, porcine, and human ova and in the porcine preimplantation embryo. MVP was identified by matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) peptide sequencing and Western blotting as a protein accumulating in porcine zygotes cultured in the presence of specific proteasomal inhibitor MG132. MVP also accumulated in poor-quality human oocytes donated by infertile couples and porcine embryos that failed to develop normally after in vitro fertilization or somatic cell nuclear transfer. Normal porcine oocytes and embryos at various stages of preimplantation development showed mostly cytoplasmic labeling, with increased accumulation of vault particles around large cytoplasmic lipid inclusions and membrane vesicles. Occasionally, MVP was associated with the nuclear envelope and nucleolus precursor bodies. Nucleotide sequences with a high degree of homology to human MVP gene sequence were identified in porcine oocyte and endometrial cell cDNA libraries. We interpret these data as the evidence for the expression and ubiquitin-proteasome-dependent turnover of MVP in the mammalian ovum. Similar to carcinoma cells, MVP could fulfill a cell-protecting function during early embryonic development.

  10. Studies on the toxic effects of periodontal sustained release drug containing ornidazole and pefloxacin mesylate on early embryonic development of SD rat

    Directory of Open Access Journals (Sweden)

    Zheng-mou DONG

    2011-01-01

    Full Text Available Objective To evaluate the toxic effects of periodontal sustained release drug containing ornidazole and pefloxacin mesylate on early embryonic development of SD rats.Methods A total of 100female SD rats were randomly divided into negative control,low-,medium-,high-dose group and intervention group(20each.Rats in low-,medium-and high-dose group were fed daily with the sustained release drug at 1,4,and 8g/kg respectively;those in negative control group were fed daily with distilled water from the 14th day before mating to the 7th day of pregnancy continuously,and those in intervention group received cyclophosphamide(40mg/kgby intraperitoneal injection for 5successive days.During this period,the general status,mating,pregnancy,coefficient of ovary and uterus,the numbers of corpus luteum,nidation,live births,stillbirths,absorbed embryo,prenidatory and postnidatory mortality,serum testosterone(Tand estradiol(E2were determined respectively.Histopathologic examination of the ovary and uterus,immunohistochemical observation of ovaries for proliferating cell nuclear antigen(PCNAand Bcl-2associated X protein(Baxwere also performed respectively.Results The general status of those rats was good except one in the low-dose group and one in the intervention group died on the 14th day of administration,and one in negative control and one in high dose group died on the 5th day of pregnancy,respectively.The body weight of animals decreased significantly(P 0.05.The serum T level in medium-and high-dose group and the E2level in high-dose group declined compared to that in negative control group(P < 0.05.Conclusions Although the periodontal sustained release drug containing ornidazole and pefloxacin mesylate shows no toxicity to the early embryonic development of SD rats,the high dose drug has certain toxicity to ovary.Declined serum concentrations of T and E2,reduced expression of PCNA,and increased Bax may be the causes of the toxicity.

  11. Expression pattern of pluripotent markers in different embryonic developmental stages of buffalo (Bubalus bubalis) embryos and putative embryonic stem cells generated by parthenogenetic activation.

    Science.gov (United States)

    Singh, Karn P; Kaushik, Ramakant; Garg, Veena; Sharma, Ruchi; George, Aman; Singh, Manoj K; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S

    2012-12-01

    In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.

  12. Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells

    DEFF Research Database (Denmark)

    Anisimov, Sergey V.; Christophersen, Nicolaj S.; Correia, Ana S.

    2011-01-01

    The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both...... the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early...... foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates....

  13. A Modified Murine Embryonic Stem Cell Test for Evaluating the Teratogenic Effects of Drugs on Early Embryogenesis.

    Directory of Open Access Journals (Sweden)

    Ruoxing Yu

    Full Text Available Mammalian fetal development is easily disrupted by exogenous agents, making it essential to test new drug candidates for embryotoxicity and teratogenicity. To standardize the testing of drugs that might be used to treat pregnant women, the U.S. Food and Drug Administration (FDA formulated special grade categories, labeled A, B, C, D and X, that define the level of risk associated with the use of a specific drug during pregnancy. Drugs in categories (Cat. D and X are those with embryotoxic and/or teratogenic effects on humans and animals. However, which stages of pregnancy are affected by these agents and their molecular mechanisms are unknown. We describe here an embryonic stem cell test (EST that classifies FDA pregnancy Cat.D and Cat.X drugs into 4 classes based on their differing effects on primitive streak formation. We show that ~84% of Cat.D and Cat.X drugs target this period of embryogenesis. Our results demonstrate that our modified EST can identify how a drug affects early embryogenesis, when it acts, and its molecular mechanism. Our test may thus be a useful addition to the drug safety testing armamentarium.

  14. Deletion of exon 20 of the Familial Dysautonomia gene Ikbkap in mice causes developmental delay, cardiovascular defects, and early embryonic lethality.

    Directory of Open Access Journals (Sweden)

    Paula Dietrich

    Full Text Available Familial Dysautonomia (FD is an autosomal recessive disorder that affects 1/3,600 live births in the Ashkenazi Jewish population, and leads to death before the age of 40. The disease is characterized by abnormal development and progressive degeneration of the sensory and autonomic nervous system. A single base pair substitution in intron 20 of the Ikbkap gene accounts for 98% of FD cases, and results in the expression of low levels of the full-length mRNA with simultaneous expression of an aberrantly spliced mRNA in which exon 20 is missing. To date, there is no animal model for the disease, and the essential cellular functions of IKAP--the protein encoded by Ikbkap--remain unknown. To better understand the normal function of IKAP and in an effort to generate a mouse model for FD, we have targeted the mouse Ikbkap gene by homologous recombination. We created two distinct alleles that result in either loss of Ikbkap expression, or expression of an mRNA lacking only exon 20. Homozygosity for either mutation leads to developmental delay, cardiovascular and brain malformations, accompanied with early embryonic lethality. Our analyses indicate that IKAP is essential for expression of specific genes involved in cardiac morphogenesis, and that cardiac failure is the likely cause of abnormal vascular development and embryonic lethality. Our results also indicate that deletion of exon 20 abolishes gene function. This implies that the truncated IKAP protein expressed in FD patients does not retain any significant biological function.

  15. The effect of excess expression of GFP in a novel heart-specific green fluorescence zebrafish regulated by nppa enhancer at early embryonic development.

    Science.gov (United States)

    Huang, Wen; Deng, Yun; Dong, Wei; Yuan, Wuzhou; Wan, Yongqi; Mo, Xiaoyan; Li, Yongqing; Wang, Zequn; Wang, Yuequn; Ocorr, Karen; Zhang, Bo; Lin, Shuo; Wu, Xiushan

    2011-02-01

    In order to study the impalpable effect of GFP in homozygous heart-specific GFP-positive zebrafish during the early stage, the researchers analyzed the heart function of morphology and physiology at the first 3 days after fertilization. This zebrafish line was produced by a large-scale Tol2 transposon mediated enhancer trap screen that generated a transgenic zebrafish with a heart-specific expression of green fluorescent protein (GFP)-tagged under control of the nppa enhancer. In situ hybridization experiments showed that the nppa:GFP line faithfully recapitulated both the spatial and temporal expressions of the endogenous nppa. Green fluorescence was intensively and specifically expressed in the myocardial cells located both in the heart chambers and in the atrioventricular canal. The embryonic heart of nppa:GFP line developed normally compared with those in the wild type. There was no difference between the nappa:GFP and wild type lines with respect to heart rate, overall size, ejection volume, and fractional shortening. Thus the excess expression of GFP in this transgenic line seemed to exert no detrimental effects on zebrafish hearts during the early stages.

  16. Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate

    Directory of Open Access Journals (Sweden)

    Chih-Hong Lou

    2016-06-01

    Full Text Available Nonsense-mediated RNA decay (NMD is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages.

  17. Isolation and purification of porcine LH for radioimmunoassay and radioreceptor assay

    International Nuclear Information System (INIS)

    Ziecik, A.; Goralska, M.; Krzymowski, T.; Pogorzelski, K.

    1979-01-01

    The procedure of isolation and purification of LH from porcine pituitary glands is described. From 1 kg of pituitary glands 150 mg of LH GPZ-1 preparation of high purity were obtained. Immunization of rabbits with the prepared hormone gave homogeneous antibodies against porcine LH with high affinity and low cross-reactions with FSH. Radioreceptor assay with the use of the prepared porcine LH demonstrated the high capacity of cell membrane receptors of the boar tests for binding this hormone. (author)

  18. Subretinally transplanted embryonic stem cells rescue photoreceptor cells from degeneration in the RCS rats.

    Science.gov (United States)

    Schraermeyer, U; Thumann, G; Luther, T; Kociok, N; Armhold, S; Kruttwig, K; Andressen, C; Addicks, K; Bartz-Schmidt, K U

    2001-01-01

    The Royal College of Surgeons (RCS) rat is an animal model for retinal degeneration such as the age-related macular degeneration. The RCS rat undergoes a progressive retinal degeneration during the early postnatal period. A potential treatment to prevent this retinal degeneration is the transplantation into the subretinal space of cells that would replace functions of the degenerating retinal pigment epithelium (RPE) cells or may form neurotrophic factors. In this study we have investigated the potential of subretinally transplanted embryonic stem cells to prevent the genetically determined photoreceptor cell degeneration in the RCS rat. Embryonic stem cells from the inner cell mass of the mouse blastocyst were allowed to differentiate to neural precursor cells in vitro and were then transplanted into the subretinal space of 20-day-old RCS rats. Transplanted and sham-operated rats were sacrificed 2 months following cell transplantation. The eyes were enucleated and photoreceptor degeneration was quantified by analyzing and determining the thickness of the outer nuclear layer by light and electron microscopy. In the eyes transplanted with embryonic cells up to 8 rows of photoreceptor cell nuclei were observed, whereas in nontreated control eyes the outer nuclear layer had degenerated completely. Transplantation of embryonic stem cells appears to delay photoreceptor cell degeneration in RCS rats.

  19. Immunological half-life of porcine proinsulin C-peptide

    Energy Technology Data Exchange (ETDEWEB)

    Oyama, H; Horino, M; Matsumura, S [Kawasaki Medical Coll., Kurashiki (Japan). Div. of Endocrinology; Kobayshi, K; Suetsugu, N [Yamaguchi Univ., Ube (Japan). School of Medicine

    1975-11-01

    Immunological half-lifes of injected porcine C-peptide and insulin with RIA were studied and calculated as 9.8 and 8.0 minutes. Higher circulating levels of C-peptide as compared to insulin in normal young swines lead to speculation about a longer half-life of C-peptide. This hypothesis was verified in this study. Immunological half-lifes of porcine proinsulin and insulin in the pig were 20 and 6 minutes, respectively.

  20. Endocrine control of embryonic diapause in the Australian sharpnose shark Rhizoprionodon taylori.

    Directory of Open Access Journals (Sweden)

    Daniela Waltrick

    Full Text Available The reproductive cycle of the Australian sharpnose shark, Rhizoprionodon taylori, includes a temporary suspension of development at the commencement of embryogenesis termed embryonic diapause. This study investigated levels of 17β-estradiol (E2, testosterone (T and progesterone (P4 in plasma samples of mature wild female R. taylori captured throughout the reproductive cycle and correlated them with internal morphological changes. Levels of T were elevated through most of the embryonic diapause period, suggesting a role of this hormone in the maintenance of this condition. Increasing plasma T concentrations from late diapause to early active development were associated with a possible role of androgens in the termination of embryonic diapause. As in other elasmobranchs, a concomitant increase of E2 with ovarian follicle size indicated a direct role of this hormone in regulating vitellogenesis, while a peak in P4 suggested this hormone is associated with preovulation and ovulation. Additionally, significant correlations between photoperiod or water temperature and maximum follicular diameter and hepatosomatic index suggest that these abiotic factors may also play a role triggering and regulating the synchrony and timing of reproductive events.

  1. Changes in the concentrations of four maternal steroids during embryonic development in the threespined stickleback (Gasterosteus aculeatus).

    Science.gov (United States)

    Paitz, Ryan Thomas; Mommer, Brett Christian; Suhr, Elissa; Bell, Alison Marie

    2015-08-01

    Embryonic exposure to steroids often leads to long-term phenotypic effects. It has been hypothesized that mothers may be able to create a steroid environment that adjusts the phenotypes of offspring to current environmental conditions. Complicating this hypothesis is the potential for developing embryos to modulate their early endocrine environment. This study utilized the threespined stickleback (Gasterosteus aculeatus) to characterize the early endocrine environment within eggs by measuring four steroids (progesterone, testosterone, estradiol, and cortisol) of maternal origin. We then examined how the concentrations of these four steroids changed over the first 12 days post fertilization (dpf). Progesterone, testosterone, estradiol, and cortisol of maternal origin could be detected within unfertilized eggs and levels of all four steroids declined in the first 3 days following fertilization. While levels of progesterone, testosterone, and estradiol remained low after the initial decline, levels of cortisol rose again by 8 dpf. These results demonstrate that G. aculeatus embryos begin development in the presence of a number of maternal steroids but levels begin to change quickly following fertilization. This suggests that embryonic processes change the early endocrine environment and hence influence the ability of maternal steroids to affect development. With these findings, G. aculeatus becomes an intriguing system in which to study how selection may act on both maternal and embryonic processes to shape the evolutionary consequence of steroid-mediated maternal effects. © 2015 Wiley Periodicals, Inc.

  2. Emerging technologies to create inducible and genetically defined porcine cancer models

    Directory of Open Access Journals (Sweden)

    Lawrence B Schook

    2016-02-01

    Full Text Available There is an emerging need for new animal models that address unmet translational cancer research requirements. Transgenic porcine models provide an exceptional opportunity due to their genetic, anatomic and physiological similarities with humans. Due to recent advances in the sequencing of domestic animal genomes and the development of new organism cloning technologies, it is now very feasible to utilize pigs as a malleable species, with similar anatomic and physiological features with humans, in which to develop cancer models. In this review, we discuss genetic modification technologies successfully used to produce porcine biomedical models, in particular the Cre-loxP System as well as major advances and perspectives the CRISPR/Cas9 System. Recent advancements in porcine tumor modeling and genome editing will bring porcine models to the forefront of translational cancer research.

  3. Emerging Technologies to Create Inducible and Genetically Defined Porcine Cancer Models.

    Science.gov (United States)

    Schook, Lawrence B; Rund, Laurie; Begnini, Karine R; Remião, Mariana H; Seixas, Fabiana K; Collares, Tiago

    2016-01-01

    There is an emerging need for new animal models that address unmet translational cancer research requirements. Transgenic porcine models provide an exceptional opportunity due to their genetic, anatomic, and physiological similarities with humans. Due to recent advances in the sequencing of domestic animal genomes and the development of new organism cloning technologies, it is now very feasible to utilize pigs as a malleable species, with similar anatomic and physiological features with humans, in which to develop cancer models. In this review, we discuss genetic modification technologies successfully used to produce porcine biomedical models, in particular the Cre-loxP System as well as major advances and perspectives the CRISPR/Cas9 System. Recent advancements in porcine tumor modeling and genome editing will bring porcine models to the forefront of translational cancer research.

  4. Identification of human embryonic progenitor cell targeting peptides using phage display.

    Directory of Open Access Journals (Sweden)

    Paola A Bignone

    Full Text Available Human pluripotent stem (hPS cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken

  5. Chelated mineral supplements for Nelore: quality and early embryonic development

    Directory of Open Access Journals (Sweden)

    Camila Pasa

    2014-01-01

    Full Text Available ABSTRACT. Pasa C., Hatamoto-Zervoudakis L.K., Zervoudakis J.T. & Soares L. [Chelated mineral supplements for Nelore: quality and early embryonic development.] Suplementos minerais quelatados para vacas Nelore: qualidade e desenvolvimento embrionário inicial. Revista Brasileira de Medicina Veterinária, 36(1:29-34, 2014. Programa de Pós-Graduação em Ciência Animal, Faculdade de Agronomia e Medicina Veterinária, Universidade Federal do Mato Grosso, Av. Fernando Corrêa da Costa, 2367, Bairro Boa Esperança, Cuiabá, MT 78060-900, Brasil. E-mail: pasa_camila@hotmail.com The objective of this study was to evaluate the quality and early development of embryos produced with oocytes of cows supplemented with copper, zinc and selenium in a non-chelated and chelated. The experiment was conducted in Cuiabá-MT during the months April to July 2009. We used 24 adult Nellore multiparous, aged, average weights of the initial 36 months, 395 kg and mean body condition score 4.8, respectively randomly divided into 2 groups: control group (CG, supplemented with conventional mineral and Supplemented Group (GS, animals supplemented with zinc, copper and selenium chelated. Each group was kept in a paddock of Brachiaria brizantha cv Marandu received 1 kg of animal per day. chelated mineral supplementation (GS and conventional mineral (GC delivered via the protein supplement was given during a period of 99 days with daily average 1kg/cabeça. During the experimental period were two follicular aspirations, one to 59 days and another at 99 days of supplementation. Every two weeks the animals were weighed and ECC evaluated. oocytes viable (grades I, II and III were used for in vitro production of embryos. The experiment was completely randomized and data were analyzed by ANOVA and a significance level of 10%. There was no effect (p> 0.10 of supplementation with chelated minerals on the percentage of cleaved oocytes, total embryos produced, percentage of produced

  6. Characterization of serotonergic receptors in rabbit, porcine and human conjunctivae.

    Science.gov (United States)

    Turner, Helen C; Alvarez, Lawrence J; Candia, Oscar A; Bernstein, Audrey M

    2003-10-01

    To characterize the serotonin (5-HT) receptors linked to the modulation of adenylyl cyclase activity in rabbit, porcine and human conjunctivae. Serotonin receptor-subtype expression was examined using reverse transcription-polymerase chain reaction (RT-PCR) and receptor subtype-specific polyclonal antibodies for the immunofluorescent labeling of conjunctival cryosections. In addition, measurements of the effects of serotonergics on the short-circuit current (I(sc)) across rabbit and porcine conjunctivae were contrasted. RT-PCR assays indicated the expression of 5-HT(1B ) and 5-HT(1D) receptors, subtypes negatively coupled to adenylyl cyclase, in the rabbit conjunctiva. This approach also suggested the co-expression of 5-HT(1B), 5-HT(1D), 5-HT(1F), 5-HT(4) and 5-HT(7) mRNA's in the porcine conjunctiva, and 5-HT( 1D), 5-HT(1F) and 5-HT(7) in the human conjunctiva. Since the 5-HT(4) and 5-HT(7) receptors are positively linked to adenylyl cyclase, these results implied that the porcine and human tissues exhibited subtypes both positively and negatively linked to the enzyme. However, immunohistochemical observations, using currently available antibodies solely localized the 5-HT(7) moiety in the porcine and human epithelia, suggested that the 1B/1D forms may be minor elements. Consistent with this prospect, 5-HT was a stimulant of the transepithelial I(sc) across the porcine conjunctiva, an opposite response from earlier findings that demonstrated inhibitory effects by 5-HT on the rabbit I(sc), which are now explained by the localization of the 1B/1D receptors in the rabbit stratified epithelium. The 5-HT receptors expressed by mammalian conjunctivae are not identical. In terms of 5-HT receptor expression, the porcine tissue may be a more appropriate model for human, than is the rabbit, in that 5-HT may serve as a secretagogue in the human epithelium.

  7. Beta-hydroxybutyrate increases reactive oxygen species in late but not in early postimplantation embryonic cells in vitro.

    Science.gov (United States)

    Forsberg, H; Eriksson, U J; Melefors, O; Welsh, N

    1998-02-01

    Embryonic dysmorphogenesis has been blocked by antioxidant treatment in vivo and in vitro, suggesting that embryonic excess of reactive oxygen species (ROS) has a role in the teratogenic process of diabetic pregnancy. We report that the basal levels of ROS in dispersed rat embryonic cells in vitro, as determined by fluorescence of dichlorofluorescein (DCF), were not different in cells from control and diabetic pregnancy at day 10 or 12. Beta-hydroxybutyrate (beta-HB) and succinic acid monomethyl ester both augmented DCF fluorescence in cells from day 12 embryos of normal and diabetic rats but not from day 10 embryos. Cells of day 10 and day 12 embryos from normal and diabetic rats responded to increasing glucose concentrations with a dosage-dependent alleviation of DCF fluorescence. Day 10 embryonic cells exhibited high glucose utilization rates and high pentose phosphate shunt rates, but low mitochondrial oxidation rates. Moreover, in vitro culture of embryos between gestational days 9 and 10 in the presence of 20% oxygen induced an increased and glucose-sensitive oxidation of glucose compared with embryos not cultured in vitro. At gestation day 12, however, pentose phosphate shunt rates showed a decrease, whereas the mitochondrial beta-HB oxidation rates were increased compared with those at gestation day 10. This was paralleled by a lower expression of glucose 6-phosphate dehydrogenase- and phosphofructokinase-mRNA levels at day 12 than at day 10. On the other hand, H-ferritin mRNA expression at day 12 was high compared with day 10. None of the mRNA species investigated were affected by the diabetic state of the mother. It was concluded that beta-HB-induced stimulation of mitochondrial oxidative events may lead to the generation of ROS at gestational day 12, but probably not at day 10, when only a minute amount of mitochondrial activity occurs. Thus our results do not support the notion of diabetes-induced mitochondrial oxidative stress before the development of

  8. Early embryonic development of the head region of Gryllus assimilis Fabricius, 1775 (Orthoptera, Insecta).

    Science.gov (United States)

    Liu, Yu; Maas, Andreas; Waloszek, Dieter

    2010-09-01

    We report our investigations on the embryonic development of Gryllus assimilis, with particular attention to the head. Significant findings revealed with scanning electron microscopy (SEM) images include: (1) the pre-antennal lobes represent the anterior-most segment that does not bear any appendages; (2) each of the lobes consists of central and marginal regions; (3) the central region thereof develops into the protocerebrum and the optic lobes, whereas the marginal region thereof becomes the anterior portion of the head capsule; (4) the initial position of the antennal segment is posterior to the mouth region; (5) appendage anlagen are transitorily present in the intercalary segment, and they later vanish together with the segment itself; (6) a bulged sternum appears to develop from the ventral surface of the mandibular, maxillary and labial segments. Embryonic features are then compared across the Insecta and further extended to the embryos of a spider (Araneae, Chelicerata). Striking similarities shared by the anterior-most region of the insect and spider embryos lead the authors to conclude that such comparison should be further undertaken to cover the entire Euarthropoda. This will help us to understand the embryology and evolution of the arthropod head. Copyright © 2010 Elsevier Ltd. All rights reserved.

  9. ZO-1 and ZO-2 are required for extra-embryonic endoderm integrity, primitive ectoderm survival and normal cavitation in embryoid bodies derived from mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Dominic C Y Phua

    Full Text Available The Zonula Occludens proteins ZO-1 and ZO-2 are cell-cell junction-associated adaptor proteins that are essential for the structural and regulatory functions of tight junctions in epithelial cells and their absence leads to early embryonic lethality in mouse models. Here, we use the embryoid body, an in vitro peri-implantation mouse embryogenesis model, to elucidate and dissect the roles ZO-1 and ZO-2 play in epithelial morphogenesis and de novo tight junction assembly. Through the generation of individual or combined ZO-1 and ZO-2 null embryoid bodies, we show that their dual deletion prevents tight junction formation, resulting in the disorganization and compromised barrier function of embryoid body epithelial layers. The disorganization is associated with poor microvilli development, fragmented basement membrane deposition and impaired cavity formation, all of which are key epithelial tissue morphogenetic processes. Expression of Podocalyxin, which positively regulates the formation of microvilli and the apical membrane, is repressed in embryoid bodies lacking both ZO-1 and ZO-2 and this correlates with an aberrant submembranous localization of Ezrin. The null embryoid bodies thus give an insight into how the two ZO proteins influence early mouse embryogenesis and possible mechanisms underlying the embryonic lethal phenotype.

  10. MiRNA-mediated regulation of cell signaling and homeostasis in the early mouse embryo.

    Science.gov (United States)

    Pernaute, Barbara; Spruce, Thomas; Rodriguez, Tristan A; Manzanares, Miguel

    2011-02-15

    At the time of implantation the mouse embryo is composed of three tissues the epiblast, trophectoderm and primitive endoderm. As development progresses the epiblast goes on to form the foetus whilst the trophectoderm and primitive endoderm give rise to extra-embryonic structures with important roles in embryo patterning and nutrition. Dramatic changes in gene expression occur during early embryo development and these require regulation at different levels. miRNAs are small non coding RNAs that have emerged over the last decade as important post-transcriptional repressors of gene expression. The roles played by miRNAs during early mammalian development are only starting to be elucidated. In order to gain insight into the function of miRNAs in the different lineages of the early mouse embryo we have analysed in depth the phenotype of embryos and extra-embryonic stem cells mutant for the miRNA maturation protein Dicer. This study revealed that miRNAs are involved in regulating cell signaling and homeostasis in the early embryo. Specifically, we identified a role for miRNAs in regulating the Erk signaling pathway in the extra-embryonic endoderm, cell cycle progression in extra-embryonic tissues and apoptosis in the epiblast.

  11. Inhibition of Rho kinase regulates specification of early differentiation events in P19 embryonal carcinoma stem cells.

    Directory of Open Access Journals (Sweden)

    Roman J Krawetz

    Full Text Available The Rho kinase pathway plays a key role in many early cell/tissue determination events that take place in embryogenesis. Rho and its downstream effector Rho kinase (ROCK play pivotal roles in cell migration, apoptosis (membrane blebbing, cell proliferation/cell cycle, cell-cell adhesion and gene regulation. We and others have previously demonstrated that inhibition of ROCK blocks endoderm differentiation in embryonal carcinoma stem cells, however, the effect of ROCK inhibition on mesoderm and ectoderm specification has not been fully examined. In this study, the role of ROCK within the specification and differentiation of all three germ layers was examined.P19 cells were treated with the specific ROCK inhibitor Y-27623, and increase in differentiation efficiency into neuro-ectodermal and mesodermal lineages was observed. However, as expected a dramatic decrease in early endodermal markers was observed when ROCK was inhibited. Interestingly, within these ROCK-inhibited RA treated cultures, increased levels of mesodermal or ectodermal markers were not observed, instead it was found that the pluripotent markers SSEA-1 and Oct-4 remained up-regulated similar to that seen in undifferentiated cultures. Using standard and widely accepted methods for reproducible P19 differentiation into all three germ layers, an enhancement of mesoderm and ectoderm differentiation with a concurrent loss of endoderm lineage specification was observed with Y-27632 treatment. Evidence would suggest that this effect is in part mediated through TGF-β and SMAD signaling as ROCK-inhibited cells displayed aberrant SMAD activation and did not return to a 'ground' state after the inhibition had been removed.Given this data and the fact that only a partial rescue of normal differentiation capacity occurred when ROCK inhibition was alleviated, the effect of ROCK inhibition on the differentiation capacity of pluripotent cell populations should be further examined to elucidate the

  12. Induction of T helper 3 regulatory cells by dendritic cells infected with porcine reproductive and respiratory syndrome virus

    International Nuclear Information System (INIS)

    Silva-Campa, Erika; Flores-Mendoza, Lilian; Resendiz, Monica; Pinelli-Saavedra, Araceli; Mata-Haro, Veronica; Mwangi, Waithaka; Hernandez, Jesus

    2009-01-01

    Delayed development of virus-specific immune response has been observed in pigs infected with the porcine reproductive and respiratory syndrome virus (PRRSV). Several studies support the hypothesis that the PRRSV is capable of modulating porcine immune system, but the mechanisms involved are yet to be defined. In this study, we evaluated the induction of T regulatory cells by PRRSV-infected dendritic cells (DCs). Our results showed that PRRSV-infected DCs significantly increased Foxp3 + CD25 + T cells, an effect that was reversible by IFN-α treatment, and this outcome was reproducible using two distinct PRRSV strains. Analysis of the expressed cytokines suggested that the induction of Foxp3 + CD25 + T cells is dependent on TGF-β but not IL-10. In addition, a significant up-regulation of Foxp3 mRNA, but not TBX21 or GATA3, was detected. Importantly, our results showed that the induced Foxp3 + CD25 + T cells were able to suppress the proliferation of PHA-stimulated PBMCs. The T cells induced by the PRRSV-infected DCs fit the Foxp3 + CD25 + T helper 3 (Th3) regulatory cell phenotype described in the literature. The induction of this cell phenotype depended, at least in part, on PRRSV viability because IFN-α treatment or virus inactivation reversed these effects. In conclusion, this data supports the hypothesis that the PRRSV succeeds to establish and replicate in porcine cells early post-infection, in part, by inducing Th3 regulatory cells as a mechanism of modulating the porcine immune system.

  13. [Characteristics of porcine thoracic arteries fixed with polyepoxy compound].

    Science.gov (United States)

    Yu, Xi-Xun; Chen, Huai-Qing

    2005-09-01

    To investigate the characteristics of porcine thoracic arteries fixed with ethylene glycol diglycidyl ether (EX-810) and to provide the proper scaffold materials for tissue-engineered blood vessel. The porcine thoracic arteries were respectively treated with 40 ml/L EX-810 and 6.25 g/L glutaraldehyde, and then they were examined with naked-eye, light microscope and scanning electron microscope. The fixation index determination, the amino acid analysis and the biomechanics test were also performed. The antigenicity of vascular tissues can be diminished by EX-810 through getting rid of cell in the vascular tissues or reducing the level of free amino groups in the vascular tissues. The structural integrity of vascular tissues can be preserved after treatment with EX-810. It was also found that the EX-810-fixed porcine vascular tissues appeared more similar to the natural vascular tissues in color and mechanical properties, and were more pliable than the glutaraldehyde-fixed tissues. The EX-810-fixed porcine thoracic arteries with low cytotoxicity and low antigenicity showed favorable characteristic similar to those of natural vessel, and it should be a promising material for fabricating scaffold of tissue-engineered blood vessel.

  14. Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin

    Indian Academy of Sciences (India)

    Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin. Qizhuang Lv Kangkang Guo Tao Wang ... Keywords. Cellular protein; FHC; ORF4 protein; porcine circovirus type 2 (PCV2); yeast two-hybrid ... Journal of Biosciences | News ...

  15. Honeybee (Apis mellifera Venom Reinforces Viral Clearance during the Early Stage of Infection with Porcine Reproductive and Respiratory Syndrome Virus through the Up-Regulation of Th1-Specific Immune Responses

    Directory of Open Access Journals (Sweden)

    Jin-A Lee

    2015-05-01

    Full Text Available Porcine reproductive and respiratory syndrome (PRRS is a chronic and immunosuppressive viral disease that is responsible for substantial economic losses for the swine industry. Honeybee venom (HBV is known to possess several beneficial biological properties, particularly, immunomodulatory effects. Therefore, this study aimed at evaluating the effects of HBV on the immune response and viral clearance during the early stage of infection with porcine reproductive and respiratory syndrome virus (PRRSV in pigs. HBV was administered via three routes of nasal, neck, and rectal and then the pigs were inoculated with PRRSV intranasally. The CD4+/CD8+ cell ratio and levels of interferon (IFN-γ and interleukin (IL-12 were significantly increased in the HBV-administered healthy pigs via nasal and rectal administration. In experimentally PRRSV-challenged pigs with virus, the viral genome load in the serum, lung, bronchial lymph nodes and tonsil was significantly decreased, as was the severity of interstitial pneumonia, in the nasal and rectal administration group. Furthermore, the levels of Th1 cytokines (IFN-γ and IL-12 were significantly increased, along with up-regulation of pro-inflammatory cytokines (TNF-α and IL-1β with HBV administration. Thus, HBV administration—especially via the nasal or rectal route—could be a suitable strategy for immune enhancement and prevention of PRRSV infection in pigs.

