WorldWideScience

Sample records for stage-specific embryonic antigen-1

  1. Stage specific requirement of platelet-derived growth factor receptor-α in embryonic development.

    Science.gov (United States)

    Qian, Chen; Wong, Carol Wing Yan; Wu, Zhongluan; He, Qiuming; Xia, Huimin; Tam, Paul Kwong Hang; Wong, Kenneth Kak Yuen; Lui, Vincent Chi Hang

    2017-01-01

    Platelet-derived growth factor receptor alpha (PDGFRα) is a cell-surface receptor tyrosine kinase for platelet-derived growth factors. Correct timing and level of Pdgfra expression is crucial for embryo development, and deletion of Pdgfra caused developmental defects of multiple endoderm and mesoderm derived structures, resulting in a complex phenotypes including orofacial cleft, spina bifida, rib deformities, and omphalocele in mice. However, it is not clear if deletion of Pdgfra at different embryonic stages differentially affects these structures. To address the temporal requirement of Pdgfra in embryonic development. We have deleted the Pdgfra in Pdgfra-expressing tissues at different embryonic stages in mice, examined and quantified the developmental anomalies. Current study showed that (i) conditional deletion of Pdgfra at different embryonic days (between E7.5 and E10.5) resulted in orofacial cleft, spina bifida, rib cage deformities, and omphalocele, and (ii) the day of Pdgfra deletion influenced the combinations, incidence and severities of these anomalies. Deletion of Pdgfra caused apoptosis of Pdgfra-expressing tissues, and developmental defects of their derivatives. Orofacial cleft, spina bifida and omphalocele are among the commonest skeletal and abdominal wall defects of newborns, but their genetic etiologies are largely unknown. The remarkable resemblance of our conditional Pdgfra knockout embryos to theses human congenital anomalies, suggesting that dysregulated PDGFRA expression could cause these anomalies in human. Future work should aim at defining (a) the regulatory elements for the expression of the human PDGFRA during embryonic development, and (b) if mutations / sequence variations of these regulatory elements cause these anomalies.

  2. Stage-specific histone modification profiles reveal global transitions in the Xenopus embryonic epigenome.

    Directory of Open Access Journals (Sweden)

    Tobias D Schneider

    Full Text Available Vertebrate embryos are derived from a transitory pool of pluripotent cells. By the process of embryonic induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While on gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue we analysed histones isolated from four different developmental stages of Xenopus laevis by mass spectrometry. In toto, we quantified 59 modification states on core histones H3 and H4 from blastula to tadpole stages. During this developmental period, we observed in general an increase in the unmodified states, and a shift from histone modifications associated with transcriptional activity to transcriptionally repressive histone marks. We also compared these naturally occurring patterns with the histone modifications of murine ES cells, detecting large differences in the methylation patterns of histone H3 lysines 27 and 36 between pluripotent ES cells and pluripotent cells from Xenopus blastulae. By combining all detected modification transitions we could cluster their patterns according to their embryonic origin, defining specific histone modification profiles (HMPs for each developmental stage. To our knowledge, this data set represents the first compendium of covalent histone modifications and their quantitative flux during normogenesis in a vertebrate model organism. The HMPs indicate a stepwise maturation of the embryonic epigenome, which may be causal to the progressing restriction of cellular potency during development.

  3. 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

  4. Laminin binding protein, 34/67 laminin receptor, carries stage-specific embryonic antigen-4 epitope defined by monoclonal antibody Raft.2

    International Nuclear Information System (INIS)

    Katagiri, Yohko U.; Kiyokawa, Nobutaka; Nakamura, Kyoko; Takenouchi, Hisami; Taguchi, Tomoko; Okita, Hajime; Umezawa, Akihiro; Fujimoto, Junichiro

    2005-01-01

    We previously produced monoclonal antibodies against the detergent-insoluble microdomain, i.e., the raft microdomain, of the human renal cancer cell line ACHN. Raft.2, one of these monoclonal antibodies, recognizes sialosyl globopentaosylceramide, which has the stage-specific embryonic antigen (SSEA)-4 epitope. Although the mouse embryonal carcinoma (EC) cell line F9 does not express SSEA-4, some F9 cells stained with Raft.2. Western analysis and matrix-assisted laser desorption ionization-time of flight mass spectrometry identified the Raft.2 binding molecule as laminin binding protein (LBP), i.e., 34/67 laminin receptor. Weak acid treatment or digestion with Clostridium perfringens sialidase reduced Raft.2 binding to LBP on nitrocellulose sheets and [ 14 C]galactose was incorporated into LBP, indicating LBP to have a sialylated carbohydrate moiety. Subcellular localization analysis by sucrose density-gradient centrifugation and examination by confocal microscopy revealed LBP to be localized on the outer surface of the plasma membrane. An SSEA-4-positive human EC cell line, NCR-G3 cells, also expressed Raft.2-binding LBP

  5. Thyroid Progenitors Are Robustly Derived from Embryonic Stem Cells through Transient, Developmental Stage-Specific Overexpression of Nkx2-1

    Directory of Open Access Journals (Sweden)

    Keri Dame

    2017-02-01

    Full Text Available The clinical importance of anterior foregut endoderm (AFE derivatives, such as thyrocytes, has led to intense research efforts for their derivation through directed differentiation of pluripotent stem cells (PSCs. Here, we identify transient overexpression of the transcription factor (TF NKX2-1 as a powerful inductive signal for the robust derivation of thyrocyte-like cells from mouse PSC-derived AFE. This effect is highly developmental stage specific and dependent on FOXA2 expression levels and precise modulation of BMP and FGF signaling. The majority of the resulting cells express thyroid TFs (Nkx2-1, Pax8, Foxe1, Hhex and thyroid hormone synthesis-related genes (Tg, Tpo, Nis, Iyd at levels similar to adult mouse thyroid and give rise to functional follicle-like epithelial structures in Matrigel culture. Our findings demonstrate that NKX2-1 overexpression converts AFE to thyroid epithelium in a developmental time-sensitive manner and suggest a general methodology for manipulation of cell-fate decisions of developmental intermediates.

  6. Thyroid Progenitors Are Robustly Derived from Embryonic Stem Cells through Transient, Developmental Stage-Specific Overexpression of Nkx2-1.

    Science.gov (United States)

    Dame, Keri; Cincotta, Steven; Lang, Alex H; Sanghrajka, Reeti M; Zhang, Liye; Choi, Jinyoung; Kwok, Letty; Wilson, Talitha; Kańduła, Maciej M; Monti, Stefano; Hollenberg, Anthony N; Mehta, Pankaj; Kotton, Darrell N; Ikonomou, Laertis

    2017-02-14

    The clinical importance of anterior foregut endoderm (AFE) derivatives, such as thyrocytes, has led to intense research efforts for their derivation through directed differentiation of pluripotent stem cells (PSCs). Here, we identify transient overexpression of the transcription factor (TF) NKX2-1 as a powerful inductive signal for the robust derivation of thyrocyte-like cells from mouse PSC-derived AFE. This effect is highly developmental stage specific and dependent on FOXA2 expression levels and precise modulation of BMP and FGF signaling. The majority of the resulting cells express thyroid TFs (Nkx2-1, Pax8, Foxe1, Hhex) and thyroid hormone synthesis-related genes (Tg, Tpo, Nis, Iyd) at levels similar to adult mouse thyroid and give rise to functional follicle-like epithelial structures in Matrigel culture. Our findings demonstrate that NKX2-1 overexpression converts AFE to thyroid epithelium in a developmental time-sensitive manner and suggest a general methodology for manipulation of cell-fate decisions of developmental intermediates. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

    DEFF Research Database (Denmark)

    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...

  8. Cell cycle synchronization of embryonic stem cells: Effect of serum deprivation on the differentiation of embryonic bodies in vitro

    International Nuclear Information System (INIS)

    Zhang Enming; Li Xiaolong; Zhang Shufang; Chen Liangqiang; Zheng Xiaoxiang

    2005-01-01

    Research on stem-cell transplantation has indicated that the success of transplantation largely depends on synchronizing donor cells into the G0/G1 phase. In this study, we investigated the profile of embryonic stem (ES) cell synchronization and its effect on the formation of embryonic bodies (EBs) using cell culture with serum deprivation. The D3 cell line of ES cells was used, and parameters such as cell proliferation and activity, EB formation, and expression of stage-specific embryonic antigen-1 and Oct-4 were investigated. Results showed that the percentage of G0/G1 stage in serum deprivation culture is significantly higher than that in culture with serum supplementation. Synchronized ES cells can reenter the normal cell cycle successfully after serum supply. EBs formed from synchronized ES cells have higher totipotency capability to differentiate into functional neuronal cells than EBs formed from unsynchronized ES cells. Our study provides a method for ES treatment before cell transplantation that possibly helps to decrease the rate of cell death after transplantation

  9. 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.

  10. Liver Stage specific response among Endemic Populations: Diet & Immunity

    Directory of Open Access Journals (Sweden)

    Sarat Kumar Dalai

    2015-03-01

    Full Text Available Developing effective anti-malarial vaccine has been a challenge for long. Various factors including complex life cycle of parasite and lack of knowledge of stage specific critical antigens are some of the reasons. Moreover, inadequate understanding of the immune responses vis-à-vis sterile protection induced naturally by Plasmodia infection has further compounded the problem. It has been shown that people living in endemic areas take years to develop protective immunity to blood stage infection. But hardly anyone believes that immunity to liver-stage infection could be developed. Various experimental model studies using attenuated parasite suggest that liver stage immunity might exist among endemic populations. This could be induced because of the attenuation of parasite in liver by various compounds present in the diet of endemic populations.