  16. Disruption of murine mp29/Syf2/Ntc31 gene results in embryonic lethality with aberrant checkpoint response.

    Directory of Open Access Journals (Sweden)

    Chia-Hsin Chen

    Full Text Available Human p29 is a putative component of spliceosomes, but its role in pre-mRNA is elusive. By siRNA knockdown and stable overexpression, we demonstrated that human p29 is involved in DNA damage response and Fanconi anemia pathway in cultured cells. In this study, we generated p29 knockout mice (mp29(GT/GT using the mp29 gene trap embryonic stem cells to study the role of mp29 in DNA damage response in vivo. Interruption of mp29 at both alleles resulted in embryonic lethality. Embryonic abnormality occurred as early as E6.5 in mp29(GT/GT mice accompanied with decreased mRNA levels of α-tubulin and Chk1. The reduction of α-tubulin and Chk1 mRNAs is likely due to an impaired post-transcriptional event. An aberrant G2/M checkpoint was found in mp29 gene trap embryos when exposed to aphidicolin and UV light. This embryonic lethality was rescued by crossing with mp29 transgenic mice. Additionally, the knockdown of zfp29 in zebrafish resulted in embryonic death at 72 hours of development postfertilization (hpf. A lower level of acetylated α-tubulin was also observed in zfp29 morphants. Together, these results illustrate an indispensable role of mp29 in DNA checkpoint response during embryonic development.

  17. Experimental Infection of Young Pigs with an Early European Strain of Porcine Epidemic Diarrhoea Virus and a Recent US Strain

    DEFF Research Database (Denmark)

    Lohse, Louise; Krog, Jesper Schak; Strandbygaard, Bertel

    2017-01-01

    Outbreaks of porcine epidemic diarrhoea (PED) were reported across Europe during the 1980s and 1990s, but only sporadic outbreaks occurred in recent years. PED virus (PEDV) spread for the first time into the USA in 2013 and has caused severe economic losses. Retrospectively, it was found that two...

  18. Anatomy and bronchoscopy of the porcine lung. A model for translational respiratory medicine.

    LENUS (Irish Health Repository)

    Judge, Eoin P

    2014-09-01

    The porcine model has contributed significantly to biomedical research over many decades. The similar size and anatomy of pig and human organs make this model particularly beneficial for translational research in areas such as medical device development, therapeutics and xenotransplantation. In recent years, a major limitation with the porcine model was overcome with the successful generation of gene-targeted pigs and the publication of the pig genome. As a result, the role of this model is likely to become even more important. For the respiratory medicine field, the similarities between pig and human lungs give the porcine model particular potential for advancing translational medicine. An increasing number of lung conditions are being studied and modeled in the pig. Genetically modified porcine models of cystic fibrosis have been generated that, unlike mouse models, develop lung disease similar to human cystic fibrosis. However, the scientific literature relating specifically to porcine lung anatomy and airway histology is limited and is largely restricted to veterinary literature and textbooks. Furthermore, methods for in vivo lung procedures in the pig are rarely described. The aims of this review are to collate the disparate literature on porcine lung anatomy, histology, and microbiology; to provide a comparison with the human lung; and to describe appropriate bronchoscopy procedures for the pig lungs to aid clinical researchers working in the area of translational respiratory medicine using the porcine model.

  19. Preservation of enucleated porcine eyes for use in a wet laboratory

    NARCIS (Netherlands)

    Nibourg, Lisanne M.; Koopmans, Steven A.

    PURPOSE: To design a method to preserve enucleated porcine eyes for use in a wet laboratory. SETTING: Laboratory of Experimental Ophthalmology, University Medical Center Groningen, the Netherlands. DESIGN: Experimental study. METHODS: Porcine eyes were preserved using 15 methods including salt

  20. Toll- like receptors expressed on embryonic macrophages couple inflammatory signals to iron metabolism during early ontogenesis

    Czech Academy of Sciences Publication Activity Database

    Balounová, Jana; Vavrochová, Tereza; Benešová, Martina; Ballek, Ondřej; Kolář, Michal; Filipp, Dominik

    2014-01-01

    Roč. 44, č. 5 (2014), s. 1491-1502 ISSN 0014-2980 R&D Projects: GA AV ČR IAA500520707 Institutional support: RVO:68378050 Keywords : Embryonic macrophages * Ferroportin * Gene expression microarray * Iron metabolism * TLR stimulation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.034, year: 2014

  1. Ofd1 controls dorso-ventral patterning and axoneme elongation during embryonic brain development.

    Directory of Open Access Journals (Sweden)

    Anna D'Angelo

    Full Text Available Oral-facial-digital type I syndrome (OFDI is a human X-linked dominant-male-lethal developmental disorder caused by mutations in the OFD1 gene. Similar to other inherited disorders associated to ciliary dysfunction OFD type I patients display neurological abnormalities. We characterized the neuronal phenotype that results from Ofd1 inactivation in early phases of mouse embryonic development and at post-natal stages. We determined that Ofd1 plays a crucial role in forebrain development, and in particular, in the control of dorso-ventral patterning and early corticogenesis. We observed abnormal activation of Sonic hedgehog (Shh, a major pathway modulating brain development. Ultrastructural studies demonstrated that early Ofd1 inactivation results in the absence of ciliary axonemes despite the presence of mature basal bodies that are correctly orientated and docked. Ofd1 inducible-mediated inactivation at birth does not affect ciliogenesis in the cortex, suggesting a developmental stage-dependent role for a basal body protein in ciliogenesis. Moreover, we showed defects in cytoskeletal organization and apical-basal polarity in Ofd1 mutant embryos, most likely due to lack of ciliary axonemes. Thus, the present study identifies Ofd1 as a developmental disease gene that is critical for forebrain development and ciliogenesis in embryonic life, and indicates that Ofd1 functions after docking and before elaboration of the axoneme in vivo.

  2. Enzyme immunoassay for the detection of porcine gelatine in edible bird's nests.

    Science.gov (United States)

    Tukiran, Nur Azira; Ismail, Amin; Mustafa, Shuhaimi; Hamid, Muhajir

    2015-01-01

    Porcine gelatine is a common adulterant found in edible bird's nests (EBNs) used to increase the net weight prior to sale. This study aimed to develop indirect enzyme-linked immunosorbent assays (ELISAs) for porcine gelatine adulteration using anti-peptide polyclonal antibodies. Three indirect ELISAs were developed (PAB1, 2 and 3), which had limits of detection (LODs) of 0.12, 0.10 and 0.11 µg g(-1), respectively. When applied to standard solutions of porcine gelatine, the inter- and intra-assays showed coefficients of variation (CVs) less than 20% and were able to detect at least 0.5 ng µg(-1) (0.05%) porcine gelatine in spiked samples. The proposed ELISA offers attractions for quality control in the EBN industry.

  3. Possibilities of microscopic detection of isolated porcine proteins in model meat products

    Directory of Open Access Journals (Sweden)

    Michaela Petrášová

    2016-05-01

    Full Text Available In recent years, various protein additives intended for manufacture of meat products have increasing importance in the food industry. These ingredients include both, plant-origin as well as animal-origin proteins. Among animal proteins, blood plasma, milk protein or collagen are used most commonly. Collagen is obtained from pork, beef, and poultry or fish skin. Collagen does not contain all the essential amino acids, thus it is not a full protein in terms of essential amino acids supply for one's organism. However, it is rather rich in amino acids of glycine, hydroxyproline and proline which are almost absent in other proteins and their synthesis is very energy intensive. Collagen, which is added to the soft and small meat products in the form of isolated porcine protein, significantly affects the organoleptic properties of these products. This work focused on detection of isolated porcine protein in model meat products where detection of isolated porcine protein was verified by histological staining and light microscopy. Seven model meat products from poultry meat and 7 model meat products from beef and pork in the ratio of 1:1, which contained 2.5% concentration of various commercially produced isolated porcine proteins, were examined. These model meat products were histologically processed by means of cryosections and stained with hematoxylin-eosin staining, toluidine blue staining and Calleja. For the validation phase, Calleja was utilized. To determine the sensitivity and specificity, five model meat products containing the addition of isolated porcine protein and five model meat products free of it were used. The sensitivity was determined for isolated porcine protein at 1.00 and specificity was determined at 1.00. The detection limit of the method was at the level of 0.001% addition. Repeatability of the method was carried out using products with addition as well as without addition of isolated porcine protein and detection was repeated

  4. Surgical induction of choroidal neovascularization in a porcine model

    DEFF Research Database (Denmark)

    Lassota, Nathan; Kiilgaard, Jens Folke; Prause, Jan Ulrik

    2007-01-01

    PURPOSE: To develop a reproducible surgical technique for the induction of choroidal neovascularization (CNV) in the subretinal space of porcine eyes and to analyse the resulting CNV clinically and histologically. METHODS: Two different modifications of a surgical technique previously described...... were compared with the original method. In ten porcine eyes retinal pigment epithelial (RPE) cells were removed using a silicone tipped cannula, in ten porcine eyes Bruch's membrane was perforated once with a retinal perforator without prior RPE removal and in ten eyes RPE removal was followed...... by a single perforation of Bruch's membrane. Fifteen of the eyes, five from each group, were enucleated 30 minutes after surgery, while the remaining eyes were enucleated after 14 days. Prior to enucleation, at day 14, fundus photographs and fluorescein angiograms were obtained. Eyes were examined by light...

  5. A porcine astrocyte/endothelial cell co-culture model of the blood-brain barrier.

    Science.gov (United States)

    Jeliazkova-Mecheva, Valentina V; Bobilya, Dennis J

    2003-10-01

    A method for the isolation of porcine atrocytes as a simple extension of a previously described procedure for isolation of brain capillary endothelial cells from adolescent pigs [Methods Cell Sci. 17 (1995) 2] is described. The obtained astroglial culture purified through two passages and by the method of the selective detachment was validated by a phase contrast microscopy and through an immunofluorescent assay for the glial fibrillary acidic protein (GFAP). Porcine astrocytes were co-cultivated with porcine brain capillary endothelial cells (PBCEC) for the development of an in vitro blood-brain barrier (BBB) model. The model was visualized by an electron microscopy and showed elevated transendothellial electrical resistance and reduced inulin permeability. To our knowledge, this is the first report for the establishment of a porcine astrocyte/endothelial cell co-culture BBB model, which avoids interspecies and age differences between the two cell types, usually encountered in the other reported co-culture BBB models. Considering the availability of the porcine brain tissue and the close physiological and anatomical relation between the human and pig brain, the porcine astrocyte/endothelial cell co-culture system can serve as a reliable and easily reproducible model for different in vitro BBB studies.

  6. Highly efficient differentiation of embryonic stem cells into adipocytes by ascorbic acid

    OpenAIRE

    Ixchelt Cuaranta-Monroy; Zoltan Simandi; Zsuzsanna Kolostyak; Quang-Minh Doan-Xuan; Szilard Poliska; Attila Horvath; Gergely Nagy; Zsolt Bacso; Laszlo Nagy

    2014-01-01

    Adipocyte differentiation and function have become the major research targets due to the increasing interest in obesity and related metabolic conditions. Although, late stages of adipogenesis have been extensively studied, the early phases remain poorly understood. Here we present that supplementing ascorbic acid (AsA) to the adipogenic differentiation cocktail enables the robust and efficient differentiation of mouse embryonic stem cells (mESCs) to mature adipocytes. Such ESC-derived adipocy...

  7. Novel porcine repetitive elements

    Directory of Open Access Journals (Sweden)

    Nonneman Dan J

    2006-12-01

    Full Text Available Abstract Background Repetitive elements comprise ~45% of mammalian genomes and are increasingly known to impact genomic function by contributing to the genomic architecture, by direct regulation of gene expression and by affecting genomic size, diversity and evolution. The ubiquity and increasingly understood importance of repetitive elements contribute to the need to identify and annotate them. We set out to identify previously uncharacterized repetitive DNA in the porcine genome. Once found, we characterized the prevalence of these repeats in other mammals. Results We discovered 27 repetitive elements in 220 BACs covering 1% of the porcine genome (Comparative Vertebrate Sequencing Initiative; CVSI. These repeats varied in length from 55 to 1059 nucleotides. To estimate copy numbers, we went to an independent source of data, the BAC-end sequences (Wellcome Trust Sanger Institute, covering approximately 15% of the porcine genome. Copy numbers in BAC-ends were less than one hundred for 6 repeat elements, between 100 and 1000 for 16 and between 1,000 and 10,000 for 5. Several of the repeat elements were found in the bovine genome and we have identified two with orthologous sites, indicating that these elements were present in their common ancestor. None of the repeat elements were found in primate, rodent or dog genomes. We were unable to identify any of the replication machinery common to active transposable elements in these newly identified repeats. Conclusion The presence of both orthologous and non-orthologous sites indicates that some sites existed prior to speciation and some were generated later. The identification of low to moderate copy number repetitive DNA that is specific to artiodactyls will be critical in the assembly of livestock genomes and studies of comparative genomics.

  8. Radiation sensitivity of bacteria and virus in porcine xenoskin for dressing agent

    International Nuclear Information System (INIS)

    Jo, Eu-Ri; Jung, Pil-Mun; Choi, Jong-il; Lee, Ju-Woon

    2012-01-01

    In this study, gamma irradiation sensitivities of bacteria and viruses in porcine skin were evaluated to establish the optimum sterilization condition for the dressing material and a xenoskin graft. Escherichia coli and Bacillus subtilis were used as model pathogens and inoculated at 10 6 –10 7 log CFU/g. As model viruses, porcine parvovirus (PPV), bovine viral diarrhea virus (BVDV), and poliovirus were used and inoculated at 10 5 –10 6 TCID 50 /g into porcine skin. The D 10 value of E. coli was found to be 0.25±0.1 kGy. B. subtilis endospores produced under stressful environmental conditions showed lower radiation sensitivity as D 10 was 3.88±0.3 kGy in porcine skin. The D 10 values of PPV, BVDV, and poliovirus were found to be 1.73±0.2, 3.81±0.2, and 6.88±0.3 kGy, respectively. These results can offer the basic information required for inactivating pathogens by gamma irradiation and achieving dressing material and porcine skin grafts.

  9. The vasa-like gene, olvas, identifies the migration path of primordial germ cells during embryonic body formation stage in the medaka, Oryzias latipes.

    Science.gov (United States)

    Shinomiya, A; Tanaka, M; Kobayashi, T; Nagahama, Y; Hamaguchi, S

    2000-08-01

    The medaka homolog of the Drosophila vasa gene, olvas (Oryzias latipes vas) was obtained using polymerase chain reaction of medaka cDNA from the testis and ovary. The spatio-temporal expression pattern of olvas transcripts was observed by in situ hybridization on gonads and embryos. The transcripts for olvas were exclusively detected in the cytoplasm of germ cells in the testis and ovary, not in gonadal somatic cells. In the early developmental stages, each blastomere possessed the maternal transcripts of olvas, which disappeared during gastrula stages. At the late gastrula stage, specific expression of olvas was observed only in germline cells located at the posterior shield. Embryos after the hybridization were examined histologically, and the distribution and migration path of primordial germ cells (PGC) during early stages of embryonic-body formation were revealed using the olvas gene as a germline cell marker. The PGC were translocated from the posterior shield to both sides of the embryonic body via the inner embryonic body in the medaka.

  10. Nodal signals mediate interactions between the extra-embryonic and embryonic tissues in zebrafish

    OpenAIRE

    Xiang, Fan; Hagos, Engda G.; Xu, Bo; Sias, Christina; Kawakami, Koichi; Burdine, Rebecca D.; Dougan, Scott T.

    2007-01-01

    In many vertebrates, extra-embryonic tissues are important signaling centers that induce and pattern the germ layers. In teleosts, the mechanism by which the extra-embryonic yolk syncytial layer (YSL) patterns the embryo is not understood. Although the Nodal-related protein Squint is expressed in the YSL, its role in this tissue is not known. We generated a series of stable transgenic lines with GFP under the control of squint genomic sequences. In all species, nodal-related genes induce thei...

  11. Porcine SOX9 Gene Expression Is Influenced by an 18 bp Indel in the 5'-Untranslated Region.

    Directory of Open Access Journals (Sweden)

    Bertram Brenig

    Full Text Available Sex determining region Y-box 9 (SOX9 is an important regulator of sex and skeletal development and is expressed in a variety of embryonal and adult tissues. Loss or gain of function resulting from mutations within the coding region or chromosomal aberrations of the SOX9 locus lead to a plethora of detrimental phenotypes in humans and animals. One of these phenotypes is the so-called male-to-female or female-to-male sex-reversal which has been observed in several mammals including pig, dog, cat, goat, horse, and deer. In 38,XX sex-reversal French Large White pigs, a genome-wide association study suggested SOX9 as the causal gene, although no functional mutations were identified in affected animals. However, besides others an 18 bp indel had been detected in the 5'-untranslated region of the SOX9 gene by comparing affected animals and controls. We have identified the same indel (Δ18 between position +247 bp and +266 bp downstream the transcription start site of the porcine SOX9 gene in four other pig breeds; i.e., German Large White, Laiwu Black, Bamei, and Erhualian. These animals have been genotyped in an attempt to identify candidate genes for porcine inguinal and/or scrotal hernia. Because the 18 bp segment in the wild type 5'-UTR harbours a highly conserved cAMP-response element (CRE half-site, we analysed its role in SOX9 expression in vitro. Competition and immunodepletion electromobility shift assays demonstrate that the CRE half-site is specifically recognized by CREB. Both binding of CREB to the wild type as well as the absence of the CRE half-site in Δ18 reduced expression efficiency in HEK293T, PK-15, and ATDC5 cells significantly. Transfection experiments of wild type and Δ18 SOX9 promoter luciferase constructs show a significant reduction of RNA and protein levels depending on the presence or absence of the 18 bp segment. Hence, the data presented here demonstrate that the 18 bp indel in the porcine SOX9 5'-UTR is of functional

  12. Induction of anchorage-independent growth of human embryonic fibroblasts with a deletion in the short arm of chromosome 11 by human papillomavirus type 16 DNA

    International Nuclear Information System (INIS)

    Smits, H.L.; Raadsheer, E.; Rood, I.; Mehendale, S.; Slater, R.M.; van der Noordaa, J.; Ter Schegget, J.

    1988-01-01

    Human embryonic fibroblasts with a large deletion (11p11.11p15.1) in the short arm of one chromosome 11 (del-11 cells) appeared to be susceptible to transformation by early human papillomavirus type 16 (HPV-16) DNA, whereas diploid human embryonic fibroblasts were not. This difference in susceptibility might be explained by the absence of a tumor suppressor gene located within the deleted part on the short arm of chromosome 11. The presence of abundant viral early-gene transcripts in transformed cells suggests that transformation was induced by an elevated level of an HPV-16 early-gene product(s). The low transcriptional activity of HPV-16 in diploid cells may indicate that cellular genes affect viral transcription. Interruption of the HPV-16 E2 early open reading frame is probably required for high-level HPV-16 early-gene expression driven from the homologous enhancer-promoter region

  13. Sequence and expression analyses of porcine ISG15 and ISG43 genes.

    Science.gov (United States)

    Huang, Jiangnan; Zhao, Shuhong; Zhu, Mengjin; Wu, Zhenfang; Yu, Mei

    2009-08-01

    The coding sequences of porcine interferon-stimulated gene 15 (ISG15) and the interferon-stimulated gene (ISG43) were cloned from swine spleen mRNA. The amino acid sequences deduced from porcine ISG15 and ISG43 genes coding sequence shared 24-75% and 29-83% similarity with ISG15s and ISG43s from other vertebrates, respectively. Structural analyses revealed that porcine ISG15 comprises two ubiquitin homologues motifs (UBQ) domain and a conserved C-terminal LRLRGG conjugating motif. Porcine ISG43 contains an ubiquitin-processing proteases-like domain. Phylogenetic analyses showed that porcine ISG15 and ISG43 were mostly related to rat ISG15 and cattle ISG43, respectively. Using quantitative real-time PCR assay, significant increased expression levels of porcine ISG15 and ISG43 genes were detected in porcine kidney endothelial cells (PK15) cells treated with poly I:C. We also observed the enhanced mRNA expression of three members of dsRNA pattern-recognition receptors (PRR), TLR3, DDX58 and IFIH1, which have been reported to act as critical receptors in inducing the mRNA expression of ISG15 and ISG43 genes. However, we did not detect any induced mRNA expression of IFNalpha and IFNbeta, suggesting that transcriptional activations of ISG15 and ISG43 were mediated through IFN-independent signaling pathway in the poly I:C treated PK15 cells. Association analyses in a Landrace pig population revealed that ISG15 c.347T>C (BstUI) polymorphism and the ISG43 c.953T>G (BccI) polymorphism were significantly associated with hematological parameters and immune-related traits.

  14. Melatonin-induced increase of lipid droplets accumulation and in vitro maturation in porcine oocytes is mediated by mitochondrial quiescence.

    Science.gov (United States)

    He, Bin; Yin, Chao; Gong, Yabin; Liu, Jie; Guo, Huiduo; Zhao, Ruqian

    2018-01-01

    Melatonin, the major pineal secretory product, has a significant impact on the female reproductive system. Recently, the beneficial effects of melatonin on mammalian oocyte maturation and embryonic development have drawn increased attention. However, the exact underlying mechanisms remain to be fully elucidated. This study demonstrates that supplementing melatonin to in vitro maturation (IVM) medium enhances IVM rate, lipid droplets (LDs) accumulation as well as triglyceride content in porcine oocytes. Decrease of mitochondrial membrane potential, mitochondrial respiratory chain complex IV activity as well as mitochondrial reactive oxygen species (mROS) content indicated that melatonin induced a decrease of mitochondrial activity. The copy number of mitochondrial DNA (mtDNA) which encodes essential subunits of oxidative phosphorylation (OXPHOS), was not affected by melatonin. However, the expression of mtDNA-encoded genes was significantly down-regulated after melatonin treatment. The DNA methyltransferase DNMT1, which regulates methylation and expression of mtDNA, was increased and translocated into the mitochondria in melatonin-treated oocytes. The inhibitory effect of melatonin on the expression of mtDNA was significantly prevented by simultaneous addition of DNMT1 inhibitor, which suggests that melatonin regulates the transcription of mtDNA through up-regulation of DNMT1 and mtDNA methylation. Increase of triglyceride contents after inhibition of OXPHOS indicated that mitochondrial quiescence is crucial for LDs accumulation in oocytes. Taken together, our results suggest that melatonin-induced reduction in mROS production and increase in IVM, and LDs accumulation in porcine oocytes is mediated by mitochondrial quiescence. © 2017 Wiley Periodicals, Inc.

  15. Effects of oestradiol-enriched diet and of feeding with porcine testicular tissue on macroscopic gonadal sex in European eels

    DEFF Research Database (Denmark)

    Andersen, D.; Boetius, I.; Larsen, L.O.

    1996-01-01

    days there were 44% with female-gonads, 40% with male+female-gonads and 16% with male-gonads. Oestradiol thus had a feminizing effect, not only on morphologically undifferentiated gonads but also on morphologically differentiated male-gonads. The presence of sex steroid hormones or their precursors...... and 78% of oestradiol-treated eels developed female-gonads. Minced porcine testicular tissue fed to 2-g eels for up to 371 days had effects similar to oestradiol-enriched food (68% with female-gonads), whereas feeding for only 91 days had no effect during the next 8 months. Oestradiol-enriched food...... in porcine testicular tissue may also exert a feminizing influence. In all experiments the hormone-fed groups showed a tendency (not significant) towards increased growth rate. In small eels early rapid growth and differentiation of female-gonads were clearly correlated, both in hormone treated...

  16. Biology of Porcine Parvovirus (Ungulate parvovirus 1

    Directory of Open Access Journals (Sweden)

    István Mészáros

    2017-12-01

    Full Text Available Porcine parvovirus (PPV is among the most important infectious agents causing infertility in pigs. Until recently, it was thought that the virus had low genetic variance, and that prevention of its harmful effect on pig fertility could be well-controlled by vaccination. However, at the beginning of the third millennium, field observations raised concerns about the effectiveness of the available vaccines against newly emerging strains. Subsequent investigations radically changed our view on the evolution and immunology of PPV, revealing that the virus is much more diverse than it was earlier anticipated, and that some of the “new” highly virulent isolates cannot be neutralized effectively by antisera raised against “old” PPV vaccine strains. These findings revitalized PPV research that led to significant advancements in the understanding of early and late viral processes during PPV infection. Our review summarizes the recent results of PPV research and aims to give a comprehensive update on the present understanding of PPV biology.

  17. Biology of Porcine Parvovirus (Ungulate parvovirus 1)

    Science.gov (United States)

    Mészáros, István; Olasz, Ferenc; Cságola, Attila; Tijssen, Peter; Zádori, Zoltán

    2017-01-01

    Porcine parvovirus (PPV) is among the most important infectious agents causing infertility in pigs. Until recently, it was thought that the virus had low genetic variance, and that prevention of its harmful effect on pig fertility could be well-controlled by vaccination. However, at the beginning of the third millennium, field observations raised concerns about the effectiveness of the available vaccines against newly emerging strains. Subsequent investigations radically changed our view on the evolution and immunology of PPV, revealing that the virus is much more diverse than it was earlier anticipated, and that some of the “new” highly virulent isolates cannot be neutralized effectively by antisera raised against “old” PPV vaccine strains. These findings revitalized PPV research that led to significant advancements in the understanding of early and late viral processes during PPV infection. Our review summarizes the recent results of PPV research and aims to give a comprehensive update on the present understanding of PPV biology. PMID:29261104

  18. Altered glucose transport to utero-embryonic unit in relation to delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx.

    Science.gov (United States)

    Arnab, Banerjee; Amitabh, Krishna

    2011-02-10

    The aim of this study was to compare the changes in concentration of glucose and glucose transporters (GLUTs) in the utero-embryonic unit, consisting of decidua, trophoblast and embryo, during delayed and non-delayed periods to understand the possible cause of delayed embryonic development in Cynopterus sphinx. The results showed a significantly decreased concentration of glucose in the utero-embryonic unit due to decline in the expression of insulin receptor (IR) and GLUT 3, 4 and 8 proteins in the utero-embryonic unit during delayed period. The in vitro study showed suppressive effect of insulin on expression of GLUTs 4 and 8 in the utero-embryonic unit and a significant positive correlation between the decreased amount of glucose consumed by the utero-embryonic unit and decreased expression of GLUTs 4 (r=0.99; psphinx. Increased supply of fatty acid to the delayed embryo may be responsible for its survival under low glucose condition but unable to promote embryonic development in C. sphinx. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  19. A porcine model of haematogenous brain infectionwith staphylococcus aureus

    DEFF Research Database (Denmark)

    Astrup, Lærke Boye; Agerholm, Jørgen Steen; Nielsen, Ole Lerberg

    2012-01-01

    A PORCINE MODEL OF HAEMATOGENOUS BRAIN INFECTION WITH STAPHYLOCOCCUS AUREUS Astrup Lærke1, Agerholm Jørgen1, Nielsen Ole1, Jensen Henrik1, Leifsson Páll1, Iburg Tine2. 1: Faculty of Health and Medical Sciences, University of Copenhagen, Denmark boye@life.ku.dk 2: National Veterinary Institute......, Uppsala, Sweden Introduction Staphylococcus aureus (S.aureus) is a common cause of sepsis and brain abscesses in man and a frequent cause of porcine pyaemia. Here we present a porcine model of haematogenous S. aureus-induced brain infection. Materials and Methods Four pigs had two intravenous catheters...... thromboemboli (two pigs). The venous catheter was used for blood sampling before, during and after inoculation. The pigs were euthanized either 24 or 48 hours after inoculation. The brains were collected and examined histologically. Results We describe unifocal suppurative encephalitis 48 hours after...

  20. Molecular characterization of the porcine surfactant, pulmonary-associated protein C gene

    DEFF Research Database (Denmark)

    Cirera, S.; Nygård, A.B.; Jensen, H.E.

    2006-01-01

    The surfactant, pulmonary-associated protein C (SFTPC) is a peptide secreted by the alveolar type II pneumocytes of the lung. We have characterized the porcine SFTPC gene at genomic, transcriptional, and protein levels. The porcine SFTPC is a single-copy gene on pig chromosome 14. Two transcripts...

  1. Secretion of pancreastatins from the porcine digestive tract

    International Nuclear Information System (INIS)

    Boerglum Jensen, T.D.; Holst, J.J.; Fahrenkrug, J.

    1994-01-01

    Pancreastatin, a 49-amino acid peptide with a COOH-terminal glycine amide, was originally isolated from porcine pancreas, but pancreastatin immunoreactivity has been found in several neuroendocrine tissues. There are strong indications that pancreastatin is derived from chromogranin A, since the amino acid sequence 240-288 in porcine chromogranin A corresponds to pancreastatin flanked by typical signals for proteolytic processing. The authors studied the effect of electric stimulation of the nervous supply to perfused porcine pancreas, antrum, nonantral stomach, and small intestine on the release of immunoreactive pancreastatin, and they have characterized the molecular nature of the secreted immunoreactivity by using a radioimmunoassay specific for the COOH-terminal glycine amide of porcine pancreastatin in combination with chromatography. In all tissues nerve stimulation significantly increased the release of immunoreactive pancreastatin. The secreted immunoreactive pancreastatin was heterogeneous, consisting of pancreastatin itself, a COOH-terminal pancreastatin fragment, and NH 2 -terminally extended pancreastatin forms. Pancreastatin predominated in the perfusate from pancreas and antrum, whereas mainly NH 2 -terminally extended molecular forms were secreted from the antrectomized stomach and small intestine. The different molecular forms of pancreastatin were secreted from the perfused organs in the same molar ratio as they occur in extracts of the corresponding tissues. Thus, pancreastatin and other chromogranin A-derived peptides in organ-specific proportions regularly accompany the secretion of the peptide hormones from the gastrointestinal tissues on appropriate stimulation. 40 refs., 5 figs

  2. First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes--Chapter 3: Porcine islet product manufacturing and release testing criteria.

    Science.gov (United States)

    Rayat, Gina R; Gazda, Lawrence S; Hawthorne, Wayne J; Hering, Bernhard J; Hosking, Peter; Matsumoto, Shinichi; Rajotte, Ray V

    2016-01-01

    In the 2009 IXA consensus, the requirements for the quality and control of manufacturing of porcine islet products were based on the U.S. regulatory framework where the porcine islet products fall within the definition of somatic cell therapy under the statutory authority of the U.S. Food and Drug Administration (FDA). In addition, porcine islet products require pre-market approval as a biologic product under the Public Health Services Act and they meet the definition of a drug under the Federal Food, Drug, and Cosmetic Act (FD&C Act). Thus, they are subject to applicable provisions of the law and as such, control of manufacturing as well as reproducibility and consistency of porcine islet products, safety of porcine islet products, and characterization of porcine islet products must be met before proceeding to clinical trials. In terms of control of manufacturing as well as reproducibility and consistency of porcine islet products, the manufacturing facility must be in compliance with current Good Manufacturing Practices (cGMP) guidelines appropriate for the initiation of Phase 1/2 clinical trials. Sponsors intending to conduct a Phase 1/2 trial of islet xenotransplantation products must be able to demonstrate the safety of the product through the establishment of particular quality assurance and quality control procedures. All materials (including animal source and pancreas) used in the manufacturing process of the porcine islet products must be free of adventitious agents. The final porcine islet product must undergo tests for the presence of these adventitious agents including sterility, mycoplasma (if they are cultured), and endotoxin. Assessments of the final product must include the safety specifications mentioned above even if the results are not available until after release as these data would be useful for patient diagnosis and treatment if necessary. In addition, a plan of action must be in place for patient notification and treatment in case the

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

    Directory of Open Access Journals (Sweden)

    Ahmed Kamel El-Sayed

    2014-11-01

    Full Text Available 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.

  4. A high-resolution comparative RH map of porcine chromosome (SSC) 2.

    NARCIS (Netherlands)

    Rattink, A.P.; Faivre, M.; Jungerius, B.J.; Groenen, M.A.M.; Harlizius, B.

    2001-01-01

    A high-resolution comparative map was constructed for porcine Chromosome (SSC) 2, where a QTL for back fat thickness (BFT) is located. A radiation hybrid (RH) map containing 33 genes and 25 microsatellite markers was constructed for this chromosome with a 3000-rad porcine RH panel. In total, 16

  5. Glycogen distribution in porcine fallopian tube epithelium during the estrus cycle.

    Science.gov (United States)

    Gregoraszczuk, E Ł; Cała, M; Witkowska, E

    2000-01-01

    Histochemical features of two different parts of the porcine Fallopian tube have been studied, with special reference to cyclic changes in the distribution of glycogen particles. Porcine Fallopian tubes were obtained from a local slaughterhouse. Slides were studied under light microscopy utilising histological and histochemical techniques. The most striking feature during the periovulatory stage of the estrus cycle was the occurrence of glycogen granules in the apical cytoplasm of epithelial cells in both the ampulla and isthmus of the Fallopian tubes. In the isthmus, cells containing numerous granules of polysaccharides aggregated into areas of different sizes were noted after ovulation. During the midluteal phase their number was minimal or were even absent. In the ampula typical extrusion of secretory granules and nuclei protruding into the tubal lumen was visible after ovulation. In the luteal phase a lot of nuclei protruded into the tubal lumen and some free in the lumen were noted. It is possible that glycogen in the preovulatory stage functions as a source of energy for ciliary movement and as a nourishment for the ovum. In the isthmus large number of aggregated glycogen particles was observed also after ovulation. In this stage of the cycle, numerous granules of polysaccharide aggregated in isthmus epithelium could be the major energy source for embriogenesis when the embryo travels down the Fallopian tubes, during the early cleavage stage.

  6. Peculiarities of Embryonic and Post-Embryonic Development of Оesophagostomum dentatum (Nematoda, Strongylidae Larvae Cultured in Vitro

    Directory of Open Access Journals (Sweden)

    Yevstafieva V. А.

    2017-02-01

    Full Text Available Morphometric peculiarities of the development of Оesophagostomum dentatum Rudolphi, 1803 from egg to infective larva were studied under laboratory conditions at various temperatures. The determined optimum temperature for embryonic and post-embryonic development of О. dentatum larvae from domestic pig (Sus scrofa domesticus Linnaeus, 1758 is 22 °С. At this temperature, 81 % of larvae develop to the third stage (L3 on the 10th day. Temperatures of 24 °С and 20 °С are less favorable for the development of the nematode, at those temperatures only 67 and 63 % of larvae, respectively, reached infective stage by the 10th day of cultivation. Embryonic development of О. dentatum eggs is characterized by their lengthening (by 8.87-9.50 %, р < 0.01 and widening (by 6.77-9.35 %, р < 0.05-0.01, and post-embryonic larval development is associated with lengthening (by 4.59-17.33 %, р < 0.01-0.001.

  7. CT radiation dose and image quality optimization using a porcine model.

    Science.gov (United States)

    Zarb, Francis; McEntee, Mark F; Rainford, Louise

    2013-01-01

    To evaluate potential radiation dose savings and resultant image quality effects with regard to optimization of commonly performed computed tomography (CT) studies derived from imaging a porcine (pig) model. Imaging protocols for 4 clinical CT suites were developed based on the lowest milliamperage and kilovoltage, the highest pitch that could be set from current imaging protocol parameters, or both. This occurred before significant changes in noise, contrast, and spatial resolution were measured objectively on images produced from a quality assurance CT phantom. The current and derived phantom protocols were then applied to scan a porcine model for head, abdomen, and chest CT studies. Further optimized protocols were developed based on the same methodology as in the phantom study. The optimization achieved with respect to radiation dose and image quality was evaluated following data collection of radiation dose recordings and image quality review. Relative visual grading analysis of image quality criteria adapted from the European guidelines on radiology quality criteria for CT were used for studies completed with both the phantom-based or porcine-derived imaging protocols. In 5 out of 16 experimental combinations, the current clinical protocol was maintained. In 2 instances, the phantom protocol reduced radiation dose by 19% to 38%. In the remaining 9 instances, the optimization based on the porcine model further reduced radiation dose by 17% to 38%. The porcine model closely reflects anatomical structures in humans, allowing the grading of anatomical criteria as part of image quality review without radiation risks to human subjects. This study demonstrates that using a porcine model to evaluate CT optimization resulted in more radiation dose reduction than when imaging protocols were tested solely on quality assurance phantoms.

  8. Dioxin exerts anti-estrogenic actions in a novel dioxin-responsive telomerase-immortalized epithelial cell line of the porcine oviduct (TERT-OPEC).

    Science.gov (United States)

    Hombach-Klonisch, Sabine; Pocar, Paola; Kauffold, Johannes; Klonisch, Thomas

    2006-04-01

    Oviduct epithelial cells are important for the nourishment and survival of ovulated oocytes and early embryos, and they respond to the steroid hormones estrogen and progesterone. Endocrine-disrupting polyhalogenated aromatic hydrocarbons (PHAH) are environmental toxins that act in part through the ligand-activated transcription factor arylhydrocarbon receptor (AhR; dioxin receptor), and exposure to PHAH has been shown to decrease fertility. To investigate effects of PHAHs on the oviduct epithelium as a potential target tissue of dioxin-type endocrine disruptors, we have established a novel telomerase-immortalized oviduct porcine epithelial cell line (TERT-OPEC). TERT-OPEC exhibited active telomerase and the immunoreactive epithelial marker cytokeratin but lacked the stromal marker vimentin. TERT-OPEC contained functional estrogen receptor (ER)-alpha and AhR, as determined by the detection of ER-alpha- and AhR-specific target molecules. Treatment of TERT-OPEC with the AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a significant increase in the production of the cytochrome P-450 microsomal enzyme CYP1A1. Activated AhR caused a downregulation of ER nuclear protein fraction and significantly decreased ER-signaling in TERT-OPEC as determined by ERE-luciferase transient transfection assays. In summary, the TCDD-induced and AhR-mediated anti-estrogenic responses by TERT-OPEC suggest that PHAH affect the predominantly estrogen-dependent differentiation of the oviduct epithelium within the fallopian tube. This action then alters the local endocrine milieu, potentially resulting in a largely unexplored cause of impaired embryonic development and female infertility.

  9. Construction of EMSC-islet co-localizing composites for xenogeneic porcine islet transplantation.

    Science.gov (United States)

    Kim, Jung-Sik; Chung, Hyunwoo; Byun, Nari; Kang, Seong-Jun; Lee, Sunho; Shin, Jun-Seop; Park, Chung-Gyu

    2018-03-04

    Pancreatic islet transplantation is an ultimate solution for treating patients with type 1 diabetes (T1D). The pig is an ideal donor of islets for replacing scarce human islets. Besides immunological hurdles, non-immunological hurdles including fragmentation and delayed engraftment of porcine islets need solutions to succeed in porcine islet xenotransplantation. In this study, we suggest a simple but effective modality, a cell/islet co-localizing composite, to overcome these challenges. Endothelial-like mesenchymal stem cells (EMSCs), differentiated from bone-marrow derived mouse mesenchymal stem cells (MSCs), and MSCs evenly coated the surface of porcine islets (>85%) through optimized culture conditions. Both MSCs and EMSCs significantly reduced the fragmentation of porcine islets and increased the islet masses, designated as islet equivalents (IEQs). In fibrin in vitro and in vivo angiogenesis analysis, constructed EMSC-islet composites showed higher angiogenic potentials than naked islets, MSC-islet composites, or human endothelial cell-islet composites. This novel delivery method of porcine islets may have beneficial effects on the engraftment of transplanted islets by prevention of fragmentation and enhancement of revascularization. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. From embryonic stem cells to testicular germ cell cancer-- should we be concerned?

    DEFF Research Database (Denmark)

    Almstrup, Kristian; Sonne, Si Brask; Hoei-Hansen, Christina E

    2006-01-01

    that initial hypothesis but also indicating that CIS cells have a striking phenotypic similarity to embryonic stem cells (ESC). Many cancers have been proposed to originate from tissue-specific stem cells [so-called 'cancer stem cells' (CSC)] and we argue that CIS may be a very good example of a CSC......, but with exceptional features due to the retention of embryonic pluripotency. In addition, considering the fact that pre-invasive CIS cells are transformed from early fetal cells, possibly due to environmentally induced alterations of the niche, we discuss potential risks linked to the uncontrolled therapeutic use......Since the discovery of testicular carcinoma in situ (CIS) -- the precursor cell for the vast majority of germ cell tumours -- it has been proposed that CIS cells could be derived from transformed primordial germ cells or gonocytes. Here, we review recent discoveries not only substantiating...

  11. Pathology and biofilm formation in a porcine model of staphylococcal osteomyelitis

    DEFF Research Database (Denmark)

    Johansen, L K; Koch, J; Frees, D

    2012-01-01

    A porcine model was used to examine the potential of human and porcine Staphylococcus aureus isolates to induce haematogenously spread osteomyelitis. Pigs were inoculated in the right femoral artery with one of the following S. aureus strains: S54F9 (from a porcine lung abscess; n = 3 animals), N...... dependent on the strain of bacteria inoculated and on the formation of a biofilm....... with colonies of S. aureus as demonstrated immunohistochemically. By peptide nucleic acid fluorescence in situ hybridization bacterial aggregates were demonstrated to be embedded in an opaque matrix, indicating that the bacteria had formed a biofilm. Development of experimental osteomyelitis was therefore...

  12. An Investigation of the Pathology and Pathogens Associated with Porcine Respiratory Disease Complex in Denmark

    DEFF Research Database (Denmark)

    Hansen, Mette Sif; Pors, S. E.; Jensen, H. E.

    2010-01-01

    ), porcine reproductive and respiratory syndrome virus (both European and US type), porcine circovirus type 2 (PCV2), porcine respiratory coronavirus, porcine cytomegalovirus, Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. All cases had cranioventral lobular bronchopneumonia consistent with PRDC....... There was a broad range of microscopical lesions and the cases were characterized as acute (n=10), subacute (n=24) or chronic (n=114) bronchopneumonia. Five bacterial species, five viruses and two Mycoplasma spp. were detected in different combinations. PCV2, M. hyopneumoniae, M. hyorhinis and Pasteurella multocida...

  13. Antimicrobial compounds of porcine mucosa

    Science.gov (United States)

    Kotenkova, E. A.; Lukinova, E. A.; Fedulova, L. V.

    2017-09-01

    The aim of the study was to investigate porcine oral cavity mucosa (OCM), nasal cavity mucosa (NCM), rectal mucosa (RM) and tongue mucosa (TM) as sources of antimicrobial compounds. Ultrafiltrates with MW >30 kDa, MW 5-30 kDa and MW 30 kDa, the zone of microbial growth inhibition was 7.5 mm, for the MW<5 kDa fraction, it was 7 mm, and for MW 5-30 kDa fraction, it was 4.5 mm. No significant differences were found in high molecular weight proteomic profile, while qualitative and quantitative differences were observed in the medium and low molecular weight areas, especially in OCM and NCM. HPLC showed 221 tissue-specific peptides in OCM, 156 in NCM, 225 in RM, but only 5 in TM. The results observed confirmed porcine mucous tissues as a good source of antimicrobial compounds, which could be an actual alternative for reduction of microbial spoilage of foods.

  14. Molecular characterization, sequence analysis and tissue expression of a porcine gene – MOSPD2

    Directory of Open Access Journals (Sweden)

    Yang Jie

    2017-01-01

    Full Text Available The full-length cDNA sequence of a porcine gene, MOSPD2, was amplified using the rapid amplification of cDNA ends method based on a pig expressed sequence tag sequence which was highly homologous to the coding sequence of the human MOSPD2 gene. Sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 491 amino acids that has high homology with the motile sperm domain-containing protein 2 (MOSPD2 of five species: horse (89%, human (90%, chimpanzee (89%, rhesus monkey (89% and mouse (85%; thus, it could be defined as a porcine MOSPD2 gene. This novel porcine gene was assigned GeneID: 100153601. This gene is structured in 15 exons and 14 introns as revealed by computer-assisted analysis. The phylogenetic analysis revealed that the porcine MOSPD2 gene has a closer genetic relationship with the MOSPD2 gene of horse. Tissue expression analysis indicated that the porcine MOSPD2 gene is generally and differentially expressed in the spleen, muscle, skin, kidney, lung, liver, fat and heart. Our experiment is the first to establish the primary foundation for further research on the porcine MOSPD2 gene.

  15. Concise classification of the genomic porcine endogenous retroviral gamma1 load to defined lineages.

    Science.gov (United States)

    Klymiuk, Nikolai; Wolf, Eckhard; Aigner, Bernhard

    2008-02-05

    We investigated the infection history of porcine endogenous retroviruses (PERV) gamma1 by analyzing published env and LTR sequences. PERV sequences from various breeds, porcine cell lines and infected human primary cells were included in the study. We identified a considerable number of retroviral lineages indicating multiple independent colonization events of the porcine genome. A recent boost of the proviral load in an isolated pig herd and exclusive occurrence of distinct lineages in single studies indicated the ongoing colonization of the porcine genome with endogenous retroviruses. Retroviral recombination between co-packaged genomes was a general factor for PERV gamma1 diversity which indicated the simultaneous expression of different proviral loci over a period of time. In total, our detailed description of endogenous retroviral lineages is the prerequisite for breeding approaches to minimize the infectious potential of porcine tissues for the subsequent use in xenotransplantation.

  16. The embryonic development of the central American wandering spider Cupiennius salei

    Directory of Open Access Journals (Sweden)

    Hilbrant Maarten

    2011-06-01

    Full Text Available Abstract Background The spider Cupiennius salei (Keyserling 1877 has become an important study organism in evolutionary and developmental biology. However, the available staging system for its embryonic development is difficult to apply to modern studies, with strong bias towards the earliest developmental stages. Furthermore, important embryonic events are poorly understood. We address these problems, providing a new description of the embryonic development of C. salei. The paper also discusses various observations that will improve our understanding of spider development. Results Conspicuous developmental events were used to define numbered stages 1 to 21. Stages 1 to 9 follow the existing staging system for the spider Achaearanea tepidariorum, and stages 10 to 21 provide a high-resolution description of later development. Live-embryo imaging shows cell movements during the earliest formation of embryonic tissue in C. salei. The imaging procedure also elucidates the encircling border between the cell-dense embryo hemisphere and the hemisphere with much lower cell density (a structure termed 'equator' in earlier studies. This border results from subsurface migration of primordial mesendodermal cells from their invagination site at the blastopore. Furthermore, our detailed successive sequence shows: 1 early differentiation of the precheliceral neuroectoderm; 2 the morphogenetic process of inversion and 3 initial invaginations of the opisthosomal epithelium for the respiratory system. Conclusions Our improved staging system of development in C. salei development should be of considerable value to future comparative studies of animal development. A dense germ disc is not evident during development in C. salei, but we show that the gastrulation process is similar to that in spider species that do have a dense germ disc. In the opisthosoma, the order of appearance of precursor epithelial invaginations provides evidence for the non-homology of the

  17. Silver nanoparticles induce developmental stage-specific embryonic phenotypes in zebrafish

    Science.gov (United States)

    Lee, Kerry J.; Browning, Lauren M.; Nallathamby, Prakash D.; Osgood, Christopher J.; Xu, Xiao-Hong Nancy

    2013-11-01

    Much is anticipated from the development and deployment of nanomaterials in biological organisms, but concerns remain regarding their biocompatibility and target specificity. Here we report our study of the transport, biocompatibility and toxicity of purified and stable silver nanoparticles (Ag NPs, 13.1 +/- 2.5 nm in diameter) upon the specific developmental stages of zebrafish embryos using single NP plasmonic spectroscopy. We find that single Ag NPs passively diffuse into five different developmental stages of embryos (cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stages), showing stage-independent diffusion modes and diffusion coefficients. Notably, the Ag NPs induce distinctive stage and dose-dependent phenotypes and nanotoxicity, upon their acute exposure to the Ag NPs (0-0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c nanotoxicity, upon their acute exposure to the Ag NPs (0-0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c << 0.02 nM) and highest percentages of cardiac abnormalities, followed by early-segmentation embryos (CNP,c < 0.02 nM), suggesting that disruption of cell differentiation by the NPs causes the most toxic effects on embryonic development. The cleavage-stage embryos treated with the NPs develop into a wide variety of phenotypes (abnormal finfold, tail/spinal cord flexure, cardiac malformation/edema, yolk sac edema, and acephaly). These organ structures are not yet developed in cleavage-stage embryos, suggesting that the earliest determinative events to create these structures are ongoing, and disrupted by NPs, which leads to the downstream effects. In contrast, the hatching embryos are most resistant to the Ag NPs, and majority of embryos (94%) develop normally, and none of them develop abnormally. Interestingly, early-gastrula embryos are less sensitive

  18. A sensitive duplex nanoparticle-assisted PCR assay for identifying porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus from clinical specimens.

    Science.gov (United States)

    Zhu, Yu; Liang, Lin; Luo, Yakun; Wang, Guihua; Wang, Chunren; Cui, Yudong; Ai, Xia; Cui, Shangjin

    2017-02-01

    In this study, a novel duplex nanoparticle-assisted polymerase chain reaction (nanoPCR) assay was developed to detect porcine epidemic diarrhea virus (PEDV) and porcine transmissible gastroenteritis virus (TGEV). Two pairs of primers were designed based on the conserved region within the N gene of PEDV and TGEV. In a screening of 114 clinical samples from four provinces in China for PEDV and TGEV, 48.2 and 3.5 % of the samples, respectively, tested positive. Under optimized conditions, the duplex nanoPCR assay had a detection limit of 7.6 × 10 1 and 8.5 × 10 1 copies μL -1 for PEDV and TGEV, respectively. The sensitivity of the duplex nanoPCR assay was ten times higher than that of a conventional PCR assay. Moreover, no fragments were amplified when the duplex nanoPCR assay was used to test samples containing other porcine viruses. Our results indicate that the duplex nanoPCR assay described here is useful for the rapid detection of PEDV and TGEV and can be applied in clinical diagnosis.

  19. Comprehensive quantitative comparison of the membrane proteome and PTM-ome of human embryonic stem cells and neural stem cells

    DEFF Research Database (Denmark)

    Braga, Marcella Nunes de Melo; Schulz, Melanie; Jakobsen, Lene

    Introduction: Human embryonic stem cells (hESCs) can differentiate into all three germ layers and self-renew. Due to its ability to differentiate in vitro into human neural stem cells (hNSCs), which can further be differentiated into motor neurons and dopaminergic neurons, these cells are potential...... identified phosphorylated and SA glycosylated proteins, respectively. This study allowed us to identify several significantly regulated proteins during the differentiation process, including proteins involved in the early embryonic development as well as in the neural development. In the latter group...... of proteins we could identify a number of proteins associated with synaptic vesicles, which are vesicles that store neurotransmitters in the nerve-terminals. An example of an upregulated protein in hESCs is the gap junction alpha 1 (GJA1), a phosphorylated protein which plays a crucial role in embryonic...

  20. Identification of estrogen target genes during zebrafish embryonic development through transcriptomic analysis.

    Directory of Open Access Journals (Sweden)

    Ruixin Hao

    Full Text Available Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17β-estradiol (E2 or vehicle from 3 hours to 4 days post fertilization (dpf, harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP. Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database. The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific.

  1. Tissue Sampling Guides for Porcine Biomedical Models.

    Science.gov (United States)

    Albl, Barbara; Haesner, Serena; Braun-Reichhart, Christina; Streckel, Elisabeth; Renner, Simone; Seeliger, Frank; Wolf, Eckhard; Wanke, Rüdiger; Blutke, Andreas

    2016-04-01

    This article provides guidelines for organ and tissue sampling adapted to porcine animal models in translational medical research. Detailed protocols for the determination of sampling locations and numbers as well as recommendations on the orientation, size, and trimming direction of samples from ∼50 different porcine organs and tissues are provided in the Supplementary Material. The proposed sampling protocols include the generation of samples suitable for subsequent qualitative and quantitative analyses, including cryohistology, paraffin, and plastic histology; immunohistochemistry;in situhybridization; electron microscopy; and quantitative stereology as well as molecular analyses of DNA, RNA, proteins, metabolites, and electrolytes. With regard to the planned extent of sampling efforts, time, and personnel expenses, and dependent upon the scheduled analyses, different protocols are provided. These protocols are adjusted for (I) routine screenings, as used in general toxicity studies or in analyses of gene expression patterns or histopathological organ alterations, (II) advanced analyses of single organs/tissues, and (III) large-scale sampling procedures to be applied in biobank projects. Providing a robust reference for studies of porcine models, the described protocols will ensure the efficiency of sampling, the systematic recovery of high-quality samples representing the entire organ or tissue as well as the intra-/interstudy comparability and reproducibility of results. © The Author(s) 2016.

  2. Purification, characterization and immunolocalization of porcine surfactant protein D

    DEFF Research Database (Denmark)

    Sørensen, C.M.; Nielsen, Ove Lilholm; Willis, A.

    2005-01-01

    in a dose and Ca2+-dependent manner with a saccharide specificity similar to rat and human SP-D. The purified protein was used for the production of a monoclonal anti-pSP-D antibody. The antibody reacted specifically with pSP-D in the reduced and unreduced state when analysed by Western blotting......Surfactant protein D (SP-D) is a collectin believed to play an important role in innate immunity. SP-D is characterized by having a collagen-like domain and a carbohydrate recognition domain (CRD), which has a specific Ca2+-dependent specificity for saccharides and thus the ability to bind complex...... glycoconjugates on micro-organisms. This paper describes the tissue immunolocalization of porcine SP-D (pSP-D) in normal slaughter pigs using a monoclonal antibody raised against purified pSP-D. Porcine SP-D was purified from porcine bronchoalveolar lavage (BAL) by maltose-agarose and immunoglobulin M affinity...

  3. A comparison of anterior-posterior development in the porcine versus the chicken embryo, using goosecoid expression as a marker

    NARCIS (Netherlands)

    Pavert, van de S.A.; Schipper, H.; Wit, de A.A.C.; Soede, N.M.; Hurk, van den R.; Taverne, M.A.M.; Boerjan, M.L.; Stroband, H.W.J.

    2001-01-01

    During early embryonic development, pig and chicken embryos share striking morphological similarities. In the present study, the timing and location of expression of mRNA for goosecoid (gsc), a gene classically expressed in the nodal region of developing embryos, was examined and compared in

  4. Gene expression heterogeneities in embryonic stem cell populations

    DEFF Research Database (Denmark)

    Martinez Arias, Alfonso; Brickman, Joshua M

    2011-01-01

    Stem and progenitor cells are populations of cells that retain the capacity to populate specific lineages and to transit this capacity through cell division. However, attempts to define markers for stem cells have met with limited success. Here we consider whether this limited success reflects...... an intrinsic requirement for heterogeneity with stem cell populations. We focus on Embryonic Stem (ES) cells, in vitro derived cell lines from the early embryo that are considered both pluripotent (able to generate all the lineages of the future embryo) and indefinitely self renewing. We examine the relevance...... of recently reported heterogeneities in ES cells and whether these heterogeneities themselves are inherent requirements of functional potency and self renewal....

  5. Identification of the porcine homologous of human disease causing trinucleotide repeat sequences

    DEFF Research Database (Denmark)

    Madsen, Lone Bruhn; Thomsen, Bo; Sølvsten, Christina Ane Elisabeth

    2007-01-01

    in this paper the identification of porcine noncoding and polyglutamine-encoding TNR regions and the comparison to the homologous TNRs from human, chimpanzee, dog, opossum, rat, and mouse. Several of the porcine TNR regions are highly polymorphic both within and between different breeds. The TNR regions...

  6. An anatomical study of porcine peripheral nerve and its potential use in nerve tissue engineering

    Science.gov (United States)

    Zilic, Leyla; Garner, Philippa E; Yu, Tong; Roman, Sabiniano; Haycock, John W; Wilshaw, Stacy-Paul

    2015-01-01

    Current nerve tissue engineering applications are adopting xenogeneic nerve tissue as potential nerve grafts to help aid nerve regeneration. However, there is little literature that describes the exact location, anatomy and physiology of these nerves to highlight their potential as a donor graft. The aim of this study was to identify and characterise the structural and extracellular matrix (ECM) components of porcine peripheral nerves in the hind leg. Methods included the dissection of porcine nerves, localisation, characterisation and quantification of the ECM components and identification of nerve cells. Results showed a noticeable variance between porcine and rat nerve (a commonly studied species) in terms of fascicle number. The study also revealed that when porcine peripheral nerves branch, a decrease in fascicle number and size was evident. Porcine ECM and nerve fascicles were found to be predominately comprised of collagen together with glycosaminoglycans, laminin and fibronectin. Immunolabelling for nerve growth factor receptor p75 also revealed the localisation of Schwann cells around and inside the fascicles. In conclusion, it is shown that porcine peripheral nerves possess a microstructure similar to that found in rat, and is not dissimilar to human. This finding could extend to the suggestion that due to the similarities in anatomy to human nerve, porcine nerves may have utility as a nerve graft providing guidance and support to regenerating axons. PMID:26200940

  7. Changes in diapause related gene expression pattern during early embryonic development in HCl-treated eggs of bivoltine silkworm Bombyx mori (Lepidoptera: Bombycidae

    Directory of Open Access Journals (Sweden)

    Sirigineedi Sasibhushan

    2013-02-01

    Full Text Available Investigation of differential expression of diapause related genes (five metabolic, five heat shock protein and one translational regulatory in HCl-treated (non-diapause and untreated (diapause eggs of B. mori during early embryogenesis (up to 48h following oviposition revealed the up-regulation of sorbitol dehydrogenase upon HCl treatment, indicating increased glycogen synthesis for further embryonic development but, down-regulation of phosphofructo kinase gene expression after 18h of oviposition indicating an arrest of glycerol and sorbitol conversion. The expression of poly A binding protein gene expression was higher upon HCl treatment, revealing the initiation of translation. The expression levels of other genes analyzed did not vary significantly, except for Hsp90 and Hsp40, which were up-regulated on acid treatment until 18h. Thus, Sorbitoldehydrogenase and phosphofructo kinasegenes have a crucial role in diapause termination as evidenced by HCl treatment, while the other genes did not have major roles.

  8. Isolation of a primate embryonic stem cell line.

    OpenAIRE

    Thomson, J A; Kalishman, J; Golos, T G; Durning, M; Harris, C P; Becker, R A; Hearn, J P

    1995-01-01

    Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. Here we report the derivation of a cloned cell line (R278.5) from a rhesus monkey blastocyst that remains undifferentiated in continuous passage for > 1 year, maintains a normal XY karyotype, and expresses the cell surface markers (alkaline phosphatase, stage-specific embryonic antigen 3, st...

  9. Structural requirements for PACSIN/Syndapin operation during zebrafish embryonic notochord development.

    Science.gov (United States)

    Edeling, Melissa A; Sanker, Subramaniam; Shima, Takaki; Umasankar, P K; Höning, Stefan; Kim, Hye Y; Davidson, Lance A; Watkins, Simon C; Tsang, Michael; Owen, David J; Traub, Linton M

    2009-12-03

    PACSIN/Syndapin proteins are membrane-active scaffolds that participate in endocytosis. The structure of the Drosophila Syndapin N-terminal EFC domain reveals a crescent shaped antiparallel dimer with a high affinity for phosphoinositides and a unique membrane-inserting prong upon the concave surface. Combined structural, biochemical and reverse genetic approaches in zebrafish define an important role for Syndapin orthologue, Pacsin3, in the early formation of the notochord during embryonic development. In pacsin3-morphant embryos, midline convergence of notochord precursors is defective as axial mesodermal cells fail to polarize, migrate and differentiate properly. The pacsin3 morphant phenotype of a stunted body axis and contorted trunk is rescued by ectopic expression of Drosophila Syndapin, and depends critically on both the prong that protrudes from the surface of the bowed Syndapin EFC domain and the ability of the antiparallel dimer to bind tightly to phosphoinositides. Our data confirm linkage between directional migration, endocytosis and cell specification during embryonic morphogenesis and highlight a key role for Pacsin3 in this coupling in the notochord.

  10. Structural requirements for PACSIN/Syndapin operation during zebrafish embryonic notochord development.

    Directory of Open Access Journals (Sweden)

    Melissa A Edeling

    2009-12-01

    Full Text Available PACSIN/Syndapin proteins are membrane-active scaffolds that participate in endocytosis. The structure of the Drosophila Syndapin N-terminal EFC domain reveals a crescent shaped antiparallel dimer with a high affinity for phosphoinositides and a unique membrane-inserting prong upon the concave surface. Combined structural, biochemical and reverse genetic approaches in zebrafish define an important role for Syndapin orthologue, Pacsin3, in the early formation of the notochord during embryonic development. In pacsin3-morphant embryos, midline convergence of notochord precursors is defective as axial mesodermal cells fail to polarize, migrate and differentiate properly. The pacsin3 morphant phenotype of a stunted body axis and contorted trunk is rescued by ectopic expression of Drosophila Syndapin, and depends critically on both the prong that protrudes from the surface of the bowed Syndapin EFC domain and the ability of the antiparallel dimer to bind tightly to phosphoinositides. Our data confirm linkage between directional migration, endocytosis and cell specification during embryonic morphogenesis and highlight a key role for Pacsin3 in this coupling in the notochord.

  11. Morphological and histomorphological structures of testes and ovaries in early developmental stages of the silkworm, Bombyx mori.

    Science.gov (United States)

    Sakai, Hiroki; Kirino, Yohei; Katsuma, Susumu; Aoki, Fugaku; Suzuki, Masataka G

    2016-01-01

    The gonad develops as a testis in male or an ovary in female. In the silkworm, B. mori , little is known about testis and ovary in the embryonic stages and early larval stages. In this study, we performed morphological and histomorphological observations of ovaries and testes from the late embryonic stage to the 1st instar larval stage. Results obtained with lack of accurate information on sex of examined individuals may be misleading, thus we performed phenotypic observations of gonads by utilizing sex-limited strain that enables us to easily discriminate female embryos from male ones based on those egg colors. In testis, four testicular follicles were clearly observed in the testis at the first instar larval stage, and boundary layers were formed between the testicular follicles. At the late embryonic stage, the testis consisted of four testicular follicles, while the boundary layers were still obscure. In ovary, four ovarioles were easily recognizable in the ovary at the first instar larval stage, and boundary layers were formed between the ovarioles. However, in the late embryonic stage, it was quite difficult to identify four ovarioles. Morphological characteristics were almost similar between testis and ovary in early developmental stages. Our present study demonstrates that the most reliable difference between testis and ovary in early developmental stages is the attaching point of the duct. Formation and development of the duct may be sensitive to the sex-determining signal and display sexual dimorphism in early embryonic stages.

  12. Porcine cluster of differentiation (CD) markers 2018 update.

    Science.gov (United States)

    Dawson, Harry D; Lunney, Joan K

    2018-06-01

    Pigs are a major source of food worldwide; preventing and treating their infectious diseases is essential, requiring a thorough understanding of porcine immunity. The use of pigs as models for human physiology is a growing area; progress in this area has been limited because the immune toolkit is not robust. The international community has established cluster of differentiation (CD) markers for assessing cells involved in immunity as well as characterizing numerous other cells like stem cells. Overall, for humans 419 proteins have been designated as CD markers, each reacting with a defined set of antibodies (Abs). This paper summarizes current knowledge of swine CD markers and identifies 359 corresponding CD proteins in pigs. A broad-based literature and vendor search was conducted to identify defined sets of monoclonal (mAbs) and polyclonal Abs (pAbs) reacting with porcine CD markers along with other reagents (fusion proteins, ELISAs, PCR assays, and gene edited cell and pig models). This process identified over 800 reagents that are reportedly reactive with 266 pig CD markers. Despite this number, there is a great need to develop and characterize additional CD marker reagents, particularly mAbs, for pig research. There are numerous high priority targets: reagents for the characterization of porcine innate lymphoid cells, polarized macrophages and T regulatory cells and for the detection of porcine CD45 isoforms. Overall, improved technologies and genomics have contributed to dramatic increases in our knowledge of the pig, its immune system, disease and vaccine responses, and utility as a biomedical model. The development of more CD reagents will clearly advance these initiatives. Published by Elsevier Ltd.

  13. Discovery of a novel putative atypical porcine pestivirus in pigs in the USA.

    Science.gov (United States)

    Hause, Ben M; Collin, Emily A; Peddireddi, Lalitha; Yuan, Fangfeng; Chen, Zhenhai; Hesse, Richard A; Gauger, Phillip C; Clement, Travis; Fang, Ying; Anderson, Gary

    2015-10-01

    Pestiviruses are some of the most significant pathogens affecting ruminants and swine. Here, we assembled a 11 276 bp contig encoding a predicted 3635 aa polyprotein from porcine serum with 68 % pairwise identity to that of a recently partially characterized Rhinolophus affinis pestivirus (RaPV) and approximately 25-28 % pairwise identity to those of other pestiviruses. The virus was provisionally named atypical porcine pestivirus (APPV). Metagenomic sequencing of 182 serum samples identified four additional APPV-positive samples. Positive samples originated from five states and ELISAs using recombinant APPV Erns found cross-reactive antibodies in 94 % of a collection of porcine serum samples, suggesting widespread distribution of APPV in the US swine herd. The molecular and serological results suggest that APPV is a novel, highly divergent porcine pestivirus widely distributed in US pigs.

  14. Porcine, murine and human sialoadhesin (Sn/Siglec-1/CD169): portals for porcine reproductive and respiratory syndrome virus entry into target cells.

    Science.gov (United States)

    Van Breedam, Wander; Verbeeck, Mieke; Christiaens, Isaura; Van Gorp, Hanne; Nauwynck, Hans J

    2013-09-01

    Porcine sialoadhesin (pSn; a sialic acid-binding lectin) and porcine CD163 (pCD163) are molecules that facilitate infectious entry of porcine reproductive and respiratory syndrome virus (PRRSV) into alveolar macrophages. In this study, it was shown that murine Sn (mSn) and human Sn (hSn), like pSn, can promote PRRSV infection of pCD163-expressing cells. Intact sialic acid-binding domains are crucial, since non-sialic acid-binding mutants of pSn, mSn and hSn did not promote infection. Endodomain-deletion mutants of pSn, mSn and hSn promoted PRRSV infection less efficiently, but also showed markedly reduced expression levels, making further research into the potential role of the Sn endodomain in PRRSV receptor activity necessary. These data further complement our knowledge on Sn as an important PRRSV receptor, and suggest - in combination with other published data - that species differences in the main PRRSV entry mediators Sn and CD163 do not account for the strict host species specificity displayed by the virus.

  15. Enteric porcine viruses in farmed shellfish in Denmark.

    Science.gov (United States)

    Krog, J S; Larsen, L E; Schultz, A C

    2014-09-01

    Bivalve shellfish are at constant risk of being exposed to pathogens as a consequence of contamination of the shellfish beds with human or animal waste originating from sewage treatment plants or slurry fertilized fields. Consumption of contaminated oysters and mussels are frequently reported as causes of disease outbreaks caused by norovirus or hepatitis A virus. Other zoonotic pathogens such as hepatitis E virus (HEV), rotavirus (RV) and Salmonella from livestock may also be transmitted to shellfish via this route. In this study, 29 pooled samples from commercial Danish blue mussels were tested for porcine pathogens and indicator bacteria Escherichia coli (E. coli). All samples tested negative for HEV, RV and Salmonella, whereas E. coli and the highly stable porcine circovirus type 2 (PCV2) were detected in eight and 12 samples, respectively. This is the first study to report the detection of PCV2 in commercial mussels. Based on the detection of PCV2 in clean areas with low prevalence of the normally applied fecal indicator E. coli, testing for PCV2 may be a more sensitive and robust specific porcine waste indicator in shellfish harvesting areas. Copyright © 2014. Published by Elsevier B.V.

  16. Carbon nanotubes as VEGF carriers to improve the early vascularization of porcine small intestinal submucosa in abdominal wall defect repair

    Directory of Open Access Journals (Sweden)

    Liu Z

    2014-03-01

    Full Text Available Zhengni Liu,1,* Xueyi Feng,2,* Huichun Wang,1 Jun Ma,1 Wei Liu,3 Daxiang Cui,4 Yan Gu,1 Rui Tang,11Department of General Surgery, Shanghai Ninth People’s Hospital, Hernia and Abdominal Wall Disease Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 2Department of General Surgery, Lu’an People’s Hospital, Lu’an Affiliated Hospital of Anhui Medical University, Lu’an, Province Anhui, People’s Republic of China; 3Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Tissue Engineering, Shanghai, People’s Republic of China; 4Institute of Nano Biomedicine and Engineering, Key Laboratory for Thin Film and Microfabrication Technology of the Ministry of Education, Research Institute of Micro/Nano Science and Technology, Bio-X Center, Shanghai Jiao Tong University, Shanghai, People's Republic of China *These authors contributed equally to this work Abstract: Insufficient early vascularization in biological meshes, resulting in limited host tissue incorporation, is thought to be the primary cause for the failure of abdominal wall defect repair after implantation. The sustained release of exogenous angiogenic factors from a biocompatible nanomaterial might be a way to overcome this limitation. In the study reported here, multiwalled carbon nanotubes (MWNT were functionalized by plasma polymerization to deliver vascular endothelial growth factor165 (VEGF165. The novel VEGF165-controlled released system was incorporated into porcine small intestinal submucosa (PSIS to construct a composite scaffold. Scaffolds incorporating varying amounts of VEGF165-loaded functionalized MWNT were characterized in vitro. At 5 weight percent MWNT, the scaffolds exhibited optimal properties and were implanted in rats to repair abdominal wall defects. PSIS scaffolds incorporating VEGF165-loaded MWNT (VEGF

  17. PXD101 significantly improves nuclear reprogramming and the in vitro developmental competence of porcine SCNT embryos

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Jun-Xue; Kang, Jin-Dan; Li, Suo; Jin, Long; Zhu, Hai-Ying; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2015-01-02

    Highlights: • First explored that the effects of PXD101 on the development of SCNT embryos in vitro. • 0.5 μM PXD101 treated for 24 h improved the development of porcine SCNT embryos. • Level of AcH3K9 was significantly higher than control group at early stages. - Abstract: In this study, we investigated the effects of the histone deacetylase inhibitor PXD101 (belinostat) on the preimplantation development of porcine somatic cell nuclear transfer (SCNT) embryos and their expression of the epigenetic markers histone H3 acetylated at lysine 9 (AcH3K9). We compared the in vitro developmental competence of SCNT embryos treated with various concentrations of PXD101 for 24 h. Treatment with 0.5 μM PXD101 significantly increased the proportion of SCNT embryos that reached the blastocyst stage, in comparison to the control group (23.3% vs. 11.5%, P < 0.05). We tested the in vitro developmental competence of SCNT embryos treated with 0.5 μM PXD101 for various amounts of times following activation. Treatment for 24 h significantly improved the development of porcine SCNT embryos, with a significantly higher proportion of embryos reaching the blastocyst stage in comparison to the control group (25.7% vs. 10.6%, P < 0.05). PXD101-treated SCNT embryos were transferred into two surrogate sows, one of whom became pregnant and four fetuses developed. PXD101 treatment significantly increased the fluorescence intensity of immunostaining for AcH3K9 in embryos at the pseudo-pronuclear and 2-cell stages. At these stages, the fluorescence intensities of immunostaining for AcH3K9 were significantly higher in PXD101-treated embryos than in control untreated embryos. In conclusion, this study demonstrates that PXD101 can significantly improve the in vitro and in vivo developmental competence of porcine SCNT embryos and can enhance their nuclear reprogramming.

  18. Loss of ATM kinase activity leads to embryonic lethality in mice

    DEFF Research Database (Denmark)

    Daniel, J.A.; Pellegrini, M.; Filsuf, D.

    2012-01-01

    whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice......, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate...

  19. Mouse survival motor neuron alleles that mimic SMN2 splicing and are inducible rescue embryonic lethality early in development but not late.

    Directory of Open Access Journals (Sweden)

    Suzan M Hammond

    Full Text Available Spinal muscular atrophy (SMA is caused by low survival motor neuron (SMN levels and patients represent a clinical spectrum due primarily to varying copies of the survival motor neuron-2 (SMN2 gene. Patient and animals studies show that disease severity is abrogated as SMN levels increase. Since therapies currently being pursued target the induction of SMN, it will be important to understand the dosage, timing and cellular requirements of SMN for disease etiology and potential therapeutic intervention. This requires new mouse models that can induce SMN temporally and/or spatially. Here we describe the generation of two hypomorphic Smn alleles, Smn(C-T-Neo and Smn(2B-Neo. These alleles mimic SMN2 exon 7 splicing, titre Smn levels and are inducible. They were specifically designed so that up to three independent lines of mice could be generated, herein we describe two. In a homozygous state each allele results in embryonic lethality. Analysis of these mutants indicates that greater than 5% of Smn protein is required for normal development. The severe hypomorphic nature of these alleles is caused by inclusion of a loxP-flanked neomycin gene selection cassette in Smn intron 7, which can be removed with Cre recombinase. In vitro and in vivo experiments demonstrate these as inducible Smn alleles. When combined with an inducible Cre mouse, embryonic lethality caused by low Smn levels can be rescued early in gestation but not late. This provides direct genetic evidence that a therapeutic window for SMN inductive therapies may exist. Importantly, these lines fill a void for inducible Smn alleles. They also provide a base from which to generate a large repertoire of SMA models of varying disease severities when combined with other Smn alleles or SMN2-containing mice.

  20. [Activation of nucleolar organizers during in vitro cultivation of mouse R1 embryonic stem cells].

    Science.gov (United States)

    Kunafina, E R; Chaplina, M V; Filiasova, E I; Gibanova, N V; Khodarovich, Iu M; Larionov, O A; Zatsepina, O V

    2005-01-01

    We studies the activities of ribosomal genes (nucleolus forming regions of chromosomes) at successive stages of cultivation of the mouse R1 embryonic stem cells. The total number and number of active nucleolar organizers were estimated by means of in situ hybridization with mouse rDNA probes and argentophilic staining of nucleolus forming chromosomes regions from the 16th until the 32nd passages. The data we obtained suggest that the total number of nucleolar organizers per metaphase plate was constant (as a rule, eight), while the mean number of active nucleolar organizers progressively increased from the early (16th) to the late (32nd) passages: 5.2 +/- 0.4 versus 7.4 +/- 0.9 argentophilic organizers per cell. Cell heterogeneity by the number of active nucleolar organizers also increased during the late passages. Taken together, these data suggest activation of DNA transcription and synthesis of ribosomes during cultivation of mouse R1 embryonic stem cells. Based on the experimental and published data, it has been proposed that activation of ribosomal genes correlates in time with a decreased capacity of embryonic stem cells for pluripotent differentiation.

  1. p53 dependent apoptotic cell death induces embryonic malformation in Carassius auratus under chronic hypoxia.

    Directory of Open Access Journals (Sweden)

    Paramita Banerjee Sawant

    Full Text Available Hypoxia is a global phenomenon affecting recruitment as well as the embryonic development of aquatic fauna. The present study depicts hypoxia induced disruption of the intrinsic pathway of programmed cell death (PCD, leading to embryonic malformation in the goldfish, Carrasius auratus. Constant hypoxia induced the early expression of pro-apoptotic/tumor suppressor p53 and concomitant expression of the cell death molecule, caspase-3, leading to high level of DNA damage and cell death in hypoxic embryos, as compared to normoxic ones. As a result, the former showed delayed 4 and 64 celled stages and a delay in appearance of epiboly stage. Expression of p53 efficiently switched off expression of the anti-apoptotic Bcl-2 during the initial 12 hours post fertilization (hpf and caused embryonic cell death. However, after 12 hours, simultaneous downregulation of p53 and Caspase-3 and exponential increase of Bcl-2, caused uncontrolled cell proliferation and prevented essential programmed cell death (PCD, ultimately resulting in significant (p<0.05 embryonic malformation up to 144 hpf. Evidences suggest that uncontrolled cell proliferation after 12 hpf may have been due to downregulation of p53 abundance, which in turn has an influence on upregulation of anti-apoptotic Bcl-2. Therefore, we have been able to show for the first time and propose that hypoxia induced downregulation of p53 beyond 12 hpf, disrupts PCD and leads to failure in normal differentiation, causing malformation in gold fish embryos.

  2. Ultrasonographically documented early pregnancy loss in an Asian elephant (Elephas maximus).

    Science.gov (United States)

    Lueders, Imke; Drews, Barbara; Niemuller, Cheryl; Gray, Charlie; Rich, Peter; Fickel, Jörns; Wibbelt, Gudrun; Göritz, Frank; Hildebrandt, Thomas B

    2010-01-01

    Early embryonic resorption or fetal loss is known to occur occasionally in captive elephants; however, this has mostly been reported anecdotally. The present study documents the case of a 24-year-old, multiparous Asian elephant cow that suffered embryonic death and resorption at around 18 weeks of gestation. From ovulation onwards, this female was sonographically examined 58 times. Blood was collected twice weekly for progestagen determination via enzyme immunoassay. On Day 42 after ovulation, a small quantity of fluid was detected in the uterine horn, which typically indicates the presence of a developing conceptus. Repeated inspections followed what appeared to be a normal pregnancy until Day 116. However, on Day 124, signs of embryonic life were absent. Progestagen concentrations started declining two weeks later, reaching baseline levels one month after embryonic death. Retrospectively, ultrasound examination revealed several abnormalities in the uterine horn. Besides an existing leiomyoma, multiple small cystic structures had formed in the endometrium at the implantation site and later in the placenta. These pathological findings were considered as possible contributors to the early pregnancy failure. PCR for endotheliotropic elephant herpes virus (EEHV) (which had occurred previously in the herd) as well as serology for other infectious organisms known to cause abortion in domestic animals did not yield any positive results. Although no definitive reason was found for this pregnancy to abort, this ultrasonographically and endocrinologically documented study of an early pregnancy loss provides important insights into the resorption process in Asian elephants.

  3. EFFECT OF NATURAL PLANT EXTRACTS ON PORCINE OVARIAN FUNCTIONS

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    Attila Kádasi

    2015-02-01

    Full Text Available This report provides information about the impact of chosen natural plant extracts on basic ovarian functions. This article summarizes our results concerning the effect of selected plant extracts on proliferation, apoptosis and hormone secretion – release of progesterone (P4, testosterone (T and leptin (L on porcine granulosa cells (GC, We analyzed effects of ginkgo (GB, rooibos (RB, flaxseed (FL, green tea polyphenols (GTPP, green tea - epigallocatechin-3-gallate (EGCG, resveratrol (RSV and curcumin (CURC (0; 1; 10 and 100 μg.ml-1 on markers of proliferation, apoptosis and secretory activity of porcine ovarian granulosa cells by using immunocytochemistry and EIA. It was demonstrated, that all these natural plants and plant molecules inhibited the accumulation of proliferation-related peptide (PCNA and apoptosis-associated peptide (Bax in cultured. Furthermore, it was observed that natural plant extracts altered progesterone, testosterone and leptin release in porcine ovarian cells. It is concluded, that GB, RB, FL, RSV, CURC, GTPP and EGCG can directly affect ovarian cells and therefore they could potentially influence ovarian functions.

  4. Human Embryonic Stem Cells: A Model for the Study of Neural Development and Neurological Diseases

    Directory of Open Access Journals (Sweden)

    Piya Prajumwongs

    2016-01-01

    Full Text Available Although the mechanism of neurogenesis has been well documented in other organisms, there might be fundamental differences between human and those species referring to species-specific context. Based on principles learned from other systems, it is found that the signaling pathways required for neural induction and specification of human embryonic stem cells (hESCs recapitulated those in the early embryo development in vivo at certain degree. This underscores the usefulness of hESCs in understanding early human neural development and reinforces the need to integrate the principles of developmental biology and hESC biology for an efficient neural differentiation.

  5. Irreversible electroporation of the pancreas is feasible and safe in a porcine survival model.

    Science.gov (United States)

    Fritz, Stefan; Sommer, Christof M; Vollherbst, Dominik; Wachter, Miguel F; Longerich, Thomas; Sachsenmeier, Milena; Knapp, Jürgen; Radeleff, Boris A; Werner, Jens

    2015-07-01

    Use of thermal tumor ablation in the pancreatic parenchyma is limited because of the risk of pancreatitis, pancreatic fistula, or hemorrhage. This study aimed to evaluate the feasibility and safety of irreversible electroporation (IRE) in a porcine model. Ten pigs were divided into 2 study groups. In the first group, animals received IRE of the pancreatic tail and were killed after 60 minutes. In the second group, animals received IRE at the head of the pancreas and were followed up for 7 days. Clinical parameters, computed tomography imaging, laboratory results, and histology were obtained. All animals survived IRE ablation, and no cardiac adverse effects were noted. Sixty minutes after IRE, a hypodense lesion on computed tomography imaging indicated the ablation zone. None of the animals developed clinical signs of acute pancreatitis. Only small amounts of ascites fluid, with a transient increase in amylase and lipase levels, were observed, indicating that no pancreatic fistula occurred. This porcine model shows that IRE is feasible and safe in the pancreatic parenchyma. Computed tomography imaging reveals significant changes at 60 minutes after IRE and therefore might serve as an early indicator of therapeutic success. Clinical studies are needed to evaluate the efficacy of IRE in pancreatic cancer.

  6. Significant Down-Regulation of “Biological Adhesion” Genes in Porcine Oocytes after IVM

    Directory of Open Access Journals (Sweden)

    Joanna Budna

    2017-12-01

    Full Text Available Proper maturation of the mammalian oocyte is a compound processes determining successful monospermic fertilization, however the number of fully mature porcine oocytes is still unsatisfactory. Since oocytes’ maturation and fertilization involve cellular adhesion and membranous contact, the aim was to investigate cell adhesion ontology group in porcine oocytes. The oocytes were collected from ovaries of 45 pubertal crossbred Landrace gilts and subjected to two BCB tests. After the first test, only granulosa cell-free BCB+ oocytes were directly exposed to microarray assays and RT-qPCR (“before IVM” group, or first in vitro matured and then if classified as BCB+ passed to molecular analyses (“after IVM” group. As a result, we have discovered substantial down-regulation of genes involved in adhesion processes, such as: organization of actin cytoskeleton, migration, proliferation, differentiation, apoptosis, survival or angiogenesis in porcine oocytes after IVM, compared to oocytes analyzed before IVM. In conclusion, we found that biological adhesion may be recognized as the process involved in porcine oocytes’ successful IVM. Down-regulation of genes included in this ontology group in immature oocytes after IVM points to their unique function in oocyte’s achievement of fully mature stages. Thus, results indicated new molecular markers involved in porcine oocyte IVM, displaying essential roles in biological adhesion processes.

  7. The basic route of the nuclear translocation porcine growth hormone (GH)-growth hormone receptor (GHR) complex (pGH/GHR) in porcine hepatocytes.

    Science.gov (United States)

    Hainan, Lan; Huilin, Liu; Khan, Mahamad; Xin, Zheng; YuJiang, Yang; Hui, Zhang; Naiquan, Yao

    2018-06-08

    Traditional views suggest that growth hormone and the growth hormone receptor (GH/GHR complex) exert their functions only on the plasma membrane. This paradigm, however, has been challenged by recent new findings that the GH/GHR complex could translocate into cell nuclei where they could still exhibit important physiological functions. We also reported the nuclear localization of porcine GH/GHR and their potential functions in porcine hepatocytes. However, the basic path of pGH/GHR's nuclear translocation remains unclear. Combining previous research results and our current findings, we proposed two basic routes of pGH/GHR's nuclear transportation as follows: 1) after pGH binding to GHR, pGH/GHR enters into the cytoplasm though clathrin- or caveolin-mediated endocytosis, then the pGH/GHR complex enters into early endosomes (Rab5-positive), and the endosome carries the GH/GHR complex to the endoplasmic reticulum (ER). After endosome docking on the ER, the endosome starts fission, and the pGH/GHR complex enters into the ER lumen. Then the pGH/GHR complex transports into the cytoplasm, possibly by the ERAD pathway. Subsequently, the pGH/GHR complex interacts with IMPα/β, which, in turn, mediates GH/GHR nuclear localization; 2) pGH binds with the GHR on the cell membrane and, subsequently, pGH/GHR internalizes into the cell and enters into the endosome (this endosome may belong to a class of endosomes called envelope-associated endosomes (NAE)). Then, the endosome carries the pGH/GHR to the nuclear membrane. After docking on the nuclear membrane, the pGH/GHR complex fuses with the nuclear membrane and then enters into the cell nucleus. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Effects of oxidative stress on human embryonic stem cells; global gene expression, advanced glycation end products and NEDD1 levels

    NARCIS (Netherlands)

    Barandalla Sobrados, M.

    2017-01-01

    A number of unfavorable conditions can affect the development of the early embryo inducing oxidative stress both in vivo, for instance in gestational diabetes, and in vitro, when embryos are derived from Assisted Reproductive Technologies (ART). Human Embryonic Stem Cells (hESCs) potentially offer a

  9. A genome-wide RNAi screen reveals MAP kinase phosphatases as key ERK pathway regulators during embryonic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Shen-Hsi Yang

    Full Text Available Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Complex interlinked transcriptional and signaling networks control the fate of these cells towards maintenance of pluripotency or differentiation. In this study we have focused on how mouse embryonic stem cells begin to differentiate and lose pluripotency and, in particular, the role that the ERK MAP kinase and GSK3 signaling pathways play in this process. Through a genome-wide siRNA screen we have identified more than 400 genes involved in loss of pluripotency and promoting the onset of differentiation. These genes were functionally associated with the ERK and/or GSK3 pathways, providing an important resource for studying the roles of these pathways in controlling escape from the pluripotent ground state. More detailed analysis identified MAP kinase phosphatases as a focal point of regulation and demonstrated an important role for these enzymes in controlling ERK activation kinetics and subsequently determining early embryonic stem cell fate decisions.

  10. Appraisal of the porcine kidney autotransplantation model

    NARCIS (Netherlands)

    Post, Ivo C. J. H.; Dirkes, Marcel C.; Heger, Michal; van Loon, Johannes P. A. M.; Swildens, Bas; Huijzer, Goos M.; van Gulik, Thomas M.

    2012-01-01

    Animal models are extensively used for transplantation related research, especially kidney transplantation. Porcine autotransplantation models are considered to be favorable regarding translatability to the human setting. The key determinants for translatability of the model are discussed,

  11. Comparison of the effect of fatty alcohols on the permeation of melatonin between porcine and human skin.

    Science.gov (United States)

    Andega, S; Kanikkannan, N; Singh, M

    2001-11-09

    Melatonin (MT) is a hormone secreted by the pineal gland that plays an important role in the regulation of the circadian sleep-wake cycle. It would be advantageous to administer MT using a transdermal delivery system for the treatment of sleep disorders such as delayed sleep syndrome, jet lag in travelers, cosmonauts and shift workers. The porcine skin has been found to have similar morphological and functional characteristics as human skin. The elastic fibres in the dermis, enzyme pattern of the epidermis, epidermal tissue turnover time, keratinous proteins and thickness of epidermis of porcine skin are similar to human skin. However, the fat deposition and vascularisation of the cutaneous glands of porcine skin are different from human skin. In addition, porcine skin has been found to have a close permeability character to human skin. However, the comparative effect of chemical penetration enhancers on the permeation of drugs between porcine and human skin has not been reported. The purpose of this study was to compare the effect of fatty alcohols on the permeability of porcine and human skin using MT as a model compound. The effect of saturated fatty alcohols (octanol, nonanol, decanol, undecanol, lauryl alcohol, tridecanol, myristyl alcohol) and unsaturated fatty alcohols (oleyl alcohol, linoleyl alcohol, linolenyl alcohol) at 5% concentration was tested across dermatomed porcine and human skin. Our studies showed a parabolic relationship between the carbon chain length of saturated fatty alcohols and permeation enhancement of MT with both porcine and human skin. Maximum permeation of MT was observed when fatty alcohol carbon chain length was 10. In general, as the level of unsaturation increased from one to two double bonds, there was an increase in the permeation of MT both in porcine and human skin. However, a decrease in the permeation was observed with three double bonds. Regression analysis using the steady state flux data showed a significant positive

  12. Bioengineering Embryonic Stem Cell Microenvironments for the Study of Breast Cancer

    Directory of Open Access Journals (Sweden)

    Yubing Xie

    2011-11-01

    Full Text Available Breast cancer is the most prevalent disease amongst women worldwide and metastasis is the main cause of death due to breast cancer. Metastatic breast cancer cells and embryonic stem (ES cells display similar characteristics. However, unlike metastatic breast cancer cells, ES cells are nonmalignant. Furthermore, embryonic microenvironments have the potential to convert metastatic breast cancer cells into a less invasive phenotype. The creation of in vitro embryonic microenvironments will enable better understanding of ES cell-breast cancer cell interactions, help elucidate tumorigenesis, and lead to the restriction of breast cancer metastasis. In this article, we will present the characteristics of breast cancer cells and ES cells as well as their microenvironments, importance of embryonic microenvironments in inhibiting tumorigenesis, convergence of tumorigenic and embryonic signaling pathways, and state of the art in bioengineering embryonic microenvironments for breast cancer research. Additionally, the potential application of bioengineered embryonic microenvironments for the prevention and treatment of invasive breast cancer will be discussed.

  13. Oocyte exposure to ZnO nanoparticles inhibits early embryonic development through the γ-H2AX and NF-κB signaling pathways.

    Science.gov (United States)

    Liu, Jing; Zhao, Yong; Ge, Wei; Zhang, Pengfei; Liu, Xinqi; Zhang, Weidong; Hao, Yanan; Yu, Shuai; Li, Lan; Chu, Meiqiang; Min, Lingjiang; Zhang, Hongfu; Shen, Wei

    2017-06-27

    The impacts of zinc oxide nanoparticles on embryonic development following oocyte stage exposure are unknown and the underlying mechanisms are sparsely understood. In the current investigation, intact nanoparticles were detected in ovarian tissue in vivo and cultured cells in vitro under zinc oxide nanoparticles treatment. Zinc oxide nanoparticles exposure during the oocyte stage inhibited embryonic development. Notably, in vitro culture data closely matched in vivo embryonic data, in that the impairments caused by Zinc oxide nanoparticles treatment passed through cell generations; and both gamma-H2AX and NF-kappaB pathways were involved in zinc oxide nanoparticles caused embryo-toxicity. Copper oxide and silicon dioxide nanoparticles have been used to confirm that particles are important for the toxicity of zinc oxide nanoparticles. The toxic effects of zinc oxide nanoparticles emanate from both intact nanoparticles and Zn2+. Our investigation along with others suggests that zinc oxide nanoparticles are toxic to the female reproductive system [ovaries (oocytes)] and subsequently embryo-toxic and that precaution should be taken regarding human exposure to their everyday use.

  14. Isolation and culture of porcine neural progenitor cells from embryos and pluripotent stem cells

    DEFF Research Database (Denmark)

    Rasmussen, Mikkel Aabech; Hall, Vanessa Jane; Hyttel, Poul

    2013-01-01

    from porcine embryos or induced pluripotent stem cells is presented. The neural induction is performed in coculture and the isolation of rosette structures is carried out manually to ensure a homogenous population of NPCs. Using this method, multipotent NPCs can be obtained in approximately 1 month......The isolation and culture of neural progenitor cells (NPCs) from pluripotent stem cells has facilitated in vitro mechanistic studies of diseases related to the nervous system, as well as discovery of new medicine. In addition, NPCs are envisioned to play a crucial role in future cell replacement...... therapy. The pig has become recognized as an important large animal model and establishment of in vitro-derived porcine NPCs would allow for preclinical safety testing by transplantation in a porcine biomedical model. In this chapter, a detailed method for isolation and in vitro culture of porcine NPCs...

  15. Transcription analysis of the porcine alveolar macrophage response to Mycoplasma hyopneumoniae.

    Directory of Open Access Journals (Sweden)

    Li Bin

    Full Text Available Mycoplasma hyopneumoniae is considered the major causative agent of porcine respiratory disease complex, occurs worldwide and causes major economic losses to the pig industry. To gain more insights into the pathogenesis of this organism, the high throughput cDNA microarray assays were employed to evaluate host responses of porcine alveolar macrophages to M. hyopneumoniae infection. A total of 1033 and 1235 differentially expressed genes were identified in porcine alveolar macrophages in responses to exposure to M. hyopneumoniae at 6 and 15 hours post infection, respectively. The differentially expressed genes were involved in many vital functional classes, including inflammatory response, immune response, apoptosis, cell adhesion, defense response, signal transduction, protein folding, protein ubiquitination and so on. The pathway analysis demonstrated that the most significant pathways were the chemokine signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, nucleotide-binding oligomerization domains (Nod-like receptor signaling pathway and apoptosis signaling pathway. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR. The expression kinetics of chemokines was further analyzed. The present study is the first to document the response of porcine alveolar macrophages to M. hyopneumoniae infection. The data further developed our understanding of the molecular pathogenesis of M. hyopneumoniae.

  16. Transcription analysis of the porcine alveolar macrophage response to Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Bin, Li; Luping, Du; Bing, Sun; Zhengyu, Yu; Maojun, Liu; Zhixin, Feng; Yanna, Wei; Haiyan, Wang; Guoqing, Shao; Kongwang, He

    2014-01-01

    Mycoplasma hyopneumoniae is considered the major causative agent of porcine respiratory disease complex, occurs worldwide and causes major economic losses to the pig industry. To gain more insights into the pathogenesis of this organism, the high throughput cDNA microarray assays were employed to evaluate host responses of porcine alveolar macrophages to M. hyopneumoniae infection. A total of 1033 and 1235 differentially expressed genes were identified in porcine alveolar macrophages in responses to exposure to M. hyopneumoniae at 6 and 15 hours post infection, respectively. The differentially expressed genes were involved in many vital functional classes, including inflammatory response, immune response, apoptosis, cell adhesion, defense response, signal transduction, protein folding, protein ubiquitination and so on. The pathway analysis demonstrated that the most significant pathways were the chemokine signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, nucleotide-binding oligomerization domains (Nod)-like receptor signaling pathway and apoptosis signaling pathway. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR. The expression kinetics of chemokines was further analyzed. The present study is the first to document the response of porcine alveolar macrophages to M. hyopneumoniae infection. The data further developed our understanding of the molecular pathogenesis of M. hyopneumoniae.

  17. DIFFERENTIATION OF EMBRYONIC STEM CELLS: LESSONS FROM EMBRYONIC DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    EMOKE PALL

    2008-05-01

    Full Text Available Embryonic stem (ES cells, the undifferentiated cells of early embryos are established as permanent lines and are characterised by their self-renewal capacity and the ability to retain their developmental capacity in vivo and in vitro. The pluripotent properties of ES cells are the basis of gene targeting technologies used to create mutant mouse strains with inactivated genes by homologous recombination. There are several methods to induce the formation of EBs. One of them the formation by aggregating ES cells in hanging drops, using gravity as an aggregation force. This method presents the advantage of obtaining well-calibrated EBs almost identical in size. We used at our experiment the mouse ES cell line KA1/11/C3/C8 with a normal karyotype, at 14th passages. Immunohistochemical examination was aimed to identify tissue-restricted proteins for the two differentiated lineages: titin as a cell-specific antigen for cardiac and skeletal muscle, betaIII-tubulin for the neuronal differentiation, cytokeratin Endo-A (TROMA for the presence of mesenchymal progenitor cells, Oct-4 for the presence of the undifferentiated ES cells. The beating cardiac muscle clumps showed more synchronous rhythm than those seen in EBs obtained from suspension culture method, where the beating cardiac muscle clumps appeared later, had a lower frequency and were uneven. The synaptic networks of neuronal cells were best developed in EBs from suspension, compared to those observed in EBs from hanging-drop method.

  18. Interdigitated electrode (IDE) for porcine detection based on titanium dioxide (TiO_2) thin films

    International Nuclear Information System (INIS)

    Nordin, N.; Azizah, N.; Hashim, U.

    2016-01-01

    Interdigited Electrode (IDE) porcine detection can be accomplished to authenticate the halal issue that has been a concern to Muslim not only in Malaysia but all around the world. The method used is photolithography that used the p-type photoresist on the spin coater with 2500 rpm. Bare IDEs device is deposited with Titanium Dioxide (TiO_2) to improve the performance of the device. The result indicates that current-voltage (I-V) measurement of porcine probe line slightly above porcine target due to negative charges repelled each other. The IDE device can detect the porcine presence in food as lowest as 1.0 µM. Better performance of the device can be achieved with the replacement of gold deposited to trigger more sensitivity of the device.

  19. Interdigitated electrode (IDE) for porcine detection based on titanium dioxide (TiO2) thin films

    Science.gov (United States)

    Nordin, N.; Hashim, U.; Azizah, N.

    2016-07-01

    Interdigited Electrode (IDE) porcine detection can be accomplished to authenticate the halal issue that has been a concern to Muslim not only in Malaysia but all around the world. The method used is photolithography that used the p-type photoresist on the spin coater with 2500 rpm. Bare IDEs device is deposited with Titanium Dioxide (TiO2) to improve the performance of the device. The result indicates that current-voltage (I-V) measurement of porcine probe line slightly above porcine target due to negative charges repelled each other. The IDE device can detect the porcine presence in food as lowest as 1.0 µM. Better performance of the device can be achieved with the replacement of gold deposited to trigger more sensitivity of the device.

  20. A new and efficient culture method for porcine bone marrow-derived M1- and M2-polarized macrophages.

    Science.gov (United States)

    Gao, Jiye; Scheenstra, Maaike R; van Dijk, Albert; Veldhuizen, Edwin J A; Haagsman, Henk P

    2018-06-01

    Macrophages play an important role in the innate immune system as part of the mononuclear phagocyte system (MPS). They have a pro-inflammatory signature (M1-polarized macrophages) or anti-inflammatory signature (M2-polarized macrophages) based on expression of surface receptors and secretion of cytokines. However, very little is known about the culture of macrophages from pigs and more specific about the M1 and M2 polarization in vitro. Porcine monocytes or mononuclear bone marrow cells were used to culture M1- and M2-polarized macrophages in the presence of GM-CSF and M-CSF, respectively. Surface receptor expression was measured with flow cytometry and ELISA was used to quantify cytokine secretion in response to LPS and PAM 3 CSK 4 stimulation. Human monocyte-derived macrophages were used as control. Porcine M1- and M2-polarized macrophages were cultured best using porcine GM-CSF and murine M-CSF, respectively. Cultures from bone marrow cells resulted in a higher yield M1- and M2-polarized macrophages which were better comparable to human monocyte-derived macrophages than cultures from porcine monocytes. Porcine M1-polarized macrophages displayed the characteristic fried egg shape morphology, lower CD163 expression and low IL-10 production. Porcine M2-polarized macrophages contained the spindle-like morphology, higher CD163 expression and high IL-10 production. Porcine M1- and M2-polarized macrophages can be most efficiently cultured from mononuclear bone marrow cells using porcine GM-CSF and murine M-CSF. The new culture method facilitates more refined studies of porcine macrophages in vitro, important for both porcine and human health since pigs are increasingly used as model for translational research. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Low impact of exposure to environmentally relevant doses of 226Ra in Atlantic cod (Gadus morhua) embryonic cells

    International Nuclear Information System (INIS)

    Olsvik, Pål A.; Berntssen, Marc H.G.; Hylland, Ketil; Eriksen, Dag Ø.; Holen, Elisabeth

    2012-01-01

    The aim of this study was to investigate whether 226 Ra, a radionuclide present in produced water from oil platforms in the North Sea and other offshore drilling areas, could affect vulnerable early life stages of Atlantic cod (Gadus morhua). Blastula-stage embryonic cells (EC) from fertilized eggs of Atlantic cod were isolated and exposed to environmental relevant concentrations of 226 Ra and transcription of selected genes quantified. The results showed a weak, but significant up-regulation of GPx3 and HSP70 transcripts after 48 h of exposure to 2.11 Bq/L. In EC exposed to three 226 Ra concentrations (2.11, 23 and 117 Bq/L) for 12 h, metallothionein, HSP90AA, thioredoxin and caspase 8 were significantly up-regulated in cells exposed to 117 Bq/L, whereas thioredoxin was also significantly up-regulated in EC exposed to 23 Bq/L. When EC were exposed to the same 226 Ra concentrations for 48 h, only heme oxygenase was significantly up-regulated in the 23 Bq/L exposure group. The results suggest that environmentally relevant activities of 226 Ra may induce oxidative stress and apoptosis in fish ECs. Exposure of Atlantic cod EC to Cd, selected as a model toxicant, supported the ability of EC around blastula stage to respond to toxicants by altered transcription. Due to dilution, environmentally relevant concentrations of radionuclides present in produced water would be expected to pose a minor threat to early life stages of fish. - Highlights: ► 226 Ra affects the transcription of genes in Atlantic cod embryonic cells. ► 226 Ra may induce oxidative stress and apoptosis in fish embryonic cells. ► 226 Ra not expected to pose a major threat to early life stages of marine fish.

  2. Embryonic development of human lice: rearing conditions and susceptibility to spinosad

    Directory of Open Access Journals (Sweden)

    Gastón Mougabure Cueto

    2006-05-01

    Full Text Available The embryonic development of human lice was evaluated according to the changes in the morphology of the embryo observed through the transparent chorion. Based on ocular and appendage development, three stages of embryogenesis were established: early, medium, and late. Influence of temperature and relative humidity (RH on the laboratory rearing of Pediculus humanus capitis eggs was assessed. The optimal ranges for temperature and RH were 27-31°C and 45-75%. The susceptibility of human louse eggs to insecticide spinosad (a macrocyclic lactone was assessed by immersion method. The results showed similar susceptibility to spinosad in early, medium, and late stages of head lice eggs. In addition, this study showed similar susceptibility of head and body lice eggs to spinosad, an insecticide that has not been used as pediculicide in Argentina (lethal concentration 50: 0.01%.

  3. Effect of zona pellucida on porcine parthenogenetically activated embryos

    DEFF Research Database (Denmark)

    Li, Rong; Liu, Ying; Li, Juan

    2011-01-01

    μg mL–1 cytochalasin B and 10 μg mL–1 cycloheximide in PZM-3 medium for 4 h. ZP was removed by 3.3 mg mL–1 pronase. Both zona-intact (PAZI) and zona-free (PAZF) embryos were cultured individually for 6 days either in time-lapse incubator (Embryoscope D, Unisense A/S, Aarhus, Denmark) for 15-min......). The timing of morulae was recorded when they completed compaction. Good blastocysts were defined when they expanded to 1.5 times larger than oocytes and formed regular blastocoel cavity with uniform colour and distribution of cells. Timing data were analysed by Student's t-test, while development rates....... 22, 234). In the present study, we expanded this study to include also the timing of early development and the resulting quality and robustness (for vitrification) of porcine PA embryos. Parthenogenetic activation was made first by an electric pulse (1.26 kV cm–1, 80 μs) and then by incubation with 5...

  4. Characterisation of the porcine eyeball as an in-vitro model for dry eye.

    Science.gov (United States)

    Menduni, Francesco; Davies, Leon N; Madrid-Costa, D; Fratini, Antonio; Wolffsohn, James S

    2018-02-01

    To characterise the anatomical parameters of the porcine eye for potentially using it as a laboratory model of dry eye. Anterior chamber depth and angle, corneal curvature, shortest and longest diameter, endothelial cell density, and pachymetry were measured in sixty freshly enucleated porcine eyeballs. Corneal steepest meridian was 7.85±0.32mm, corneal flattest meridian was 8.28±0.32mm, shortest corneal diameter was 12.69±0.58mm, longest corneal diameter was 14.88±0.66mm and central corneal ultrasonic pachymetry was 1009±1μm. Anterior chamber angle was 28.83±4.16°, anterior chamber depth was 1.77±0.27mm, and central corneal thickness measured using OCT was 1248±144μm. Corneal endothelial cell density was 3250±172 cells/mm 2 . Combining different clinical techniques produced a pool of reproducible data on the porcine eye anatomy, which can be used by researchers to assess the viability of using the porcine eye as an in-vitro/ex-vivo model for dry eye. Due to the similar morphology with the human eye, porcine eyeballs may represent a useful and cost effective model to individually study important key factors in the development of dry eye, such as environmental and mechanical stresses. Copyright © 2017 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

  5. Purification and characterization of glutaryl-CoA dehydrogenase from porcine and human liver

    International Nuclear Information System (INIS)

    Lenich, A.C.

    1985-01-01

    Glutaryl-CoA dehydrogenase (GCDH) was purified from porcine liver mitochondria by pH and ammonium sulfate fractionations followed by a series of column chromatographies. The purified porcine enzyme was found by sodium dodecyl-sulfate polyacrylamide gel electrophoresis to have a subunit molecular weight of 47,800 and by gradient polyacrylamide gel electrophoresis (PAGE) to have a native molecular weight of approximately 186,000. The product of the GCDH reaction with its primary substrate, glutaryl-CoA, was investigated by radio-gas chromatography and found to be crotonyl-CoA. Alternate substrates as well as crotonyl-CoA, the glutaryl-CoA reaction end product, demonstrated competitive inhibition when incubated with (1,5- 14 C)-glutaryl-CoA in the presence of porcine GCDH. Kinetic parameters for the interaction of both ETF and glutaryl-CoA with porcine GCDH were determined. Purified porcine GCDH was used to produce an antiserum which cross-reacted with human liver GCDH with a reaction of partial identity, but proved too insensitive to detect GCDH in control human fibroblasts. As a result of these negative findings, GCDH was purified by a series of column chromatographies from human liver. The purified human enzyme was found by SDS-PAGE and gel filtration to have subunit and native molecular weights of 58,800 and 256,000 respectively

  6. Embryonic, Larval, and Early Juvenile Development of the Tropical Sea Urchin, Salmacis sphaeroides (Echinodermata: Echinoidea

    Directory of Open Access Journals (Sweden)

    M. Aminur Rahman

    2012-01-01

    Full Text Available Salmacis sphaeroides (Linnaeus, 1758 is one of the regular echinoids, occuring in the warm Indo-West Pacific, including Johor Straits, between Malaysia and Singapore. In order to investigate the developmental basis of morphological changes in embryos and larvae, we documented the ontogeny of S. sphaeroides in laboratory condition. Gametes were obtained from adult individuals by 0.5 M KCl injection into the coelomic cavity. Fertilization rate at limited sperm concentration (10−5 dilution was 96.6±1.4% and the resulting embryos were reared at 24°C. First cleavage (2-cell, 4-cell, 8-cell, 16-cell, 32-cell, and multicell (Morulla stages were achieved 01.12, 02.03, 02.28, 02.51, 03.12, and 03.32 h postfertilization. Ciliated blastulae with a mean length of 174.72±4.43 μm hatched 08.45 h after sperm entry. The gastrulae formed 16.15 h postfertilization and the archenteron elongated constantly while ectodermal red-pigmented cells migrated synchronously to the apical plate. Pluteus larva started to feed unicellular algae in 2 d, grew continuously, and finally attained metamorphic competence in 35 d after fertilization. Metamorphosis took approximately 1 h 30 min from attachment to the complete resorption of larval tissues and the development of complete juvenile structure with adult spines, extended tubefeet and well-developed pedicellaria, the whole event of which usually took place within 1 d postsettlement. This study represents the first successful investigation on embryonic, larval, and early juvenile development of S. sphaeroides. The findings would greatly be helpful towards the understanding of ontogeny and life-history strategies, which will facilitate us to develop the breeding, seed production, and culture techniques of sea urchins in captive condition.

  7. Ultrasound Backscatter Microscopy Image-Guided Intraventricular Gene Delivery at Murine Embryonic Age 9.5 and 10.5 Produces Distinct Transgene Expression Patterns at the Adult Stage

    Directory of Open Access Journals (Sweden)

    Jiwon Jang

    2013-11-01

    Full Text Available In utero injection of a retroviral vector into the embryonic telencephalon aided by ultrasound backscatter microscopy permits introduction of a gene of interest at an early stage of development. In this study, we compared the tissue distribution of gene expression in adult mice injected with retroviral vectors at different embryonic ages in utero. Following ultrasound image-guided gene delivery (UIGD into the embryonic telencephalon, adult mice were subjected to whole-body luciferase imaging and immunohistochemical analysis at 6 weeks and 1 year postinjection. Luciferase activity was observed in a wide range of tissues in animals injected at embryonic age 9.5 (E9.5, whereas animals injected at E10.5 showed brain-localized reporter gene expression. These results suggest that mouse embryonic brain creates a closed and impermeable structure around E10. Therefore, by injecting a transgene before or after E10, transgene expression can be manipulated to be local or systemic. Our results also provide information that widens the applicability of UIGD beyond neuroscience studies.

  8. Cloning and expression of porcine SRPK1 gene

    African Journals Online (AJOL)

    Academic Journals

    2012-01-10

    Jan 10, 2012 ... different porcine tissue and skeletal muscle repair processes. ... Biology Engineering Technology Service Co., Ltd; while ethanol, agarose gel DNA ..... muscle fiber regeneration after bupivacaine hydrochloride-and acid.

  9. Inhibition of NF-κB promotes autophagy via JNK signaling pathway in porcine granulosa cells

    International Nuclear Information System (INIS)

    Gao, Hui; Lin, Lu; Haq, Ihtesham Ul; Zeng, Shen-ming

    2016-01-01

    The transcription factor nuclear factor-κB (NF-κB) plays an important role in diverse processes, including cell proliferation and differentiation, apoptosis and inflammation. However, the role of NF-κB in porcine follicle development is not clearly elucidated. In this study, we demonstrated that follicle stimulating hormone (FSH) increased the level of inhibitor of NF-κB (IκB) protein and promoted the cytoplasmic localization of p65, indicating that FSH inhibits the activation of NF-κB in porcine granulosa cells. Moreover, inhibition of NF-κB by FSH or another specific inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), could activate JNK signaling and enhance autophagic activity in porcine granulosa cells. Knockdown of RelA (p65) Subunit of NF-κB by RNA interference abrogated the activation of JNK signaling pathway and the increase of autophagic protein expression by FSH. Meanwhile, the functional significance of FSH or PDTC-mediated autophagy were further investigated. Our results demonstrated that the increased autophagy promoted progesterone secretion in porcine granulosa cells. Blockage of autophagy by chloroquine obviated the FSH or PDTC-induced progesterone production. Taken together, these results indicate that inhibition of NF-κB increased autophagy via JNK signaling, and promote steroidogenesis in porcine granulosa cells. Our results provide new insights into the regulation and function of autophagy in mammalian follicle development. - Highlights: • FSH inhibits the activation of NF-κB in porcine primary granulosa cells. • Inhibition of NF-κB by FSH promotes autophagy via JNK signaling in granulosa cells. • Increased autophagy contributes to progesterone production in granulosa cells. • This is the first report against beclin1 regulation in porcine granulosa cells.

  10. Inhibition of NF-κB promotes autophagy via JNK signaling pathway in porcine granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Hui; Lin, Lu; Haq, Ihtesham Ul; Zeng, Shen-ming, E-mail: zengshenming@gmail.com

    2016-04-22

    The transcription factor nuclear factor-κB (NF-κB) plays an important role in diverse processes, including cell proliferation and differentiation, apoptosis and inflammation. However, the role of NF-κB in porcine follicle development is not clearly elucidated. In this study, we demonstrated that follicle stimulating hormone (FSH) increased the level of inhibitor of NF-κB (IκB) protein and promoted the cytoplasmic localization of p65, indicating that FSH inhibits the activation of NF-κB in porcine granulosa cells. Moreover, inhibition of NF-κB by FSH or another specific inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), could activate JNK signaling and enhance autophagic activity in porcine granulosa cells. Knockdown of RelA (p65) Subunit of NF-κB by RNA interference abrogated the activation of JNK signaling pathway and the increase of autophagic protein expression by FSH. Meanwhile, the functional significance of FSH or PDTC-mediated autophagy were further investigated. Our results demonstrated that the increased autophagy promoted progesterone secretion in porcine granulosa cells. Blockage of autophagy by chloroquine obviated the FSH or PDTC-induced progesterone production. Taken together, these results indicate that inhibition of NF-κB increased autophagy via JNK signaling, and promote steroidogenesis in porcine granulosa cells. Our results provide new insights into the regulation and function of autophagy in mammalian follicle development. - Highlights: • FSH inhibits the activation of NF-κB in porcine primary granulosa cells. • Inhibition of NF-κB by FSH promotes autophagy via JNK signaling in granulosa cells. • Increased autophagy contributes to progesterone production in granulosa cells. • This is the first report against beclin1 regulation in porcine granulosa cells.

  11. Genetic Characterization of porcine circovirus type 2 isolated from different pig-farms in Croatia

    DEFF Research Database (Denmark)

    Rudan, Nevenka; Hjulsager, Charlotte Kristiane; Dupont, Kitt

    2009-01-01

    Histopathological fifi ndings in 25 pig tissue samples, which indicated PCVD (porcine circovirus diseases), were studied. Pig tissue samples originated from 5 different pig-farms in the north-west part of Croatia. Histopathological lesions showed two clinical pictures of the disease: porcine...

  12. The primary role of zebrafish nanog is in extra-embryonic tissue.

    Science.gov (United States)

    Gagnon, James A; Obbad, Kamal; Schier, Alexander F

    2018-01-09

    The role of the zebrafish transcription factor Nanog has been controversial. It has been suggested that Nanog is primarily required for the proper formation of the extra-embryonic yolk syncytial layer (YSL) and only indirectly regulates gene expression in embryonic cells. In an alternative scenario, Nanog has been proposed to directly regulate transcription in embryonic cells during zygotic genome activation. To clarify the roles of Nanog, we performed a detailed analysis of zebrafish nanog mutants. Whereas zygotic nanog mutants survive to adulthood, maternal-zygotic (MZ nanog ) and maternal mutants exhibit developmental arrest at the blastula stage. In the absence of Nanog, YSL formation and epiboly are abnormal, embryonic tissue detaches from the yolk, and the expression of dozens of YSL and embryonic genes is reduced. Epiboly defects can be rescued by generating chimeric embryos of MZ nanog embryonic tissue with wild-type vegetal tissue that includes the YSL and yolk cell. Notably, cells lacking Nanog readily respond to Nodal signals and when transplanted into wild-type hosts proliferate and contribute to embryonic tissues and adult organs from all germ layers. These results indicate that zebrafish Nanog is necessary for proper YSL development but is not directly required for embryonic cell differentiation. © 2018. Published by The Company of Biologists Ltd.

  13. Pathways in pluripotency and differentiation of embryonic cells

    NARCIS (Netherlands)

    du Puy, L.

    2010-01-01

    Pluripotency - the potential to differentiate into derivatives of the three embryonic germ layers endoderm, ectoderm and mesoderm - is the main characteristic of embryonic stem (ES) cells. ES cells are derived from the inner cell mass (ICM) of a pre-implantation blastocyst and can self-renew

  14. Adrenal hormones interact with sympathetic innervation to modulate growth of embryonic heart in oculo.

    Science.gov (United States)

    Tucker, D C; Torres, A

    1992-02-01

    To allow experimental manipulation of adrenal hormone and autonomic influences on developing myocardium without alteration of hemodynamic load, embryonic rat heart was cultured in the anterior eye chamber of an adult rat. Sympathetic innervation of embryonic day 12 heart grafts was manipulated by surgical sympathectomy of one eye chamber in each host rat. Adrenal hormone exposure was manipulated by host adrenal medullectomy (MEDX) in experiment 1 and by host adrenalectomy (ADX) in experiment 2. In experiment 1, whole heart grafts were larger in MEDX than in sham-operated hosts by 8 wk in oculo (6.14 +/- 0.71 vs. 5.09 +/- 0.69 mm2 with innervation intact and 7.97 +/- 2.07 vs. 3.09 +/- 0.63 mm2 with sympathetic innervation prevented). In experiment 2, host ADX increased growth of embryonic day 12 ventricles grafted into sympathectomized eye chambers (0.69 +/- 0.10 vs. 0.44 +/- 0.04 mm2) but did not affect growth of grafts in intact eye chambers (0.85 +/- 0.09 vs. 1.05 +/- 0.15 mm2). Corticosterone replacement (4 mg/day) entirely reversed the effect of host ADX on graft growth (superior cervical ganglionectomy, 0.47 +/- 0.03 mm2; intact eye chambers, 0.90 +/- 0.91 mm2). Beating rate of grafts was not affected by adrenal hormone manipulations. These experiments indicate that the compromised growth of embryonic heart grafts placed in sympathectomized eye chambers requires exposure to adult levels of glucocorticoids during the early days after grafting. These results suggest that interactions between neural and hormonal stimulation influence cardiac growth in the in oculo culture system and during normal development.

  15. Porcine head response to blast.

    Science.gov (United States)

    Shridharani, Jay K; Wood, Garrett W; Panzer, Matthew B; Capehart, Bruce P; Nyein, Michelle K; Radovitzky, Raul A; Bass, Cameron R 'dale'

    2012-01-01

    Recent studies have shown an increase in the frequency of traumatic brain injuries related to blast exposure. However, the mechanisms that cause blast neurotrauma are unknown. Blast neurotrauma research using computational models has been one method to elucidate that response of the brain in blast, and to identify possible mechanical correlates of injury. However, model validation against experimental data is required to ensure that the model output is representative of in vivo biomechanical response. This study exposes porcine subjects to primary blast overpressures generated using a compressed-gas shock tube. Shock tube blasts were directed to the unprotected head of each animal while the lungs and thorax were protected using ballistic protective vests similar to those employed in theater. The test conditions ranged from 110 to 740 kPa peak incident overpressure with scaled durations from 1.3 to 6.9 ms and correspond approximately with a 50% injury risk for brain bleeding and apnea in a ferret model scaled to porcine exposure. Instrumentation was placed on the porcine head to measure bulk acceleration, pressure at the surface of the head, and pressure inside the cranial cavity. Immediately after the blast, 5 of the 20 animals tested were apneic. Three subjects recovered without intervention within 30 s and the remaining two recovered within 8 min following respiratory assistance and administration of the respiratory stimulant doxapram. Gross examination of the brain revealed no indication of bleeding. Intracranial pressures ranged from 80 to 390 kPa as a result of the blast and were notably lower than the shock tube reflected pressures of 300-2830 kPa, indicating pressure attenuation by the skull up to a factor of 8.4. Peak head accelerations were measured from 385 to 3845 G's and were well correlated with peak incident overpressure (R(2) = 0.90). One SD corridors for the surface pressure, intracranial pressure (ICP), and head acceleration are

  16. Nuclear vlimata and aneuploidy in embryonic cells is caused by meiosis. Behaviour and properties of meiotic cells

    OpenAIRE

    Logothetou-Rella, H.

    1995-01-01

    This study demonstrates that human embryonic cells divide by meiosis. The use of trophoblastic tissue cells (early embryo) and amniotic cells (late embryo) exhibited the following characteristic events of meiosis: nuclear (NVs) and nucleolar (NuVs) vlimata formation; NV invasion in host cells; extrusion of chromosomes; nuclear fusion; metaphase fusion; hybrid cell formation; nuclear, nucleolar and cytoplasmic bridges, chromosomal transfer, variablesized nuc...

  17. Evaluation of biological effects of intermediate frequency magnetic field on differentiation of embryonic stem cell.

    Science.gov (United States)

    Yoshie, Sachiko; Ogasawara, Yuki; Ikehata, Masateru; Ishii, Kazuyuki; Suzuki, Yukihisa; Wada, Keiji; Wake, Kanako; Nakasono, Satoshi; Taki, Masao; Ohkubo, Chiyoji

    2016-01-01

    The embryotoxic effect of intermediate frequency (IF) magnetic field (MF) was evaluated using murine embryonic stem (ES) cells and fibroblast cells based on the embryonic stem cell test (EST). The cells were exposed to 21 kHz IF-MF up to magnetic flux density of 3.9 mT during the cell proliferation process (7 days) or the cell differentiation process (10 days) during which an embryonic body differentiated into myocardial cells. As a result, there was no significant difference in the cell proliferation between sham- and IF-MF-exposed cells for both ES and fibroblast cells. Similarly, the ratio of the number of ES-derived cell aggregates differentiated to myocardial cells to total number of cell aggregates was not changed by IF-MF exposure. In addition, the expressions of a cardiomyocytes-specific gene, Myl2 , and an early developmental gene, Hba-x , in the exposed cell aggregate were not altered. Since the magnetic flux density adopted in this study is much higher than that generated by an inverter of the electrical railway, an induction heating (IH) cooktop, etc . in our daily lives, these results suggested that IF-MF in which the public is exposed to in general living environment would not have embryotoxic effect.

  18. Inter-laboratory study to characterize the detection of serum antibodies against porcine epidemic diarrhoea virus.

    Science.gov (United States)

    Strandbygaard, Bertel; Lavazza, Antonio; Lelli, Davide; Blanchard, Yannick; Grasland, Béatrice; Poder, Sophie Le; Rose, Nicolas; Steinbach, Falko; van der Poel, Wim H M; Widén, Frederik; Belsham, Graham J; Bøtner, Anette

    2016-12-25

    Porcine epidemic diarrhea virus (PEDV) has caused extensive economic losses to pig producers in many countries. It was recently introduced, for the first time, into North America and outbreaks have occurred again in multiple countries within Europe as well. To assess the properties of various diagnostic assays for the detection of PEDV infection, multiple panels of porcine sera have been shared and tested for the presence of antibodies against PEDV in an inter-laboratory ring trial. Different laboratories have used a variety of "in house" ELISAs and also one commercial assay. The sensitivity and specificity of each assay has been estimated using a Bayesian analysis applied to the ring trial results obtained with the different assays in the absence of a gold standard. Although different characteristics were found, it can be concluded that each of the assays used can detect infection of pigs at a herd level by either the early European strains of PEDV or the recently circulating strains (INDEL and non-INDEL). However, not all the assays seem suitable for demonstrating freedom from disease in a country. The results from individual animals, especially when the infection has occurred within an experimental situation, show more variation. Copyright © 2016. Published by Elsevier B.V.

  19. Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay.

    Science.gov (United States)

    Pegels, N; González, I; Fernández, S; García, T; Martín, R

    2012-01-01

    A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79 bp fragment of the mitochondrial (mt) 12 S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18 S rRNA gene system, yielding a 77 bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.

  20. Ascorbic acid as a free radical scavenger in porcine and bovine aqueous humour.

    Science.gov (United States)

    Erb, Carl; Nau-Staudt, Kerstin; Flammer, Josef; Nau, Werner

    2004-01-01

    To study the antioxidant activity, UV absorption, concentration and stability of ascorbic acid (AA) in porcine and bovine aqueous humour (AH). Porcine and bovine AH was taken within 5 min after death and frozen at -70 degrees C. The characteristic UV absorption band of AA and the concentration of AA in AH was determined by UV spectrophotometry. The antioxidant activity of AA to serve as a free radical scavenger in AH has been determined by using a novel fluorescent probe for antioxidants, the azoalkane 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO). The fluorescence lifetime and intensity of this probe reflect the concentration of dissolved antioxidants. The time-resolved fluorescence of DBO (laser excitation at 351 nm) in AH and in a neutral phosphate-buffered saline (PBS) solution containing only the natural amount of AA as an additive were measured. The characteristic UV absorption band of AA has its maximum at 266 nm in AH. The concentration of AA in porcine and bovine AH was found to be 0.547 +/- 0.044 and 1.09 +/- 0.16 mM, respectively, by spectrophotometry. The fluorescence lifetime of the probe DBO was reduced from 320 +/- 5 ns in pure aerated PBS to 205 +/- 5 ns in porcine AH and 165 +/- 3 ns in bovine AH. A detailed kinetic analysis of the lifetime shortening suggests that AA contributes approximately 75 and 85% to the antioxidant activity of porcine and bovine AH, respectively. Our experiments suggest that AA is the major contributor to the antioxidant activity of porcine and bovine AH. The role of AA to serve as an antioxidant in AH is discussed. In addition, UV spectrophotometry is established as an alternative method to determine the concentration of AA in AH. Copyright 2004 S. Karger AG, Basel

  1. Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments.

    Science.gov (United States)

    Yamakoshi, Yasuo; Nagano, Takatoshi; Hu, Jan Cc; Yamakoshi, Fumiko; Simmer, James P

    2011-02-03

    Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB) and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG) attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr200, Thr216 and Thr

  2. Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments

    Directory of Open Access Journals (Sweden)

    Yamakoshi Fumiko

    2011-02-01

    Full Text Available Abstract Background Dentin sialophosphoprotein (Dspp is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp, the N-terminal domain of dentin sialophosphoprotein (Dspp, is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. Results To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were

  3. Notch signaling activation in human embryonic stem cells is required for embryonic but not trophoblastic lineage commitment

    OpenAIRE

    Yu, Xiaobing; Zou, Jizhong; Ye, Zhaohui; Hammond, Holly; Chen, Guibin; Tokunaga, Akinori; Mali, Prashant; Li, Yue-Ming; Civin, Curt; Gaiano, Nicholas; Cheng, Linzhao

    2008-01-01

    The Notch signaling pathway plays important roles in cell fate determination during embryonic development and adult life. In this study, we focus on the role of Notch signaling in governing cell fate choices in human embryonic stem (hES) cells. Using genetic and pharmacological approaches, we achieved both blockade and conditional activation of Notch signaling in several hES cell lines. We report here that activation of Notch signaling is required for undifferentiated hES cells to form the pr...

  4. Genomic and proteomic analyses of Prdm5 reveal interactions with insulator binding proteins in embryonic stem cells

    DEFF Research Database (Denmark)

    Galli, Giorgio Giacomo; Carrara, Matteo; Francavilla, Chiara

    2013-01-01

    PRDM proteins belong to the SET- domain protein family involved in the regulation of gene expression. Although few PRDM members possess histone methyltransferase activity, the molecular mechanisms by which the other members exert transcriptional regulation remain to be delineated. In this study, we...... find that Prdm5 is highly expressed in mouse embryonic stem cells (mES) and exploit this cellular system to characterize molecular functions of Prdm5. By combining proteomics and next generation sequencing technologies we identify Prdm5 interaction partners and genomic occupancy. We demonstrate that......, despite Prdm5 is dispensable for mES cell maintenance, it directly targets genomic regions involved in early embryonic development and affects the expression of a subset of developmental regulators during cell differentiation. Importantly, Prdm5 interacts with Ctcf, Cohesin and TFIIIC and co...

  5. Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells

    DEFF Research Database (Denmark)

    Melo-Braga, Marcella Nunes; Schulz, Melanie; Liu, Qiuyue

    2014-01-01

    Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important......ESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well...... in which 78% of phosphopeptides were identified with ≥99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development...

  6. GLI1 is involved in cell cycle regulation and proliferation of NT2 embryonal carcinoma stem cells

    DEFF Research Database (Denmark)

    Vestergaard, Janni; Lind-Thomsen, Allan; Pedersen, Mikkel W.

    2008-01-01

    of altered HH signaling are interpreted by specific cell types. We have investigated the role of the HH transcription factor glioma-associated oncogene homolog 1 (GLI1) in the human Ntera2=D1 (NT2) embryonal carcinoma stem cell line. The study revealed that expression of GLI1 and its direct transcriptional......1 phase cyclins. In conclusion, our results suggest that GLI1 is involved in cell cycle and proliferation control in the embryonal carcinoma stem cell line NT2....... target Patched (PTCH) is downregulated in the early stages of retinoic acid-induced neuronal differentiation of NT2 cells. To identify transcriptional targets of the HH transcription factor GLI1 in NT2 cells, we performed global expression profiling following GLI1 RNA interference (RNAi). Of the similar...

  7. First identification of porcine parvovirus 7 in China.

    Science.gov (United States)

    Xing, Xiulin; Zhou, Han; Tong, Ling; Chen, Yao; Sun, Yankuo; Wang, Heng; Zhang, Guihong

    2018-01-01

    Porcine parvovirus (PPV) are small, non-enveloped and single-stranded DNA viruses, taxonomically classifiable within the family Parvoviridae. Seven PPV genotypes (PPV1 to PPV7) have been identified to date. PPV7, the most recently discovered PPV genotype, was first reported in US pigs in 2016. To explore PPV7 status in Chinese pig populations a total of 64 serum samples collected from two commercial farms in Guangdong province in 2014 were analyzed. PPV7 DNA was detected in 32.8% (21/64) of tested samples. On the porcine circovirus type 2 (PCV2) positive farm, the prevalence rate of PPV7 was 65.5% (19/29) which was significantly higher than that on the PCV2 negative farm (2/35, 5.7%), indicating a possible association between PCV2 and PPV7 infections. The sequences of three PPV7 strains were determined. Phylogenetic analysis revealed that the identified PPV7 strains circulating in China shared 98.7%-99.7% nucleotide homology with the US strain. Further sequence comparison analysis indicated that GD-2014-2 and GD-2014-3 possess a consecutive 9-nt deletion in the VP gene. This is the first report of the existence of PPV7 in China and this finding will strengthen understanding of the epidemiology of porcine parvovirus in Chinese pigs.

  8. Perspectives on the Evolution of Porcine Parvovirus.

    Science.gov (United States)

    Oh, Woo-Taek; Kim, Ri-Yeon; Nguyen, Van-Giap; Chung, Hee-Chun; Park, Bong-Kyun

    2017-07-26

    Porcine parvovirus (PPV) is one of the main causes of porcine reproductive failure. It is important for swine industries to understand the recent trends in PPV evolution. Previous data show that PPV has two genetic lineages originating in Germany. In this study, two more genetic lineages were defined, one of which was distinctly Asian. Additionally, amino acid substitutions in European strains and Asian strains showed distinct differences in several regions of the VP2 gene. The VP1 gene of the recent PPV isolate (T142_South Korea) was identical to that of Kresse strain isolated in the USA in 1985, indicating that modern PPV strains now resemble the original strains (Kresse and NADL-2). In this study, we compared strains isolated in the 20th century to recent isolates and confirmed the trend that modern strains are becoming more similar to previous strains.

  9. Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

    Science.gov (United States)

    Inoue, Kimiko; Ogura, Atsuo

    2013-01-01

    The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (Pcloning efficiency using SCNT. PMID:24146866

  10. Preparation and Characterization of an Antibody Antagonist That Targets the Porcine Growth Hormone Receptor

    Directory of Open Access Journals (Sweden)

    Huanzhong Cui

    2016-10-01

    Full Text Available A series of antagonists specifically targeting growth hormone receptors (GHR in different species, such as humans, rats, bovines, and mice, have been designed; however, there are currently no antagonists that target the porcine growth hormone (GH. Therefore, in this study, we developed and characterized a porcine GHR (pGHR antibody antagonist (denoted by AN98 via the hybridoma technique. The results from enzyme-linked immunosorbent assay, fluorescence activated cell sorter, indirect immunoinfluscent assay, and competitive receptor binding analysis showed that AN98 could specifically recognize pGHR, and further experiments indicated that AN98 could effectively inhibit pGH-induced signalling in CHO-pGHR cells and porcine hepatocytes. In addition, AN98 also inhibited GH-induced insulin-like growth factor-1 (IGF-1 secretion in porcine hepatocytes. In summary, these findings indicated that AN98, as a pGHR-specific antagonist, has potential applications in pGH-pGHR-related research on domestic pigs.

  11. Immobilization of pseudorabies virus in porcine tracheal respiratory mucus revealed by single particle tracking.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Yang

    Full Text Available Pseudorabies virus (PRV initially replicates in the porcine upper respiratory tract. It easily invades the mucosae and submucosae for subsequent spread throughout the body via blood vessels and nervous system. In this context, PRV developed ingenious processes to overcome different barriers such as epithelial cells and the basement membrane. Another important but often overlooked barrier is the substantial mucus layer which coats the mucosae. However, little is known about how PRV particles interact with porcine respiratory mucus. We therefore measured the barrier properties of porcine tracheal respiratory mucus, and investigated the mobility of nanoparticles including PRV in this mucus. We developed an in vitro model utilizing single particle tracking microscopy. Firstly, the mucus pore size was evaluated with polyethylene glycol coupled (PEGylated nanoparticles and atomic force microscope. Secondly, the mobility of PRV in porcine tracheal respiratory mucus was examined and compared with that of negative, positive and PEGylated nanoparticles. The pore size of porcine tracheal respiratory mucus ranged from 80 to 1500 nm, with an average diameter of 455±240 nm. PRV (zeta potential: -31.8±1.5 mV experienced a severe obstruction in porcine tracheal respiratory mucus, diffusing 59-fold more slowly than in water. Similarly, the highly negatively (-49.8±0.6 mV and positively (36.7±1.1 mV charged nanoparticles were significantly trapped. In contrast, the nearly neutral, hydrophilic PEGylated nanoparticles (-9.6±0.8 mV diffused rapidly, with the majority of particles moving 50-fold faster than PRV. The mobility of the particles measured was found to be related but not correlated to their surface charge. Furthermore, PEGylated PRV (-13.8±0.9 mV was observed to diffuse 13-fold faster than native PRV. These findings clearly show that the mobility of PRV was significantly hindered in porcine tracheal respiratory mucus, and that the obstruction of PRV

  12. Left-Right Asymmetry of Maturation Rates in Human Embryonic Neural Development.

    Science.gov (United States)

    de Kovel, Carolien G F; Lisgo, Steven; Karlebach, Guy; Ju, Jia; Cheng, Gang; Fisher, Simon E; Francks, Clyde

    2017-08-01

    Left-right asymmetry is a fundamental organizing feature of the human brain, and neuropsychiatric disorders such as schizophrenia sometimes involve alterations of brain asymmetry. As early as 8 weeks postconception, the majority of human fetuses move their right arms more than their left arms, but because nerve fiber tracts are still descending from the forebrain at this stage, spinal-muscular asymmetries are likely to play an important developmental role. We used RNA sequencing to measure gene expression levels in the left and right spinal cords, and the left and right hindbrains, of 18 postmortem human embryos aged 4 to 8 weeks postconception. Genes showing embryonic lateralization were tested for an enrichment of signals in genome-wide association data for schizophrenia. The left side of the embryonic spinal cord was found to mature faster than the right side. Both sides transitioned from transcriptional profiles associated with cell division and proliferation at earlier stages to neuronal differentiation and function at later stages, but the two sides were not in synchrony (p = 2.2 E-161). The hindbrain showed a left-right mirrored pattern compared with the spinal cord, consistent with the well-known crossing over of function between these two structures. Genes that showed lateralization in the embryonic spinal cord were enriched for association signals with schizophrenia (p = 4.3 E-05). These are the earliest stage left-right differences of human neural development ever reported. Disruption of the lateralized developmental program may play a role in the genetic susceptibility to schizophrenia. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  13. Stoichiometric iodination and purification of porcine insulin with chloramine T for radioimmunoassay

    International Nuclear Information System (INIS)

    Toledo e Souza, I.T. de; Giannella Neto, D.; Wajchenberg, B.L.

    1986-01-01

    Stoichiometric iodination and purification of porcine insulin was performed to the general method of Hunter and Greenwood (classical chloramine T) with modifications recommended by Roth (chloramine T is added in limiting amounts in multiple small additions). Satisfactory specific activity of the labeled hormone was obtained and the characteristics of the radioimmunoassay, based on the competition of the 125-I labeled porcine and cold insulin for specific antibody were studied. (Author) [pt

  14. Embryonic Methamphetamine Exposure Inhibits Methamphetamine Cue Conditioning and Reduces Dopamine Concentrations in Adult N2 Caenorhabditis elegans.

    Science.gov (United States)

    Katner, Simon N; Neal-Beliveau, Bethany S; Engleman, Eric A

    2016-01-01

    Methamphetamine (MAP) addiction is substantially prevalent in today's society, resulting in thousands of deaths and costing billions of dollars annually. Despite the potential deleterious consequences, few studies have examined the long-term effects of embryonic MAP exposure. Using the invertebrate nematode Caenorhabditis elegans allows for a controlled analysis of behavioral and neurochemical changes due to early developmental drug exposure. The objective of the current study was to determine the long-term behavioral and neurochemical effects of embryonic exposure to MAP in C. elegans. In addition, we sought to improve our conditioning and testing procedures by utilizing liquid filtration, as opposed to agar, and smaller, 6-well testing plates to increase throughput. Wild-type N2 C. elegans were embryonically exposed to 50 μM MAP. Using classical conditioning, adult-stage C. elegans were conditioned to MAP (17 and 500 μM) in the presence of either sodium ions (Na+) or chloride ions (Cl-) as conditioned stimuli (CS+/CS-). Following conditioning, a preference test was performed by placing worms in 6-well test plates spotted with the CS+ and CS- at opposite ends of each well. A preference index was determined by counting the number of worms in the CS+ target zone divided by the total number of worms in the CS+ and CS- target zones. A food conditioning experiment was also performed in order to determine whether embryonic MAP exposure affected food conditioning behavior. For the neurochemical experiments, adult worms that were embryonically exposed to MAP were analyzed for dopamine (DA) content using high-performance liquid chromatography. The liquid filtration conditioning procedure employed here in combination with the use of 6-well test plates significantly decreased the time required to perform these experiments and ultimately increased throughput. The MAP conditioning data found that pairing an ion with MAP at 17 or 500 μM significantly increased the preference

  15. Enhanced synaptic activity and epileptiform events in the embryonic Kcc2 deficient hippocampus

    Directory of Open Access Journals (Sweden)

    Ilgam eKhalilov

    2011-11-01

    Full Text Available The neuronal potassium-chloride co-transporter Kcc2 is thought to play an important role in the post natal excitatory to inhibitory switch of GABA actions in the rodent hippocampus. Here, by studying hippocampi of wild-type (Kcc2+/+ and Kcc2 deficient (Kcc2-/- mouse embryos, we unexpectedly found increased spontaneous neuronal network activity at E18.5, a developmental stage when Kcc2 is thought not to be functional in the hippocampus. Embryonic Kcc2-/- hippocampi have also an augmented synapse density and a higher frequency of spontaneous glutamatergic and GABAergic postsynaptic currents (PSCs than naïve age matched neurons. However, intracellular chloride concentration ([Cl-]i and the reversal potential of GABA-mediated currents (EGABA were similar in embryonic Kcc2+/+ and Kcc2-/- CA3 neurons. In addition, Kcc2 immuno-labelling was cytoplasmic in the majority of neurons suggesting that the molecule is not functional as a plasma membrane chloride co-transporter. Collectively, our results show that already at an embryonic stage, Kcc2 controls the formation of synapses and, when deleted, the hippocampus has a higher density of GABAergic and glutamatergic synapses and generates spontaneous and evoked epileptiform activities. These results may be explained either by a small population of orchestrating neurons in which Kcc2 operates early as a chloride exporter or by transporter independent actions of Kcc2 that are instrumental in synapses formation and networks construction.

  16. Mechanics of fresh, frozen-thawed and heated porcine liver tissue.

    Science.gov (United States)

    Wex, Cora; Stoll, Anke; Fröhlich, Marlen; Arndt, Susann; Lippert, Hans

    2014-06-01

    For a better understanding of the effects of thermally altered soft tissue, the biothermomechanics of these tissues need to be studied. Without the knowledge of the underlying physical processes and the parameters that can be controlled clinically, thermal treatment of cancerous hepatic tissue or the preservation of liver grafts are based primarily on trial and error. Thus, this study is concerned with the investigation of the influence of temperature on the rheological properties and the histological properties of porcine liver. Heating previously cooled porcine liver tissue above 40 °C leads to significant, irreversible stiffness changes observed in the amplitude sweep. The increase of the complex shear module of healthy porcine liver from room temperature to 70 °C is approximately 9-fold. Comparing the temperatures -20 °C and 20 °C, no significant difference of the mechanical properties was observed. Furthermore, there is a strong relation between the mechanical and histological properties of the porcine liver. Temperatures above 40 °C destroy the collagen matrix within the liver tissue. This results in the alteration of the biomechanical properties. The time-temperature superposition principle is applied to generate temperature-dependent shift factors that can be described by a two-part exponential function model with an inflection temperature of 45 °C. Tumor ablation techniques such as heating or freezing have a significant influence on the histology of liver tissue. However, only for temperatures above body temperature an influence on the mechanical properties of hepatic tissues was noticeable. Freezing up to -20 °C did not affect the liver mechanics.

  17. Left ventricular mass regression after porcine versus bovine aortic valve replacement: a randomized comparison.

    Science.gov (United States)

    Suri, Rakesh M; Zehr, Kenton J; Sundt, Thoralf M; Dearani, Joseph A; Daly, Richard C; Oh, Jae K; Schaff, Hartzell V

    2009-10-01

    It is unclear whether small differences in transprosthetic gradient between porcine and bovine biologic aortic valves translate into improved regression of left ventricular (LV) hypertrophy after aortic valve replacement. We investigated transprosthetic gradient, aortic valve orifice area, and LV mass in patients randomized to aortic valve replacement with either the Medtronic Mosaic (MM) porcine or an Edwards Perimount (EP) bovine pericardial bioprosthesis. One hundred fifty-two patients with aortic valve disease were randomly assigned to receive either the MM (n = 76) or an EP prosthesis. There were 89 men (59%), and the mean age was 76 years. Echocardiograms from preoperative, postoperative, predismissal, and 1-year time points were analyzed. Baseline characteristics and preoperative echocardiograms were similar between the two groups. The median implant size was 23 mm for both. There were no early deaths, and 10 patients (7%) died after dismissal. One hundred seven of 137 patients (78%) had a 1-year echocardiogram, and none required aortic valve reoperation. The mean aortic valve gradient at dismissal was 19.4 mm Hg (MM) versus13.5 mm Hg (EP; p regression of LV mass index (MM, -32.4 g/m(2) versus EP, -27.0 g/m(2); p = 0.40). Greater preoperative LV mass index was the sole independent predictor of greater LV mass regression after surgery (p regression of LV mass during the first year after aortic valve replacement.

  18. Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

    Science.gov (United States)

    Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik

    2012-01-01

    Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

  19. Characterization of porcine MMP-2 and its association with immune traits

    DEFF Research Database (Denmark)

    Huang, Honggang; Zhao, Weimin; Tang, Zhonglin

    2009-01-01

    cloned the 5'-upstream sequence, 3'-downstream sequence as well as other missed genomic sequences of porcine MMP-2, the genomic structure and the promotor sequence were analyzed and found to share high similarity with those of human MMP-2. Porcine MMP-2 was assigned to SSC6p14-p15, and closely linked......Matrix metalloproteinase-2 (MMP-2) plays important roles in inflammation and immunity besides its basic role in degrading and remodelling extracellular matrix (ECM). The expression of MMP-2 is up-regulated in many human as well as animal models of inflammatory and immune diseases. In this study, we...... to microsatellite SW1108 (53cR, LOD score 7.59) by IMpRH panel. Real-time PCR analysis revealed that the expression of porcine MMP-2 was remarkably different in diverse tissues, a high level expression was observed in the testis and uterus, relatively low expression in other tissues. Allele frequencies...

  20. An in-depth comparison of the porcine, murine and human inflammasomes; lessons from the porcine genome and transcriptome.

    Science.gov (United States)

    Dawson, Harry D; Smith, Allen D; Chen, Celine; Urban, Joseph F

    2017-04-01

    Emerging evidence suggests that swine are a scientifically acceptable intermediate species between rodents and humans to model immune function relevant to humans. The swine genome has recently been sequenced and several preliminary structural and functional analysis of the porcine immunome have been published. Herein we provide an expanded in silico analysis using an improved assembly of the porcine transcriptome that provides an in depth analysis of genes that are related to inflammasomes, responses to Toll-like receptor ligands, and M1 macrophage polarization and Escherichia coli as a model organism. Comparisons of the expansion or contraction of orthologous gene families indicated more similar rates and classes of genes in humans and pigs than in mice; however several novel porcine or artiodactyl-specific paralogs or pseudogenes were identified. Conservation of homology and structural motifs of orthologs revealed that the overall similarity to human proteins was significantly higher for pigs compared to mouse. Despite these similarities, two out of four canonical inflammasome pathways, Absent in melanoma 2 (AIM2) and NLR family and CARD domain containing 4 (NLRC4), were found to be missing in pigs. Pig M1 Mφ polarization in response to interferon-γ (IFN-γ) and lipopolysaccharide (LPS) was assessed, via the transcriptome, using next generation sequencing. Our analysis revealed predominantly human-like responses however some, mouse-like responses were observed, as well as induction of numerous pig or artiodactyl-specific genes. This work supports using swine to model both human immunological and inflammatory responses to infection. However, caution must be exercised as pigs differ from humans in several fundamental pathways. Published by Elsevier B.V.

  1. A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Mummery, C.L.; Krijgsveld, J.; Heck, A.

    2008-01-01

    The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological

  2. Childhood Central Nervous System Embryonal Tumors (PDQ®)—Health Professional Version

    Science.gov (United States)

    Pediatric CNS embryonal tumors are a collection of heterogeneous lesions (medulloblastoma, and nonmedulloblastoma). Molecular genetic studies are used to classify embryonal tumors, stratify risk, and plan treatment. Get detailed information about tumor biology, diagnosis, prognosis, and treatment of untreated and recurrent CNS embryonal tumors in this summary for clinicians.

  3. Porcine Is a Positional Candidate Gene Associated with Growth and Fat Deposition

    Directory of Open Access Journals (Sweden)

    Bong Hwan Choi

    2012-12-01

    Full Text Available Crosses between Korean and Landrace pigs have revealed a large quantitative trait loci (QTL region for fat deposition in a region (89 cM of porcine chromosome 4 (SSC4. To more finely map this QTL region and identify candidate genes for this trait, comparative mapping of pig and human chromosomes was performed in the present study. A region in the human genome that corresponds to the porcine QTL region was identified in HSA1q21. Furthermore, the LMNA gene, which is tightly associated with fat augmentation in humans, was localized to this region. Radiation hybrid (RH mapping using a Sus scrofa RH panel localized LMNA to a region of 90.3 cM in the porcine genome, distinct from microsatellite marker S0214 (87.3 cM. Two-point analysis showed that LMNA was linked to S0214, SW1996, and S0073 on SSC4 with logarithm (base 10 of odds scores of 20.98, 17.78, and 16.73, respectively. To clone the porcine LMNA gene and to delineate the genomic structure and sequences, including the 3′untranslated region (UTR, rapid amplification of cDNA ends was performed. The coding sequence of porcine LMNA consisted of 1,719 bp, flanked by a 5’UTR and a 3’UTR. Two synonymous single nucleotide polymorphisms (SNPs were identified in exons 3 and 7. Association tests showed that the SNP located in exon 3 (A193A was significantly associated with weight at 30 wks (p<0.01 and crude fat content (p<0.05. This association suggests that SNPs located in LMNA could be used for marker-assisted selection in pigs.

  4. Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Imai

    Full Text Available Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.

  5. Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers.

    Science.gov (United States)

    Imai, Hiroyuki; Kano, Kiyoshi; Fujii, Wataru; Takasawa, Ken; Wakitani, Shoichi; Hiyama, Masato; Nishino, Koichiro; Kusakabe, Ken Takeshi; Kiso, Yasuo

    2015-01-01

    Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.

  6. Heterochromatin position effects on circularized sex chromosomes cause filicidal embryonic lethality in Drosophila melanogaster.

    Science.gov (United States)

    Ferree, Patrick M; Gomez, Karina; Rominger, Peter; Howard, Dagnie; Kornfeld, Hannah; Barbash, Daniel A

    2014-04-01

    Some circularized X-Y chromosomes in Drosophila melanogaster are mitotically unstable and induce early embryonic lethality, but the genetic basis is unknown. Our experiments suggest that a large region of X-linked satellite DNA causes anaphase bridges and lethality when placed into a new heterochromatic environment within certain circularized X-Y chromosomes. These results reveal that repetitive sequences can be incompatible with one another in cis. The lethal phenotype also bears a remarkable resemblance to a case of interspecific hybrid lethality.

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    Lifescience Database Archive (English)

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  11. Porcine prion protein amyloid.

    Science.gov (United States)

    Hammarström, Per; Nyström, Sofie

    2015-01-01

    Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat, elk, deer) and largely prion resistant species (pig) or susceptible and resistant pets (cat and dogs, respectively). PrPs from these species have been characterized using biochemistry, biophysics and neurobiology. Recently we studied PrPs from several mammals in vitro and found evidence for generic amyloidogenicity as well as cross-seeding fibril formation activity of all PrPs on the human PrP sequence regardless if the original species was resistant or susceptible to prion disease. Porcine PrP amyloidogenicity was among the studied. Experimentally inoculated pigs as well as transgenic mouse lines overexpressing porcine PrP have, in the past, been used to investigate the possibility of prion transmission in pigs. The pig is a species with extraordinarily wide use within human daily life with over a billion pigs harvested for human consumption each year. Here we discuss the possibility that the largely prion disease resistant pig can be a clinically silent carrier of replicating prions.

  12. Reproductive effects of two neonicotinoid insecticides on mouse sperm function and early embryonic development in vitro.

    Directory of Open Access Journals (Sweden)

    Yi-Hua Gu

    Full Text Available Acetamiprid (ACE and imidacloprid (IMI are two major members in the family of neonicotinoid pesticides, which are synthesized with a higher selectivity to insects. The present study determined and compared in vitro effects of ACE, IMI and nicotine on mammalian reproduction by using an integrated testing strategy for reproductive toxicology, which covered sperm quality, sperm penetration into oocytes and preimplantation embryonic development. Direct chemical exposure (500 µM or 5 mM on spermatozoa during capacitation was performed, and in vitro fertilization (IVF process, zygotes and 2-cell embryos were respectively incubated with chemical-supplemented medium until blastocyst formation to evaluate the reproductive toxicity of these chemicals and monitor the stages mainly affected. Generally, treatment of 500 µM or 5 mM chemicals for 30 min did not change sperm motility and DNA integrity significantly but the fertilization ability in in vitro fertilization (IVF process, indicating that IVF process could detect and distinguish subtle effect of spermatozoa exposed to different chemicals. Culture experiment in the presence of chemicals in medium showed that fertilization process and zygotes are adversely affected by direct exposure of chemicals (PIMI>ACE, whereas developmental progression of 2-cell stage embryos was similar to controls (P>0.05. These findings unveiled the hazardous effects of neonicotinoid pesticides exposure on mammalian sperm fertilization ability as well as embryonic development, raising the concerns that neonicotinoid pesticides may pose reproductive risks on human reproductive health, especially in professional populations.

  13. miR-26b promotes granulosa cell apoptosis by targeting ATM during follicular atresia in porcine ovary.

    Directory of Open Access Journals (Sweden)

    Fei Lin

    Full Text Available More than 99% of ovarian follicles undergo atresia in mammals, but the mechanism of follicular atresia remains to be elucidated. In this study, we explored microRNA (miRNA regulation of follicular atresia in porcine ovary. A miRNA expression profile was constructed for healthy, early atretic, and progressively atretic follicles, and the differentially expressed miRNAs were selected and analyzed. We found that miR-26b, which was upregulated during follicular atresia, increased the number of DNA breaks and promoted granulosa cell apoptosis by targeting the ataxia telangiectasia mutated gene directly in vitro.

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  16. In vivo survival of [14C]sucrose-loaded porcine carrier erythrocytes

    International Nuclear Information System (INIS)

    DeLoach, J.R.

    1983-01-01

    Porcine carrier erythrocyte survival was measured in adult pigs. [14C]Sucrose-loaded erythrocytes had a biphasic survival curve, with as much as 50% of the cells removed from circulation in the first 24 hours. The remaining cells had a 35-day half-life. Encapsulation values were measured for porcine erythrocytes and entrapment of [14C]sucrose was greater than 45%. Addition of inosine and glucose to the dialyzed cells and to the final wash buffer before reinjection of autologous cells did not improve their survival

  17. Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains

    Directory of Open Access Journals (Sweden)

    K.C. Médici

    2010-01-01

    Full Text Available Group B rotaviruses (RV-B were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8 were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7% nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8% identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8%. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.

  18. File list: Oth.Emb.10.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.10.AllAg.Embryonic_palates mm9 TFs and others Embryo Embryonic palates http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.10.AllAg.Embryonic_palates.bed ...

  19. File list: Oth.Emb.20.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.20.AllAg.Embryonic_palates mm9 TFs and others Embryo Embryonic palates http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.20.AllAg.Embryonic_palates.bed ...

  20. File list: Oth.Emb.50.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.50.AllAg.Embryonic_palates mm9 TFs and others Embryo Embryonic palates http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.50.AllAg.Embryonic_palates.bed ...

  1. File list: Oth.Emb.05.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.05.AllAg.Embryonic_palates mm9 TFs and others Embryo Embryonic palates http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.05.AllAg.Embryonic_palates.bed ...

  2. File list: Pol.Emb.20.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.20.AllAg.Embryonic_pancreas mm9 RNA polymerase Embryo Embryonic pancreas ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.20.AllAg.Embryonic_pancreas.bed ...

  3. File list: Pol.Emb.10.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.10.AllAg.Embryonic_pancreas mm9 RNA polymerase Embryo Embryonic pancreas ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.10.AllAg.Embryonic_pancreas.bed ...

  4. File list: Pol.Emb.05.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.05.AllAg.Embryonic_pancreas mm9 RNA polymerase Embryo Embryonic pancreas ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.05.AllAg.Embryonic_pancreas.bed ...

  5. Optimal doses of EGF and GDNF act as biological response modifiers to improve porcine oocyte maturation and quality

    DEFF Research Database (Denmark)

    Valleh, Mehdi Vafaye; Zandi, Nahid Karimi; Rasmussen, Mikkel Aabech

    2017-01-01

    It is well documented that both epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) are critical for porcine oocyte maturation, however, little information is known about their mechanism of action in vitro. To gain insight into the mechanisms of action of the opti......It is well documented that both epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) are critical for porcine oocyte maturation, however, little information is known about their mechanism of action in vitro. To gain insight into the mechanisms of action...... of the optimum doses of EGF and GDNF on porcine oocyte maturation, porcine cumulus-oocyte complexes (COCs) were matured in defined porcine oocyte medium supplemented with EGF, GDNF or a combination of both at varying concentrations (0-100 ng/ml) for 44 h. Nuclear and cytoplasmic maturation were determined...

  6. Simulated Microgravity Modulates Differentiation Processes of Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Vaibhav Shinde

    2016-04-01

    Full Text Available Background/Aims: Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of altered gravity on the embryonic development processes we established an in vitro methodology allowing differentiation of mouse embryonic stem cells (mESCs under simulated microgravity within a fast-rotating clinostat (clinorotation and capture of microarray-based gene signatures. Methods: The differentiating mESCs were cultured in a 2D pipette clinostat. The microarray and bioinformatics tools were used to capture genes that are deregulated by simulated microgravity and their impact on developmental biological processes. Results: The data analysis demonstrated that differentiation of mESCs in pipettes for 3 days resultet to early germ layer differentiation and then to the different somatic cell types after further 7 days of differentiation in the Petri dishes. Clinorotation influences differentiation as well as non-differentiation related biological processes like cytoskeleton related 19 genes were modulated. Notably, simulated microgravity deregulated genes Cyr61, Thbs1, Parva, Dhrs3, Jun, Tpm1, Fzd2 and Dll1 are involved in heart morphogenesis as an acute response on day 3. If the stem cells were further cultivated under normal gravity conditions (1 g after clinorotation, the expression of cardiomyocytes specific genes such as Tnnt2, Rbp4, Tnni1, Csrp3, Nppb and Mybpc3 on day 10 was inhibited. This correlated well with a decreasing beating activity of the 10-days old embryoid bodies (EBs. Finally, we captured Gadd45g, Jun, Thbs1, Cyr61and Dll1 genes whose expressions were modulated by simulated microgravity and by real microgravity in various reported studies. Simulated microgravity also deregulated genes belonging to the MAP kinase and focal dhesion signal transduction pathways. Conclusion: One of the most prominent biological processes affected by simulated microgravity was the process of cardiomyogenesis. The

  7. Storage of Hatching Eggs : Effects of storage and early incubation conditions on egg characteristics, embryonic development, hatchability, and chicken quality

    NARCIS (Netherlands)

    Reijrink, I.A.M.

    2010-01-01

    Key words: egg storage, embryonic development, albumen quality, hatchability, chick quality

    It is well known that an increase in the storage duration increases incubation duration and decreases hatchability and chick quality. The negative effects of prolonged egg storage (> 7 days)

  8. Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization

    OpenAIRE

    Jin, Muzi; Wu, Asga; Dorzhin, Sergei; Yue, Qunhua; Ma, Yuzhen; Liu, Dongjun

    2012-01-01

    Although isolation and characterization of embryonic stem cells have been successful in cattle, maintenance of bovine embryonic stem cells in culture remains difficult. In this study, we compared different methods of cell passaging, feeder cell layers and medium conditions for bovine embryonic stem cell-like cells. We found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells than bovine embryonic fibroblasts. When murine embryonic fibroblasts we...

  9. File list: DNS.Emb.20.AllAg.Embryonic_testis [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Emb.20.AllAg.Embryonic_testis mm9 DNase-seq Embryo Embryonic testis SRX1156635 ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Emb.20.AllAg.Embryonic_testis.bed ...

  10. Interdigitated electrode (IDE) for porcine detection based on titanium dioxide (TiO{sub 2}) thin films

    Energy Technology Data Exchange (ETDEWEB)

    Nordin, N.; Azizah, N. [Institute of Electronic Engineering (INEE), Universiti Malaysia Perlis, 01000 Kangar Perlis (Malaysia); Hashim, U., E-mail: uda@unimap.edu.my [Institute of Electronic Engineering (INEE), Universiti Malaysia Perlis, 01000 Kangar Perlis (Malaysia); School of Microelctronic Engineering, Universiti Malaysia Perlis, 01000 Kangar Perlis (Malaysia)

    2016-07-06

    Interdigited Electrode (IDE) porcine detection can be accomplished to authenticate the halal issue that has been a concern to Muslim not only in Malaysia but all around the world. The method used is photolithography that used the p-type photoresist on the spin coater with 2500 rpm. Bare IDEs device is deposited with Titanium Dioxide (TiO{sub 2}) to improve the performance of the device. The result indicates that current-voltage (I-V) measurement of porcine probe line slightly above porcine target due to negative charges repelled each other. The IDE device can detect the porcine presence in food as lowest as 1.0 µM. Better performance of the device can be achieved with the replacement of gold deposited to trigger more sensitivity of the device.

  11. Porcine semen as a vector for transmission of viral pathogens.

    Science.gov (United States)

    Maes, Dominiek; Van Soom, Ann; Appeltant, Ruth; Arsenakis, Ioannis; Nauwynck, Hans

    2016-01-01

    Different viruses have been detected in porcine semen. Some of them are on the list of the World Organization for Animal Health (OIE), and consequently, these pathogens are of socioeconomic and/or public health importance and are of major importance in the international trade of animals and animal products. Artificial insemination (AI) is one of the most commonly used assisted reproductive technologies in pig production worldwide. This extensive use has enabled pig producers to benefit from superior genetics at a lower cost compared to natural breeding. However, the broad distribution of processed semen doses for field AI has increased the risk of widespread transmission of swine viral pathogens. Contamination of semen can be due to infections of the boar or can occur during semen collection, processing, and storage. It can result in reduced semen quality, embryonic mortality, endometritis, and systemic infection and/or disease in the recipient female. The presence of viral pathogens in semen can be assessed by demonstration of viable virus, nucleic acid of virus, or indirectly by measuring serum antibodies in the boar. The best way to prevent disease transmission via the semen is to assure that the boars in AI centers are free from the disease, to enforce very strict biosecurity protocols, and to perform routine health monitoring of boars. Prevention of viral semen contamination should be the primary focus because it is easier to prevent contamination than to eliminate viruses once present in semen. Nevertheless, research and development of novel semen processing treatments such as single-layer centrifugation is ongoing and may allow in the future to decontaminate semen. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. File list: ALL.Emb.05.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.05.AllAg.Embryonic_palates mm9 All antigens Embryo Embryonic palates ERX650...310 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.05.AllAg.Embryonic_palates.bed ...

  13. File list: ALL.Emb.20.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.20.AllAg.Embryonic_palates mm9 All antigens Embryo Embryonic palates ERX650...310 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.20.AllAg.Embryonic_palates.bed ...

  14. File list: ALL.Emb.50.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.50.AllAg.Embryonic_palates mm9 All antigens Embryo Embryonic palates ERX650...310 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.50.AllAg.Embryonic_palates.bed ...

  15. File list: Pol.Emb.20.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.20.AllAg.Embryonic_heart mm9 RNA polymerase Embryo Embryonic heart SRX11293...9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.20.AllAg.Embryonic_heart.bed ...

  16. File list: Pol.Emb.10.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.10.AllAg.Embryonic_heart mm9 RNA polymerase Embryo Embryonic heart SRX11293...9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.10.AllAg.Embryonic_heart.bed ...

  17. File list: Pol.Emb.05.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.05.AllAg.Embryonic_heart mm9 RNA polymerase Embryo Embryonic heart SRX11293...9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.05.AllAg.Embryonic_heart.bed ...

  18. File list: Pol.Emb.50.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.50.AllAg.Embryonic_heart mm9 RNA polymerase Embryo Embryonic heart SRX11293...9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.50.AllAg.Embryonic_heart.bed ...

  19. File list: DNS.Emb.20.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Emb.20.AllAg.Embryonic_trunk mm9 DNase-seq Embryo Embryonic trunk SRX191030 htt...p://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Emb.20.AllAg.Embryonic_trunk.bed ...

  20. Mapping the stem cell state: eight novel human embryonic stem and embryonal carcinoma cell antibodies

    DEFF Research Database (Denmark)

    Wright, A; Andrews, N; Bardsley, K

    2011-01-01

    The antigenic profile of human embryonic stem (ES) and embryonal carcinoma (EC) cells has served as a key element of their characterization, with a common panel of surface and intracellular markers now widely used. Such markers have been used to identify cells within the 'undifferentiated state...... of reactivity for all antibodies against both ES and EC cells, suggesting that these markers will afford recognition of unique sub-states within the undifferentiated stem cell compartment....... and EC cells, and herein describe their characterization. The reactivity of these antibodies against a range of cell lines is reported, as well as their developmental regulation, basic biochemistry and reactivity in immunohistochemistry of testicular germ cell tumours. Our data reveal a range...

  1. File list: His.Emb.10.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.10.AllAg.Embryonic_trunk mm9 Histone Embryo Embryonic trunk SRX093317,SRX09...3316 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.10.AllAg.Embryonic_trunk.bed ...

  2. File list: His.Emb.50.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.50.AllAg.Embryonic_trunk mm9 Histone Embryo Embryonic trunk SRX093317,SRX09...3316 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.50.AllAg.Embryonic_trunk.bed ...

  3. Mechanical control of notochord morphogenesis by extra-embryonic tissues in mouse embryos.

    Science.gov (United States)

    Imuta, Yu; Koyama, Hiroshi; Shi, Dongbo; Eiraku, Mototsugu; Fujimori, Toshihiko; Sasaki, Hiroshi

    2014-05-01

    Mammalian embryos develop in coordination with extraembryonic tissues, which support embryonic development by implanting embryos into the uterus, supplying nutrition, providing a confined niche, and also providing patterning signals to embryos. Here, we show that in mouse embryos, the expansion of the amniotic cavity (AC), which is formed between embryonic and extraembryonic tissues, provides the mechanical forces required for a type of morphogenetic movement of the notochord known as convergent extension (CE) in which the cells converge to the midline and the tissue elongates along the antero-posterior (AP) axis. The notochord is stretched along the AP axis, and the expansion of the AC is required for CE. Both mathematical modeling and physical simulation showed that a rectangular morphology of the early notochord caused the application of anisotropic force along the AP axis to the notochord through the isotropic expansion of the AC. AC expansion acts upstream of planar cell polarity (PCP) signaling, which regulates CE movement. Our results highlight the importance of extraembryonic tissues as a source of the forces that control the morphogenesis of embryos. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  4. Early gene regulation of osteogenesis in embryonic stem cells

    KAUST Repository

    Kirkham, Glen R.; Lovrics, Anna; Byrne, Helen M.; Jensen, Oliver E.; King, John R.; Shakesheff, Kevin M.; Buttery, Lee D. K.

    2012-01-01

    The early gene regulatory networks (GRNs) that mediate stem cell differentiation are complex, and the underlying regulatory associations can be difficult to map accurately. In this study, the expression profiles of the genes Dlx5, Msx2 and Runx2

  5. Porcine Tricuspid Valve Anatomy and Human Compatibility

    DEFF Research Database (Denmark)

    Waziri, Farhad; Lyager Nielsen, Sten; Hasenkam, J. Michael

    2016-01-01

    before clinical use. The study aim was to evaluate and compare the tricuspid valve anatomy of porcine and human hearts. METHODS: The anatomy of the tricuspid valve and the surrounding structures that affect the valve during a cardiac cycle were examined in detail in 100 fresh and 19 formalin...

  6. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

    DEFF Research Database (Denmark)

    Kvisgaard, Lise Kirstine

    This PhD thesis presents the diversity of Porcine Reproductive and Respiratory Syndrome viruses (PRRSV) circulating in the Danish pig population. PRRS is a disease in pigs caused by the PRRS virus resulting in reproductive failures in sows and gilts and respiratory diseases in pigs . Due to genetic...

  7. Activin Signals through SMAD2/3 to Increase Photoreceptor Precursor Yield during Embryonic Stem Cell Differentiation.

    Science.gov (United States)

    Lu, Amy Q; Popova, Evgenya Y; Barnstable, Colin J

    2017-09-12

    In vitro differentiation of mouse embryonic stem cells (ESCs) into retinal fates can be used to study the roles of exogenous factors acting through multiple signaling pathways during retina development. Application of activin A during a specific time frame that corresponds to early embryonic retinogenesis caused increased generation of CRX + photoreceptor precursors and decreased PAX6 + retinal progenitor cells (RPCs). Following activin A treatment, SMAD2/3 was activated in RPCs and bound to promoter regions of key RPC and photoreceptor genes. The effect of activin on CRX expression was repressed by pharmacological inhibition of SMAD2/3 phosphorylation. Activin signaling through SMAD2/3 in RPCs regulates expression of transcription factors involved in cell type determination and promotes photoreceptor lineage specification. Our findings can contribute to the production of photoreceptors for cell replacement therapy. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Sulfated N-linked carbohydrate chains in porcine thyroglobulin

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Kamerling, J.P.; Rijkse, I.; Maas, A.A.M.; Kuik, J.A. van

    1988-01-01

    N-linked carbohydrate chains of porcine thyroglobulin were released by the hydrazinolysis procedure. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis, the acidic fractions were further separated by high-performance liquid chromatography on

  9. File list: Oth.Emb.50.AllAg.embryonic_skin [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.50.AllAg.embryonic_skin mm9 TFs and others Embryo embryonic skin SRX1062971...,SRX1062970 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.50.AllAg.embryonic_skin.bed ...

  10. File list: Oth.Emb.20.AllAg.embryonic_skin [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.20.AllAg.embryonic_skin mm9 TFs and others Embryo embryonic skin SRX1062971...,SRX1062970 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.20.AllAg.embryonic_skin.bed ...

  11. File list: Oth.Emb.10.AllAg.embryonic_skin [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.10.AllAg.embryonic_skin mm9 TFs and others Embryo embryonic skin SRX1062971...,SRX1062970 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.10.AllAg.embryonic_skin.bed ...

  12. File list: Oth.Emb.05.AllAg.embryonic_skin [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.05.AllAg.embryonic_skin mm9 TFs and others Embryo embryonic skin SRX1062971...,SRX1062970 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.05.AllAg.embryonic_skin.bed ...

  13. File list: DNS.Emb.10.AllAg.Embryonic_limb [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Emb.10.AllAg.Embryonic_limb mm9 DNase-seq Embryo Embryonic limb SRX191032,SRX19...1037 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Emb.10.AllAg.Embryonic_limb.bed ...

  14. File list: Oth.Emb.05.AllAg.Embryonic_face [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.05.AllAg.Embryonic_face mm9 TFs and others Embryo Embryonic face SRX330164,...SRX139877 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.05.AllAg.Embryonic_face.bed ...

  15. Improved cell line IPEC-J2, characterized as a model for porcine jejunal epithelium.

    Directory of Open Access Journals (Sweden)

    Silke S Zakrzewski

    Full Text Available Cell lines matching the source epithelium are indispensable for investigating porcine intestinal transport and barrier properties on a subcellular or molecular level and furthermore help to reduce animal usage. The porcine jejunal cell line IPEC-J2 is established as an in vitro model for porcine infection studies but exhibits atypically high transepithelial resistances (TER and only low active transport rates so that the effect of nutritional factors cannot be reliably investigated. This study aimed to properly remodel IPEC-J2 and then to re-characterize these cells regarding epithelial architecture, expression of barrier-relevant tight junction (TJ proteins, adequate TER and transport function, and reaction to secretagogues. For this, IPEC-J2 monolayers were cultured on permeable supports, either under conventional (fetal bovine serum, FBS or species-specific (porcine serum, PS conditions. Porcine jejunal mucosa was analyzed for comparison. Main results were that under PS conditions (IPEC-J2/PS, compared to conventional FBS culture (IPEC-J2/FBS, the cell height increased 6-fold while the cell diameter was reduced by 50%. The apical cell membrane of IPEC-J2/PS exhibited typical microvilli. Most importantly, PS caused a one order of magnitude reduction of TER and of trans- and paracellular resistance, and a 2-fold increase in secretory response to forskolin when compared to FBS condition. TJ ultrastructure and appearance of TJ proteins changed dramatically in IPEC-J2/PS. Most parameters measured under PS conditions were much closer to those of typical pig jejunocytes than ever reported since the cell line's initial establishment in 1989. In conclusion, IPEC-J2, if cultured under defined species-specific conditions, forms a suitable model for investigating porcine paracellular intestinal barrier function.

  16. Embryonic cerebellar neurons accumulate [3H-gamma-aminobutyric acid: visualization of developing gamma-aminobutyric acid-utilizing neurons in vitro and in vivo

    International Nuclear Information System (INIS)

    Hatten, M.E.; Francois, A.M.; Napolitano, E.; Roffler-Tarlov, S.

    1984-01-01

    gamma-Aminobutyric acid (GABA) is the proposed neurotransmitter for four types of cerebellar neurons-Purkinje, Golgi, basket, and stellate neurons. With this investigation we have begun studies to establish when these neurons acquire their neurotransmitter ''identification''. Autoradiographic studies of both cultured embryonic (embryonic day 13) cerebellar cells and of intact embryonic cerebellum (embryonic day 13) were conducted with tritiated GABA. Two to 5% of the embryonic cerebellar cells accumulated [ 3 H]GABA in vitro. By morphological and immunocytochemical criteria, labeled cells were large neurons with either a thick, apical process, a multipolar shape, or were bipolar with longer processes. The identification of cells which accumulated [ 3 H]GABA as neuronal precursors was supported by the differential sensitivity to drugs that preferentially inhibit accumulation of [ 3 H]GABA by neurons and glia. The results of the in vitro experiments were confirmed and extended with in vivo experiments. When intact cerebellar tissue was removed at embryonic day 13, stripped of meninges and choroid plexus, exposed to low concentrations of [ 3 H]GABA, and processed for light microscopic autoradiography, heavily labeled cells were seen in the middle of the cerebellar anlage. Labeled cells were not seen in the ventricular zone of proliferating neuroblasts lining the fourth ventricle or in the external granular layer emerging at the lateral aspect of the pial surface. The accumulation of [ 3 H]GABA by these cells also showed the pharmacological characteristics of uptake by neurons. This study shows that among migrating, immature forms of the larger neurons of the embryonic cerebellum, there is a select group which accumulates [ 3 H]GABA and other classes of cells which do not. These results indicate very early acquisition of transmitter expression by cerebellar neurons, far in advance of their final positioning and establishment of synapses

  17. A Bacterial Glycoengineered Antigen for Improved Serodiagnosis of Porcine Brucellosis.

    Science.gov (United States)

    Cortina, María E; Balzano, Rodrigo E; Rey Serantes, Diego A; Caillava, Ana J; Elena, Sebastián; Ferreira, A C; Nicola, Ana M; Ugalde, Juan E; Comerci, Diego J; Ciocchini, Andrés E

    2016-06-01

    Brucellosis is a highly zoonotic disease that affects animals and human beings. Brucella suis is the etiological agent of porcine brucellosis and one of the major human brucellosis pathogens. Laboratory diagnosis of porcine brucellosis mainly relies on serological tests, and it has been widely demonstrated that serological assays based on the detection of anti O-polysaccharide antibodies are the most sensitive tests. Here, we validate a recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide-protein conjugate (OAg-AcrA), for diagnosis of porcine brucellosis. An indirect immunoassay based on the detection of anti-O-polysaccharide IgG antibodies was developed coupling OAg-AcrA to enzyme-linked immunosorbent assay plates (glyco-iELISA). To validate the assay, 563 serum samples obtained from experimentally infected and immunized pigs, as well as animals naturally infected with B. suis biovar 1 or 2, were tested. A receiver operating characteristic (ROC) analysis was performed, and based on this analysis, the optimum cutoff value was 0.56 (relative reactivity), which resulted in a diagnostic sensitivity and specificity of 100% and 99.7%, respectively. A cutoff value of 0.78 resulted in a test sensitivity of 98.4% and a test specificity of 100%. Overall, our results demonstrate that the glyco-iELISA is highly accurate for diagnosis of porcine brucellosis, improving the diagnostic performance of current serological tests. The recombinant glycoprotein OAg-AcrA can be produced in large homogeneous batches in a standardized way, making it an ideal candidate for further validation as a universal antigen for diagnosis of "smooth" brucellosis in animals and humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. File list: Unc.Emb.20.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Emb.20.AllAg.Embryonic_heart mm9 Unclassified Embryo Embryonic heart SRX248279,...SRX190172,SRX112936,SRX022494 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Emb.20.AllAg.Embryonic_heart.bed ...

  19. File list: Unc.Emb.50.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Emb.50.AllAg.Embryonic_heart mm9 Unclassified Embryo Embryonic heart SRX248279,...SRX190172,SRX112936,SRX022494 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Emb.50.AllAg.Embryonic_heart.bed ...

  20. Generation of hematopoietic lineage cells from embryonic like cells

    Directory of Open Access Journals (Sweden)

    Gholam Reza Khamisipour

    2014-10-01

    Full Text Available Background: Epigenetic reprogramming of somatic cells into embryonic stem cells has attracted much attention, because of the potential for stem cell transplantation and compatibility with recipient. However, the therapeutic application of either nuclear transfer or nuclear fusion of somatic cell has been hindered by technical complications as well as ethical objections. Recently, a new method is reported whereby ectopic expression of embryonic specific transcription factors was shown to induce fibroblasts to become embryonic like SCs (induced pluripotent stem cells. A major limitation of this method is the use of potentially harmful genome integrating viruses such as reto- or lentivirus. The main aim of this investigation was generation of human hematopoietic stem cells from induced fibroblasts by safe adenovectors carrying embryonically active genes. Material and Methods: Isolated fibroblasts from foreskin were expanded and recombinant adenoviruses carrying human Sox2, Oct4, Klf4, cMyc genes were added to culture. After formation of embryonic like colonies and cell expansion, they were transferred to embryonic media without bFGF, and embryoid bodies were cultured on stromal and non-stromal differentiation media for 14 days. Results: Expression of CD34 gene and antigenic markers, CD34, CD38 & CD133 in stromal culture showed significant difference with non-differentiation and non-stromal media. Conclusion: These findings show high hematopoietic differentiation rate of Adeno-iPS cells in stromal culture and no need to use growth factors. While, there was no difference between non-differentiation and non-stromal media.

  1. Virulence-associated gene pattern of porcine and human Yersinia enterocolitica biotype 4 isolates.

    Science.gov (United States)

    Schneeberger, M; Brodard, I; Overesch, G

    2015-04-02

    Yersinia enterocolitica 4/O:3 is the most important human pathogenic bioserotype in Europe and the predominant pathogenic bioserotype in slaughter pigs. Although many studies on the virulence of Y. enterocolitica strains have showed a broad spectrum of detectable factors in pigs and humans, an analysis based on a strict comparative approach and serving to verify the virulence capability of porcine Y. enterocolitica as a source for human yersiniosis is lacking. Therefore, in the present study, strains of biotype (BT) 4 isolated from Swiss slaughter pig tonsils and feces and isolates from human clinical cases were compared in terms of their spectrum of virulence-associated genes (yadA, virF, ail, inv, rovA, ymoA, ystA, ystB and myfA). An analysis of the associated antimicrobial susceptibility pattern completed the characterization. All analyzed BT 4 strains showed a nearly similar pattern, comprising the known fundamental virulence-associated genes yadA, virF, ail, inv, rovA, ymoA, ystA and myfA. Only ystB was not detectable among all analyzed isolates. Importantly, neither the source of the isolates (porcine tonsils and feces, humans) nor the serotype (ST) had any influence on the gene pattern. From these findings, it can be concluded that the presence of the full complement of virulence genes necessary for human infection is common among porcine BT 4 strains. Swiss porcine BT 4 strains not only showed antimicrobial susceptibility to chloramphenicol, cefotaxime, ceftazidime, ciprofloxacin, colistin, florfenicol, gentamicin, kanamycin, nalidixic acid, sulfamethoxazole, streptomycin, tetracycline and trimethoprim but also showed 100% antibiotic resistance to ampicillin. The human BT 4 strains revealed comparable results. However, in addition to 100% antibiotic resistance to ampicillin, 2 strains were resistant to chloramphenicol and nalidixic acid. Additionally, 1 of these strains was resistant to sulfamethoxazole. The results demonstrated that Y. enterocolitica BT 4

  2. Embryonic stem cells in scaffold-free three-dimensional cell culture: osteogenic differentiation and bone generation.

    Science.gov (United States)

    Handschel, Jörg; Naujoks, Christian; Depprich, Rita; Lammers, Lydia; Kübler, Norbert; Meyer, Ulrich; Wiesmann, Hans-Peter

    2011-07-14

    Extracorporeal formation of mineralized bone-like tissue is still an unsolved challenge in tissue engineering. Embryonic stem cells may open up new therapeutic options for the future and should be an interesting model for the analysis of fetal organogenesis. Here we describe a technique for culturing embryonic stem cells (ESCs) in the absence of artificial scaffolds which generated mineralized miromasses. Embryonic stem cells were harvested and osteogenic differentiation was stimulated by the addition of dexamethasone, ascorbic acid, and ß-glycerolphosphate (DAG). After three days of cultivation microspheres were formed. These spherical three-dimensional cell units showed a peripheral zone consisting of densely packed cell layers surrounded by minerals that were embedded in the extracellular matrix. Alizarine red staining confirmed evidence of mineralization after 10 days of DAG stimulation in the stimulated but not in the control group. Transmission electron microscopy demonstrated scorching crystallites and collagenous fibrils as early indication of bone formation. These extracellular structures resembled hydroxyl apatite-like crystals as demonstrated by distinct diffraction patterns using electron diffraction analysis. The micromass culture technique is an appropriate model to form three-dimensional bone-like micro-units without the need for an underlying scaffold. Further studies will have to show whether the technique is applicable also to pluripotent stem cells of different origin. © 2011 Handschel et al; licensee BioMed Central Ltd.

  3. Embryonic stem cells in scaffold-free three-dimensional cell culture: osteogenic differentiation and bone generation

    Directory of Open Access Journals (Sweden)

    Meyer Ulrich

    2011-07-01

    Full Text Available Abstract Extracorporeal formation of mineralized bone-like tissue is still an unsolved challenge in tissue engineering. Embryonic stem cells may open up new therapeutic options for the future and should be an interesting model for the analysis of fetal organogenesis. Here we describe a technique for culturing embryonic stem cells (ESCs in the absence of artificial scaffolds which generated mineralized miromasses. Embryonic stem cells were harvested and osteogenic differentiation was stimulated by the addition of dexamethasone, ascorbic acid, and ß-glycerolphosphate (DAG. After three days of cultivation microspheres were formed. These spherical three-dimensional cell units showed a peripheral zone consisting of densely packed cell layers surrounded by minerals that were embedded in the extracellular matrix. Alizarine red staining confirmed evidence of mineralization after 10 days of DAG stimulation in the stimulated but not in the control group. Transmission electron microscopy demonstrated scorching crystallites and collagenous fibrils as early indication of bone formation. These extracellular structures resembled hydroxyl apatite-like crystals as demonstrated by distinct diffraction patterns using electron diffraction analysis. The micromass culture technique is an appropriate model to form three-dimensional bone-like micro-units without the need for an underlying scaffold. Further studies will have to show whether the technique is applicable also to pluripotent stem cells of different origin.

  4. Corneal epithelial cell viability of an ex vivo porcine eye model.

    Science.gov (United States)

    Chan, Ka Yin; Cho, Pauline; Boost, Maureen

    2014-07-01

    The aim was to assess the consistency of corneal epithelial cell viability of an ex vivo porcine eye model. Six porcine eye models (four test and two control) were prepared for each experiment. The model has a computer-controlled mechanical arm, which could move the eyelid of the porcine eye and apply phosphate buffered saline to simulate blinking and lacrimation. The four test eyes were set up to simulate evaporative dry eyes with simulated lacrimation and blinking (one blink and one drop of buffered saline per minute) over three hours. Control A models were set up to collect pre-experimental baseline data, while those of control B were the same as the test eyes but without lacrimation and blinking simulation. All porcine eyes were kept in a closed chamber with temperature and humidity well controlled. After three hours, the cells of all eyes (except control A, which were assessed immediately before commencement of the experiment) were assessed. The eyes were first dipped into 0.4 per cent trypan blue solution. Following the dissection and separation of the cells, the number of dead cells were then counted under the microscope with a field size of 0.25 mm(2). The experiment was repeated 11 times. No significant differences were found in the number of dead cells among the four test eyes in both the central and peripheral cornea. There were significantly more dead cells in the test eyes compared to control A but significantly less when compared to control B. More dead cells were found in the central cornea than the peripheral cornea in the test eyes but the difference was not observed in controls A and B. Epithelial cell viabilities among the four porcine eye models with simulated lacrimation and blinking were consistent. The majority of cells were viable before the experiment and simulated lacrimation and blinking maintained more viable cells over time. © 2014 The Authors. Clinical and Experimental Optometry © 2014 Optometrists Association Australia.

  5. Cardiomyocytes derived from embryonic stem cells resemble cardiomyocytes of the embryonic heart tube

    NARCIS (Netherlands)

    Fijnvandraat, Arnoud C.; van Ginneken, Antoni C. G.; de Boer, Piet A. J.; Ruijter, Jan M.; Christoffels, Vincent M.; Moorman, Antoon F. M.; Lekanne Deprez, Ronald H.

    2003-01-01

    OBJECTIVE: After formation of the linear heart tube a chamber-specific program of gene expression becomes active that underlies the formation of the chamber myocardium. To assess whether this program is recapitulated in in vitro differentiated embryonic stem cells, we performed qualitative and

  6. CNS embryonal tumours: WHO 2016 and beyond.

    Science.gov (United States)

    Pickles, J C; Hawkins, C; Pietsch, T; Jacques, T S

    2018-02-01

    Embryonal tumours of the central nervous system (CNS) present a significant clinical challenge. Many of these neoplasms affect young children, have a very high mortality and therapeutic strategies are often aggressive with poor long-term outcomes. There is a great need to accurately diagnose embryonal tumours, predict their outcome and adapt therapy to the individual patient's risk. For the first time in 2016, the WHO classification took into account molecular characteristics for the diagnosis of CNS tumours. This integration of histological features with genetic information has significantly changed the diagnostic work-up and reporting of tumours of the CNS. However, this remains challenging in embryonal tumours due to their previously unaccounted tumour heterogeneity. We describe the recent revisions made to the 4th edition of the WHO classification of CNS tumours and review the main changes, while highlighting some of the more common diagnostic testing strategies. © 2017 British Neuropathological Society.

  7. Physicochemical characterization of porcine bone-derived grafting material and comparison with bovine xenografts for dental applications.

    Science.gov (United States)

    Lee, Jung Heon; Yi, Gyu Sung; Lee, Jin Woong; Kim, Deug Joong

    2017-12-01

    The physicochemical properties of a xenograft are very important because they strongly influence the bone regeneration capabilities of the graft material. Even though porcine xenografts have many advantages, only a few porcine xenografts are commercially available, and most of their physicochemical characteristics have yet to be reported. Thus, in this work we aimed to investigate the physicochemical characteristics of a porcine bone grafting material and compare them with those of 2 commercially available bovine xenografts to assess the potential of xenogenic porcine bone graft materials for dental applications. We used various characterization techniques, such as scanning electron microscopy, the Brunauer-Emmett-Teller adsorption method, atomic force microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, and others, to compare the physicochemical properties of xenografts of different origins. The porcine bone grafting material had relatively high porosity (78.4%) and a large average specific surface area (SSA; 69.9 m 2 /g), with high surface roughness (10-point average roughness, 4.47 µm) and sub-100-nm hydroxyapatite crystals on the surface. Moreover, this material presented a significant fraction of sub-100-nm pores, with negligible amounts of residual organic substances. Apart from some minor differences, the overall characteristics of the porcine bone grafting material were very similar to those of one of the bovine bone grafting material. However, many of these morphostructural properties were significantly different from the other bovine bone grafting material, which exhibited relatively smooth surface morphology with a porosity of 62.0% and an average SSA of 0.5 m 2 /g. Considering that both bovine bone grafting materials have been successfully used in oral surgery applications in the last few decades, this work shows that the porcine-derived grafting material possesses most of the key physiochemical characteristics required for its

  8. Detection of porcine DNA in gelatine and gelatine-containing processed food products-Halal/Kosher authentication.

    Science.gov (United States)

    Demirhan, Yasemin; Ulca, Pelin; Senyuva, Hamide Z

    2012-03-01

    A commercially available real-time PCR, based on a multi-copy target cytochrome b (cyt b) using porcine specific primers, has been validated for the Halal/Kosher authentication of gelatine. Extraction and purification of DNA from gelatine were successfully achieved using the SureFood® PREP Animal system, and real-time PCR was carried out using SureFood® Animal ID Pork Sens kit. The minimum level of adulteration that could be detected was 1.0% w/w for marshmallows and gum drops. A small survey was undertaken of processed food products such as gum drops, marshmallows and Turkish delight, believed to contain gelatine. Of fourteen food products from Germany, two samples were found to contain porcine gelatine, whereas of twenty-nine samples from Turkey twenty-eight were negative. However, one product from Turkey contained porcine DNA and thus was not Halal, and neither was the use of porcine gelatine indicated on the product label. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Cardiac Dysfunction in a Porcine Model of Pediatric Malnutrition

    DEFF Research Database (Denmark)

    Fabiansen, Christian; Lykke, Mikkel; Hother, Anne-Louise

    2015-01-01

    BACKGROUND: Half a million children die annually of severe acute malnutrition and cardiac dysfunction may contribute to the mortality. However, cardiac function remains poorly examined in cases of severe acute malnutrition. OBJECTIVE: To determine malnutrition-induced echocardiographic disturbances...... and longitudinal changes in plasma pro-atrial natriuretic peptide and cardiac troponin-T in a pediatric porcine model. METHODS AND RESULTS: Five-week old piglets (Duroc-x-Danish Landrace-x-Yorkshire) were fed a nutritionally inadequate maize-flour diet to induce malnutrition (MAIZE, n = 12) or a reference diet...... groups. The myocardial performance index was 86% higher in MAIZE vs AGE-REF (pMalnutrition associates with cardiac dysfunction in a pediatric porcine model by increased myocardial performance index and pro-atrial natriuretic peptide...

  10. File list: His.Emb.20.AllAg.embryonic_skin [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.20.AllAg.embryonic_skin mm9 Histone Embryo embryonic skin SRX1062969,SRX106...2968,SRX1062966,SRX1062965 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.20.AllAg.embryonic_skin.bed ...

  11. File list: His.Emb.50.AllAg.embryonic_skin [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.50.AllAg.embryonic_skin mm9 Histone Embryo embryonic skin SRX1062969,SRX106...2968,SRX1062966,SRX1062965 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.50.AllAg.embryonic_skin.bed ...

  12. File list: His.Emb.05.AllAg.embryonic_skin [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.05.AllAg.embryonic_skin mm9 Histone Embryo embryonic skin SRX1062965,SRX106...2966,SRX1062968,SRX1062969 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.05.AllAg.embryonic_skin.bed ...

  13. Tension (re)builds: Biophysical mechanisms of embryonic wound repair.

    Science.gov (United States)

    Zulueta-Coarasa, Teresa; Fernandez-Gonzalez, Rodrigo

    2017-04-01

    Embryonic tissues display an outstanding ability to rapidly repair wounds. Epithelia, in particular, serve as protective layers that line internal organs and form the skin. Thus, maintenance of epithelial integrity is of utmost importance for animal survival, particularly at embryonic stages, when an immune system has not yet fully developed. Rapid embryonic repair of epithelial tissues is conserved across species, and involves the collective migration of the cells around the wound. The migratory cell behaviours associated with wound repair require the generation and transmission of mechanical forces, not only for the cells to move, but also to coordinate their movements. Here, we review the forces involved in embryonic wound repair. We discuss how different force-generating structures are assembled at the molecular level, and the mechanisms that maintain the balance between force-generating structures as wounds close. Finally, we describe the mechanisms that cells use to coordinate the generation of mechanical forces around the wound. Collective cell movements and their misregulation have been associated with defective tissue repair, developmental abnormalities and cancer metastasis. Thus, we propose that understanding the role of mechanical forces during embryonic wound closure will be crucial to develop therapeutic interventions that promote or prevent collective cell movements under pathological conditions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Porcine Circovirus Diseases: A review of PMWS

    DEFF Research Database (Denmark)

    Baekbo, P.; Kristensen, C. S.; Larsen, L. E.

    2012-01-01

    Porcine Circo Virus type 2 have been coming on the market and many studies have shown great benefits of these to control PMWS. Today, sow vaccines as well as piglet vaccines are available in most countries. An extensive meta‐analysis of many of the vaccines has shown a comparable good efficacy...

  15. A Live-Attenuated Chimeric Porcine Circovirus Type 2 (PCV2) Vaccine Is Transmitted to Contact Pigs but Is Not Upregulated by Concurrent Infection with Porcine Parvovirus (PPV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Is Efficacious in a PCV2b-PRRSV-PPV Challenge Model▿

    OpenAIRE

    Opriessnig, T.; Shen, H. G.; Pal, N.; Ramamoorthy, S.; Huang, Y. W.; Lager, K. M.; Beach, N. M.; Halbur, P. G.; Meng, X. J.

    2011-01-01

    The live chimeric porcine circovirus type 2 (PCV2) vaccine with the capsid gene of the emerging subtype 2b cloned in the genomic backbone of the nonpathogenic PCV1 is attenuated in vivo and induces protective immunity against PCV2. To further determine the safety and efficacy of this experimental vaccine, we tested for evidence of pig-to-pig transmission by commingling nonvaccinated and vaccinated pigs, determined potential upregulation by simultaneous vaccination and infection with porcine p...

  16. Embryonic Heart Morphogenesis from Confocal Microscopy Imaging and Automatic Segmentation

    Directory of Open Access Journals (Sweden)

    Hongda Mao

    2013-01-01

    Full Text Available Embryonic heart morphogenesis (EHM is a complex and dynamic process where the heart transforms from a single tube into a four-chambered pump. This process is of great biological and clinical interest but is still poorly understood for two main reasons. On the one hand, the existing imaging modalities for investigating EHM suffered from either limited penetration depth or limited spatial resolution. On the other hand, current works typically adopted manual segmentation, which was tedious, subjective, and time consuming considering the complexity of developing heart geometry and the large size of images. In this paper, we propose to utilize confocal microscopy imaging with tissue optical immersion clearing technique to image the heart at different stages of development for EHM study. The imaging method is able to produce high spatial resolution images and achieve large penetration depth at the same time. Furthermore, we propose a novel convex active contour model for automatic image segmentation. The model has the ability to deal with intensity fall-off in depth which is characterized by confocal microscopy images. We acquired the images of embryonic quail hearts from day 6 to day 14 of incubation for EHM study. The experimental results were promising and provided us with an insight view of early heart growth pattern and also paved the road for data-driven heart growth modeling.

  17. Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

    Directory of Open Access Journals (Sweden)

    Han Yong-Mahn

    2009-07-01

    Full Text Available Abstract Background Interspecies somatic cell nuclear transfer (iSCNT has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. Results No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB-iSCNT, and bovine-bovine (BB-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM, we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively. Conclusion The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

  18. Retinol improves bovine embryonic development in vitro

    Directory of Open Access Journals (Sweden)

    Edwards J Lannett

    2004-12-01

    Full Text Available Abstract Retinoids are recognized as important regulators of vertebrate development, cell differentiation, and tissue function. Previous studies, performed both in vivo and in vitro, indicate that retinoids influence several reproductive events, including follicular development, oocyte maturation and early embryonic development. The present study evaluated in vitro effects of retinol addition to media containing maturing bovine oocytes and developing embryos in both a low oxygen atmosphere (7% and under atmospheric oxygen conditions (20%. In the first experiment, abbatoir collected bovine oocytes were matured in the presence or absence of varying concentrations of retinol. After a 22–24 hour maturation period the oocytes were fertilized, denuded 18 hours later and cultured in a modified synthetic oviductal fluid (mSOF in a humidified atmosphere at 38.5 degrees C, 5% CO2, 7% O2 and 88% N2. Cleavage rates did not differ among control and retinol-treated oocytes in all three experiments. Addition of 5 micromolar retinol to the maturation medium (IVM tended (p

  19. Retention of gene expression in porcine islets after agarose encapsulation and long-term culture

    International Nuclear Information System (INIS)

    Dumpala, Pradeep R.; Holdcraft, Robert W.; Martis, Prithy C.; Laramore, Melissa A.; Parker, Thomas S.; Levine, Daniel M.; Smith, Barry H.; Gazda, Lawrence S.

    2016-01-01

    Agarose encapsulation of porcine islets allows extended in vitro culture, providing ample time to determine the functional capacity of the islets and conduct comprehensive microbiological safety testing prior to implantation as a treatment for type 1 diabetes mellitus. However, the effect that agarose encapsulation and long-term culture may have on porcine islet gene expression is unknown. The aim of the present study was to compare the transcriptome of encapsulated porcine islets following long-term in vitro culture against free islets cultured overnight. Global gene expression analysis revealed no significant change in the expression of 98.47% of genes. This indicates that the gene expression profile of free islets is highly conserved following encapsulation and long-term culture. Importantly, the expression levels of genes that code for critical hormones secreted by islets (insulin, glucagon, and somatostatin) as well as transcripts encoding proteins involved in their packaging and secretion are unchanged. While a small number of genes known to play roles in the insulin secretion and insulin signaling pathways are differentially expressed, our results show that overall gene expression is retained following islet isolation, agarose encapsulation, and long-term culture. - Highlights: • Effect of agarose encapsulation and 8 week culture on porcine islets was analyzed. • Transcriptome analysis revealed no significant change in a majority (98%) of genes. • Agarose encapsulation allows for long-term culture of porcine islets. • Islet culture allows for functional and microbial testing prior to clinical use.

  20. Retention of gene expression in porcine islets after agarose encapsulation and long-term culture

    Energy Technology Data Exchange (ETDEWEB)

    Dumpala, Pradeep R., E-mail: pdumpala@rixd.org [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States); Holdcraft, Robert W.; Martis, Prithy C.; Laramore, Melissa A. [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States); Parker, Thomas S.; Levine, Daniel M. [The Rogosin Institute, 505 East 70th Street, New York, NY 10021 (United States); Smith, Barry H. [The Rogosin Institute, 505 East 70th Street, New York, NY 10021 (United States); NewYork-Presbyterian Hospital, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021 (United States); Gazda, Lawrence S. [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States)

    2016-08-05

    Agarose encapsulation of porcine islets allows extended in vitro culture, providing ample time to determine the functional capacity of the islets and conduct comprehensive microbiological safety testing prior to implantation as a treatment for type 1 diabetes mellitus. However, the effect that agarose encapsulation and long-term culture may have on porcine islet gene expression is unknown. The aim of the present study was to compare the transcriptome of encapsulated porcine islets following long-term in vitro culture against free islets cultured overnight. Global gene expression analysis revealed no significant change in the expression of 98.47% of genes. This indicates that the gene expression profile of free islets is highly conserved following encapsulation and long-term culture. Importantly, the expression levels of genes that code for critical hormones secreted by islets (insulin, glucagon, and somatostatin) as well as transcripts encoding proteins involved in their packaging and secretion are unchanged. While a small number of genes known to play roles in the insulin secretion and insulin signaling pathways are differentially expressed, our results show that overall gene expression is retained following islet isolation, agarose encapsulation, and long-term culture. - Highlights: • Effect of agarose encapsulation and 8 week culture on porcine islets was analyzed. • Transcriptome analysis revealed no significant change in a majority (98%) of genes. • Agarose encapsulation allows for long-term culture of porcine islets. • Islet culture allows for functional and microbial testing prior to clinical use.

  1. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... hepatocyte transplantation therapy and toxicity screening in drug discovery. Key words: Embryonic stem cells, hepatic-like cells, in vitro differentiation, sodium butyrate, ... from embryonic stem (ES) cell or induced pluripotent.

  2. Tlx3 exerts context-dependent transcriptional regulation and promotes neuronal differentiation from embryonic stem cells

    OpenAIRE

    Kondo, Takako; Sheets, Patrick L.; Zopf, David A.; Aloor, Heather L.; Cummins, Theodore R.; Chan, Rebecca J.; Hashino, Eri

    2008-01-01

    The T cell leukemia 3 (Tlx3) gene has been implicated in specification of glutamatergic sensory neurons in the spinal cord. In cranial sensory ganglia, Tlx3 is highly expressed in differentiating neurons during early embryogenesis. To study a role of Tlx3 during neural differentiation, mouse embryonic stem (ES) cells were transfected with a Tlx3 expression vector. ES cells stably expressing Tlx3 were grown in the presence or absence of a neural induction medium. In undifferentiated ES cells, ...

  3. REST/NRSF Knockdown Alters Survival, Lineage Differentiation and Signaling in Human Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Kaushali Thakore-Shah

    Full Text Available REST (RE1 silencing transcription factor, also known as NRSF (neuron-restrictive silencer factor, is a well-known transcriptional repressor of neural genes in non-neural tissues and stem cells. Dysregulation of REST activity is thought to play a role in diverse diseases including epilepsy, cancer, Down's syndrome and Huntington's disease. The role of REST/NRSF in control of human embryonic stem cell (hESC fate has never been examined. To evaluate the role of REST in hESCs we developed an inducible REST knockdown system and examined both growth and differentiation over short and long term culture. Interestingly, we have found that altering REST levels in multiple hESC lines does not result in loss of self-renewal but instead leads to increased survival. During differentiation, REST knockdown resulted in increased MAPK/ERK and WNT signaling and increased expression of mesendoderm differentiation markers. Therefore we have uncovered a new role for REST in regulation of growth and early differentiation decisions in human embryonic stem cells.

  4. Characterization of porcine eyes based on autofluorescence lifetime imaging

    Science.gov (United States)

    Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Morgado, António Miguel; König, Karsten

    2015-03-01

    Multiphoton microscopy is a non-invasive imaging technique with ideal characteristics for biological applications. In this study, we propose to characterize three major structures of the porcine eye, the cornea, crystalline lens, and retina using two-photon excitation fluorescence lifetime imaging microscopy (2PE-FLIM). Samples were imaged using a laser-scanning microscope, consisting of a broadband sub-15 femtosecond (fs) near-infrared laser. Signal detection was performed using a 16-channel photomultiplier tube (PMT) detector (PML-16PMT). Therefore, spectral analysis of the fluorescence lifetime data was possible. To ensure a correct spectral analysis of the autofluorescence lifetime data, the spectra of the individual endogenous fluorophores were acquired with the 16-channel PMT and with a spectrometer. All experiments were performed within 12h of the porcine eye enucleation. We were able to image the cornea, crystalline lens, and retina at multiple depths. Discrimination of each structure based on their autofluorescence intensity and lifetimes was possible. Furthermore, discrimination between different layers of the same structure was also possible. To the best of our knowledge, this was the first time that 2PE-FLIM was used for porcine lens imaging and layer discrimination. With this study we further demonstrated the feasibility of 2PE-FLIM to image and differentiate three of the main components of the eye and its potential as an ophthalmologic technique.

  5. Factors influencing transmission of porcine cysticercosis in Tanzania

    DEFF Research Database (Denmark)

    Braae, Uffe Christian; Wendy, Harrison; Magnussen, Pascal

    Understanding the factors contributing to the transmission of Taenia solium in sub-Saharan Africa is essential for control. This study aimed to elucidate factors concerning the transmission of porcine cysticercosis in an endemic area. A longitudinal study composed of three cross-sectional surveys...

  6. Porcine circovirus: transcription and rolling-circle DNA replication

    Science.gov (United States)

    This review summarizes the molecular studies pertaining to porcine circovirus (PCV) transcription and DNA replication. The genome of PCV is circular, single-stranded DNA and contains 1759-1768 nucleotides. Both the genome-strand (packaged in the virus particle) and the complementary-strand (synthesi...

  7. Self-organization phenomena in embryonic stem cell-derived embryoid bodies: axis formation and breaking of symmetry during cardiomyogenesis.

    Science.gov (United States)

    Fuchs, Christiane; Scheinast, Matthias; Pasteiner, Waltraud; Lagger, Sabine; Hofner, Manuela; Hoellrigl, Alexandra; Schultheis, Martina; Weitzer, Georg

    2012-01-01

    Aggregation of embryonic stem cells gives rise to embryoid bodies (EBs) which undergo developmental processes reminiscent of early eutherian embryonic development. Development of the three germ layers suggests that gastrulation takes place. In vivo, gastrulation is a highly ordered process but in EBs only few data support the hypothesis that self-organization of differentiating cells leads to morphology, reminiscent of the early gastrula. Here we demonstrate that a timely implantation-like process is a prerequisite for the breaking of the radial symmetry of suspended EBs. Attached to a surface, EBs develop a bilateral symmetry and presumptive mesodermal cells emerge between the center of the EBs and a horseshoe-shaped ridge of cells. The development of an epithelial sheet of cells on one side of the EBs allows us to define an 'anterior' and a 'posterior' end of the EBs. In the mesodermal area, first cardiomyocytes (CMCs) develop mainly next to this epithelial sheet of cells. Development of twice as many CMCs at the 'left' side of the EBs breaks the bilateral symmetry and suggests that cardiomyogenesis reflects a local or temporal asymmetry in EBs. The asymmetric appearance of CMCs but not the development of mesoderm can be disturbed by ectopic expression of the muscle-specific protein Desmin. Later, the bilateral morphology becomes blurred by an apparently chaotic differentiation of many cell types. The absence of comparable structures in aggregates of cardiovascular progenitor cells isolated from the heart demonstrates that the self-organization of cells during a gastrulation-like process is a unique feature of embryonic stem cells. Copyright © 2011 S. Karger AG, Basel.

  8. Substitution of porcine small intestinal submucosa for rabbit Achilles tendon, an experimental study.

    Science.gov (United States)

    Gu, Yan; Dai, Kerong

    2002-09-25

    To study the effect of substitution of porcine small intestinal submucosa (SIS) for rabbit Achilles tendon. Porcine SIS was taken out and processed. Part of Achilles tendons of 20 rabbits' right legs were removed and substituted by porcine SIS and the Achilles tendon of the left legs were used as controls. One, four, eight, twelve, and sixteen weeks after the operation 4 rabbits were killed and their right Achilles tendons were taken out to be examined histologically and their maximum load was tested. One week after the operation, the porcine SIS was already fused with the remaining part of rabbit Achilles tendon. Sixteen weeks after all the Achilles tendons looked like normal one. The maximum load of experimental Achilles tendon was 48 N +/- 9 N one week after the operation, and increased gradually. In the 16th week after the operation, the maximum load was 178 N +/- 6 N for the experimental Achilles tendon and 174 N +/- 10 N for the control tendon. The differences of maximum load between different weeks after operation, except that between one week and 4 weeks after, were statistically significant (P Achilles tendon is effective, thus proving the feasibility of in vivo tissue engineering technology.

  9. File list: ALL.Emb.20.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.20.AllAg.Embryonic_pancreas mm9 All antigens Embryo Embryonic pancreas SRX2...87023,SRX287022,SRX287021,SRX287020,SRX287016,SRX287026,SRX287017 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.20.AllAg.Embryonic_pancreas.bed ...

  10. File list: Oth.Emb.05.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.05.AllAg.Embryonic_pancreas mm9 TFs and others Embryo Embryonic pancreas SR...X287017,SRX287023,SRX287022,SRX287021,SRX287020,SRX287016 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.05.AllAg.Embryonic_pancreas.bed ...

  11. File list: ALL.Emb.50.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.50.AllAg.Embryonic_pancreas mm9 All antigens Embryo Embryonic pancreas SRX2...87021,SRX287020,SRX287023,SRX287016,SRX287022,SRX287026,SRX287017 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.50.AllAg.Embryonic_pancreas.bed ...

  12. File list: Oth.Emb.50.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.50.AllAg.Embryonic_pancreas mm9 TFs and others Embryo Embryonic pancreas SR...X287021,SRX287020,SRX287023,SRX287016,SRX287022,SRX287017 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.50.AllAg.Embryonic_pancreas.bed ...

  13. File list: Oth.Emb.20.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.20.AllAg.Embryonic_pancreas mm9 TFs and others Embryo Embryonic pancreas SR...X287023,SRX287022,SRX287021,SRX287020,SRX287016,SRX287017 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.20.AllAg.Embryonic_pancreas.bed ...

  14. File list: Oth.Emb.10.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.10.AllAg.Embryonic_pancreas mm9 TFs and others Embryo Embryonic pancreas SR...X287023,SRX287022,SRX287020,SRX287021,SRX287016,SRX287017 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.10.AllAg.Embryonic_pancreas.bed ...

  15. File list: ALL.Emb.10.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.10.AllAg.Embryonic_pancreas mm9 All antigens Embryo Embryonic pancreas SRX2...87023,SRX287022,SRX287020,SRX287021,SRX287016,SRX287017,SRX287026 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.10.AllAg.Embryonic_pancreas.bed ...

  16. File list: ALL.Emb.05.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.05.AllAg.Embryonic_pancreas mm9 All antigens Embryo Embryonic pancreas SRX2...87017,SRX287023,SRX287022,SRX287021,SRX287026,SRX287020,SRX287016 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.05.AllAg.Embryonic_pancreas.bed ...

  17. Embryonic kidney function in a chronic renal failure model in rodents.

    Science.gov (United States)

    Fujimoto, Eisuke; Yamanaka, Shuichiro; Kurihara, Sho; Tajiri, Susumu; Izuhara, Luna; Katsuoka, Yuichi; Yokote, Shinya; Matsumoto, Kei; Kobayashi, Eiji; Okano, Hirotaka James; Chikaraishi, Tatsuya; Yokoo, Takashi

    2017-08-01

    Rapid advancements have been made in alternative treatments for renal diseases. Our goal for renal regeneration is to establish a kidney graft derived from human embryonic tissues. In this study, we investigated the effects of host renal failure on the structure and activity of transplanted embryonic kidney and bladder, and found that diuretics effectively induced urine production in the transplanted kidney. Uremic conditions were reproduced using a 5/6 renal infarction rat model. An embryonic kidney plus bladder (embryonic day 15) was isolated from a pregnant Lewis rat and transplanted into the para-aortic area of a 5/6 renal-infarcted Lewis rat. Following growth, the embryonic bladder was successfully anastomosed to the host ureter. We assessed graft function in terms of survival rates and found no differences between normal (n = 5) and renal failure (n = 8) groups (median survival: 70.5 vs 74.5 h; p = 0.331) in terms of survival, indicating that the grafts prolonged rat survival, even under renal failure conditions. Furosemide (n = 9) significantly increased urine volume compared with saline-treated controls (n = 7; p < 0.05), confirming that the grafts were functional. We also demonstrated the possibilities of an in vivo imaging system for determining the viability of transplanted embryonic kidney with bladder. The results of this study demonstrate that transplanted embryonic kidney and bladder can grow and function effectively, even under uremic conditions.

  18. Aquaporin expression in the fetal porcine urinary tract changes during gestation

    DEFF Research Database (Denmark)

    Jakobsen, L K; Trelborg, K; Kingo, P S

    2018-01-01

    The expression of aquaporins (AQPs) in the fetal porcine urinary tract and its relation to gestational age has not been established. Tissue samples from the renal pelvis, ureter, bladder and urethra were obtained from porcine fetuses. Samples were examined by RT-PCR (AQPs 1-11), QPCR (AQPs positive....... Immunohistochemistry showed AQP1 staining in sub-urothelial vessels at all locations. Western blotting analysis confirmed increased AQP1 protein levels in bladder samples during gestation. Expression levels of AQP1, 3, 5, 9 and 11 in the urinary tract change during gestation, and further studies are needed to provide...

  19. Establishment of a nanoparticle-assisted RT-PCR assay to distinguish field strains and attenuated strains of porcine epidemic diarrhea virus.

    Science.gov (United States)

    Zhu, Yu; Wang, Gui-Hua; Cui, Yu-Dong; Cui, Shang-Jin

    2016-09-01

    Porcine epidemic diarrhea virus (PEDV) can cause serious disease and even death in neonatal piglets, resulting in serious damage to the swine industry worldwide. Open reading frame 3 (ORF3) is the only accessory gene in the PEDV genome. Previous studies have indicated that PEDV vaccine strains have a partial deletion in ORF3. In this study, a nanoparticle-assisted polymerase chain reaction (nanoparticle-assisted RT-PCR) assay targeting the ORF3 of PEDV was developed to distinguish PEDV field strains from attenuated strains by using a specific pair of primers. The PCR products of field strains and attenuated strains were 264 bp and 215 bp in length, respectively. The sensitivity and specificity of this assay were also assessed. The nanoparticle-assisted RT-PCR assay was 10-100 times more sensitive than the conventional RT-PCR assay, with no cross-reactions when amplifying porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (RV), and porcine transmissible gastroenteritis virus (TGEV). The nanoparticle-assisted RT-PCR assay we describe here can be used to distinguish field strains from vaccine strains of PEDV, and it shows promise for reducing economic loss due to PEDV infection.

  20. The ethics of patenting human embryonic stem cells.

    Science.gov (United States)

    Chapman, Audrey R

    2009-09-01

    Just as human embryonic stem cell research has generated controversy about the uses of human embryos for research and therapeutic applications, human embryonic stem cell patents raise fundamental ethical issues. The United States Patent and Trademark Office has granted foundational patents, including a composition of matter (or product) patent to the Wisconsin Alumni Research Foundation (WARF), the University of Wisconsin-Madison's intellectual property office. In contrast, the European Patent Office rejected the same WARF patent application for ethical reasons. This article assesses the appropriateness of these patents placing the discussion in the context of the deontological and consequentialist ethical issues related to human embryonic stem cell patenting. It advocates for a patent system that explicitly takes ethical factors into account and explores options for new types of intellectual property arrangements consistent with ethical concerns.

  1. Similarities in the immunoglobulin response and VH gene usage in rhesus monkeys and humans exposed to porcine hepatocytes

    Directory of Open Access Journals (Sweden)

    Borie Dominic C

    2006-03-01

    Full Text Available Abstract Background The use of porcine cells and organs as a source of xenografts for human patients would vastly increase the donor pool; however, both humans and Old World primates vigorously reject pig tissues due to xenoantibodies that react with the polysaccharide galactose α (1,3 galactose (αGal present on the surface of many porcine cells. We previously examined the xenoantibody response in patients exposed to porcine hepatocytes via treatment(s with bioartficial liver devices (BALs, composed of porcine cells in a support matrix. We determined that xenoantibodies in BAL-treated patients are predominantly directed at porcine αGal carbohydrate epitopes, and are encoded by a small number of germline heavy chain variable region (VH immunoglobulin genes. The studies described in this manuscript were designed to identify whether the xenoantibody responses and the IgVH genes encoding antibodies to porcine hepatocytes in non-human primates used as preclinical models are similar to those in humans. Adult non-immunosuppressed rhesus monkeys (Macaca mulatta were injected intra-portally with porcine hepatocytes or heterotopically transplanted with a porcine liver lobe. Peripheral blood leukocytes and serum were obtained prior to and at multiple time points after exposure, and the immune response was characterized, using ELISA to evaluate the levels and specificities of circulating xenoantibodies, and the production of cDNA libraries to determine the genes used by B cells to encode those antibodies. Results Xenoantibodies produced following exposure to isolated hepatocytes and solid organ liver grafts were predominantly encoded by genes in the VH3 family, with a minor contribution from the VH4 family. Immunoglobulin heavy-chain gene (VH cDNA library screening and gene sequencing of IgM libraries identified the genes as most closely-related to the IGHV3-11 and IGHV4-59 germline progenitors. One of the genes most similar to IGHV3-11, VH3-11cyno, has

  2. Prevention of primary vascular graft infection with silver-coated polyester graft in a porcine model

    DEFF Research Database (Denmark)

    Gao, H; Sandermann, J; Prag, J

    2010-01-01

    To evaluate the efficacy of a silver-coated vascular polyester graft in the prevention of graft infection after inoculation with Staphylococcus aureus in a porcine model.......To evaluate the efficacy of a silver-coated vascular polyester graft in the prevention of graft infection after inoculation with Staphylococcus aureus in a porcine model....

  3. File list: ALL.Emb.20.AllAg.Embryonic_testis [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.20.AllAg.Embryonic_testis mm9 All antigens Embryo Embryonic testis SRX14917...57137,SRX1156635,SRX149168,SRX149172,SRX1175150,SRX149166,SRX149170,SRX1175149 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.20.AllAg.Embryonic_testis.bed ...

  4. File list: ALL.Emb.10.AllAg.Embryonic_testis [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.10.AllAg.Embryonic_testis mm9 All antigens Embryo Embryonic testis SRX14917...9169,SRX149166,SRX1175147,SRX957137,SRX1175148,SRX149170,SRX1175150,SRX1175149 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.10.AllAg.Embryonic_testis.bed ...

  5. File list: ALL.Emb.50.AllAg.embryonic_skin [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.50.AllAg.embryonic_skin mm9 All antigens Embryo embryonic skin SRX1062969,S...RX1062968,SRX1062966,SRX1062967,SRX1062972,SRX1062971,SRX1062970,SRX1062965 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.50.AllAg.embryonic_skin.bed ...

  6. File list: ALL.Emb.20.AllAg.embryonic_skin [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.20.AllAg.embryonic_skin mm9 All antigens Embryo embryonic skin SRX1062969,S...RX1062968,SRX1062966,SRX1062971,SRX1062967,SRX1062972,SRX1062965,SRX1062970 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.20.AllAg.embryonic_skin.bed ...

  7. File list: ALL.Emb.10.AllAg.embryonic_skin [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.10.AllAg.embryonic_skin mm9 All antigens Embryo embryonic skin SRX1062972,S...RX1062971,SRX1062966,SRX1062969,SRX1062968,SRX1062965,SRX1062967,SRX1062970 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.10.AllAg.embryonic_skin.bed ...

  8. Investigation of SNPs in the porcine desmoglein 1 gene

    DEFF Research Database (Denmark)

    Daugaard, L.; Andresen, Lars Ole; Fredholm, M.

    2007-01-01

    epidermitis were diagnosed clinically as affected or unaffected. Two regions of the desmoglein I gene were sequenced and genotypes of the SNPs were established. Seven SNPs (823T>C, 828A>G, 829A>G, 830A>T, 831A>T, 838A>C and 1139C>T) were found in the analysed sequences and the allele frequencies were...... the location of single nucleotide polymorphisms (SNPs) in the porcine desmoglein I gene (PIG)DSGI in correlation to the cleavage site as well as if the genotype of the SNPs is correlated to susceptibility or resistance to the disease. Results: DNA from 32 affected and 32 unaffected piglets with exudative...... the genotypes of two out of seven SNPs found in the porcine desmoglein I gene and the susceptibility to exudative epidermitis....

  9. Release of galanin from isolated perfused porcine adrenal glands

    DEFF Research Database (Denmark)

    Holst, J J; Ehrhart-Bornstein, M; Messell, T

    1991-01-01

    We found a high concentration of galanin in extracts of porcine adrenal glands (114 pmol/g). By immunohistochemistry, galanin was localized to groups of medullary cells previously shown to produce norepinephrine. To study mechanisms for the release of galanin, we developed the following in vitro...... model: isolated perfused porcine adrenals with intact splanchnic nerve supply. When the nerves were electrically stimulated, epinephrine and norepinephrine secretion increased 276- and 291-fold, respectively, and galanin release increased up to 1,300-fold. Acetylcholine at 10(-6) M stimulated galanin...... release, and hexamethonium almost abolished the response to nerve stimulation. Galanin infusions had no effect on epinephrine and norepinephrine secretion in concentrations of 10(-8) and 10(-7) M, but increased both cortisol and aldosterone secretion (P less than 0.05). Splanchnic nerve stimulation...

  10. File list: InP.Emb.05.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.05.AllAg.Embryonic_palates mm9 Input control Embryo Embryonic palates http:...//dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.05.AllAg.Embryonic_palates.bed ...

  11. File list: InP.Emb.10.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.10.AllAg.Embryonic_palates mm9 Input control Embryo Embryonic palates http:...//dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.10.AllAg.Embryonic_palates.bed ...

  12. File list: InP.Emb.20.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.20.AllAg.Embryonic_palates mm9 Input control Embryo Embryonic palates http:...//dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.20.AllAg.Embryonic_palates.bed ...

  13. File list: InP.Emb.50.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.50.AllAg.Embryonic_palates mm9 Input control Embryo Embryonic palates http:...//dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.50.AllAg.Embryonic_palates.bed ...

  14. A novel porcine cell culture based protocol for the propagation of hepatitis E virus

    Directory of Open Access Journals (Sweden)

    Walter Chingwaru

    2016-08-01

    Full Text Available Objective: To present a comprehensive protocol for the processing of hepatitis E virus (HEV infected samples and propagation of the virus in primary cell cultures. Methods: Hepatitis E was extracted from porcine liver and faecal samples following standard protocols. The virus was then allowed to attach in the presence of trypsin to primary cells that included porcine and bovine intestinal epithelial cells and macrophages over a period of up to 3 h. The virus was propagated by rotational passaging through the cell cultures. Propagation was confirmed by immunoblotting. Results: We developed a comprehensive protocol to propagate HEV in porcine cell model that includes (i rotational culturing of the virus between porcine cell types, (ii pre-incubation of infected cells for 210 min, (iii use of a semi-complete cell culture medium supplemented with trypsin (0.33 µg/mL and (iv the use of simple immunoblot technique to detect the amplified virus based on the open reading frame 2/3. Conclusions: This protocol opens doors towards systematic analysis of the mechanisms that underlie the pathogenesis of HEV in vitro. Using our protocol, one can complete the propagation process within 6 to 9 d.

  15. Immunobiotic Lactobacillus strains augment NLRP3 expression in newborn and adult porcine gut-associated lymphoid tissues.

    Science.gov (United States)

    Tohno, Masanori; Shimosato, Takeshi; Aso, Hisashi; Kitazawa, Haruki

    2011-12-15

    We isolated cDNA encoding porcine nucleotide-binding domain-like receptor family, pryin domain containing 3 (NLRP3) from Peyer's patches. The complete nucleotide open reading frame of porcine NLRP3 contains 3108-bp encoding a deduced polypeptide of 1036-amino acid residues. The porcine NLRP3 amino acid sequence is more similar to the longest isoform of human than the mouse counterpart. The predicted amino acid sequence of porcine NLRP3 presented nine C-terminal leucine-rich repeat domains. In newborn swine, the expression of NLRP3 was detected at higher levels in spleen and mesenteric lymph nodes, while lower levels were observed in intestinal tissues. In adult swine, NLRP3 was strongly expressed in Peyer's patches and the mesenteric lymph nodes, and the expression level in the lower intestinal tissues was comparable to that in spleen. Toll-like receptor and nucleotide-binding domain ligands, as well as Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus gasseri, enhanced NLRP3 expression in gut-associated lymphoid tissues (GALT) of newborn and adult swine. Our results should aid in understanding the intestinal immunoregulatory mechanisms underlying NLRP3 activation and the priming ability of immunobiotic lactic acid bacteria in porcine GALT. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. File list: NoD.Emb.10.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: NoD.Emb.05.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: NoD.Emb.20.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.20.AllAg.Embryonic_pancreas mm9 No description Embryo Embryonic pancreas ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.20.AllAg.Embryonic_pancreas.bed ...

  19. File list: NoD.Emb.50.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.50.AllAg.Embryonic_pancreas mm9 No description Embryo Embryonic pancreas ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.50.AllAg.Embryonic_pancreas.bed ...

  20. File list: ALL.Emb.20.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.20.AllAg.Embryonic_heart mm9 All antigens Embryo Embryonic heart SRX112938,...7,SRX967654,SRX967653,SRX1100404,SRX244285,SRX112936,SRX1100405,SRX022494,SRX337963 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.20.AllAg.Embryonic_heart.bed ...