  11. Gelatin–PMVE/MA composite scaffold promotes expansion of embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chhabra, Hemlata [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); Gupta, Priyanka [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); IITB-Monash Research Academy, Mumbai (India); Department of Chemical Engineering, Monash University, Melbourne (Australia); Verma, Paul J. [Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, South Australia (Australia); Jadhav, Sameer; Bellare, Jayesh R. [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India)

    2014-04-01

    We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2–1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin–PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering. - Highlights: • Composite scaffolds of gelatin and PMVE/MA were prepared by freeze-drying method. • SEM micrographs showed porous structure in all scaffolds of varying pore dimension. • GP-2 composite exhibited better cellular response in comparison to other scaffolds. • mESCs proliferated and expressed Oct-4 and SSEA-1, when cultured on GP-2 scaffold.

  12. Protein modeling of apical membrane antigen-1(AMA-1) of ...

    African Journals Online (AJOL)

    Apical membrane Antigen-1(AMA-1), an asexual blood stage antigen of Plasmodium cynomolgi, is an important candidate for testing as a component of malarial vaccine. The degree of conservation of. AMA-1 sequences implies a conserved function for this molecule across different species of Plasmodium. Since the AMA-1 ...

  13. Stage-specific functions of the small Rho GTPases Cdc42 and Rac1 for adult hippocampal neurogenesis

    DEFF Research Database (Denmark)

    Vadodaria, Krishna C; Brakebusch, Cord; Suter, Ueli

    2013-01-01

    The molecular mechanisms underlying the generation, maturation, and integration of new granule cells generated throughout life in the mammalian hippocampus remain poorly understood. Small Rho GTPases, such as Cdc42 and Rac1, have been implicated previously in neural stem/progenitor cell (NSPC......) proliferation and neuronal maturation during embryonic development. Here we used conditional genetic deletion and virus-based loss-of-function approaches to identify temporally distinct functions for Cdc42 and Rac1 in adult hippocampal neurogenesis. We found that Cdc42 is involved in mouse NSPC proliferation......, initial dendritic development, and dendritic spine maturation. In contrast, Rac1 is dispensable for early steps of neuronal development but is important for late steps of dendritic growth and spine maturation. These results establish cell-autonomous and stage-specific functions for the small Rho GTPases...

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

    Science.gov (United States)

    Li, Dong; Secher, Jan O; Juhl, Morten; Mashayekhi, Kaveh; Nielsen, Troels T; Holst, Bjørn; Hyttel, Poul; Freude, Kristine K; Hall, Vanessa J

    2017-06-03

    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, reprogramming of SSEA-1+ sorted pEFs led to higher reprogramming efficiency. Subsequent transcriptome profiling of the SSEA-1+ vs. the SSEA-1neg cell fraction revealed highly comparable gene signatures. However several genes that were found to be upregulated in the SSEA-1+ cells were similarly expressed in mesenchymal stem cells (MSCs). We therefore termed these cells SSEA-1 Expressing Enhanced Reprogramming (SEER) cells. Interestingly, SEER cells were more effective at differentiating into osteocytes and chondrocytes in vitro. We conclude that SEER cells are more amenable for reprogramming and that the expression of mesenchymal stem cell genes is advantageous in the reprogramming process. This data provides evidence supporting the elite theory and helps to delineate which cell types and specific genes are important for reprogramming in the pig.

  15. Granulosa cells and retinoic acid co-treatment enrich potential germ cells from manually selected Oct4-EGFP expressing human embryonic stem cells.

    Science.gov (United States)

    Chen, Hsin-Fu; Jan, Pey-Shynan; Kuo, Hung-Chih; Wu, Fang-Chun; Lan, Chen-Wei; Huang, Mei-Chi; Chien, Chung-Liang; Ho, Hong-Nerng

    2014-09-01

    Differentiation of human embryonic stem (HES) cells to germ cells may become clinically useful in overcoming diseases related to germ-cell development. Niches were used to differentiate HES cell lines, NTU1 and H9 Oct4-enhanced green fluorescence protein (EGFP), including laminin, granulosa cell co-culture or conditioned medium, ovarian stromal cell co-culture or conditioned medium, retinoic acid, stem cell factor (SCF) and BMP4-BMP7-BMP8b treatment. Flow cytometry showed that granulosa cell co-culture (P cells expressing early germ cell marker stage-specific embryonic antigen 1(SSEA1); sorted SSEA1[+] cells did not express higher levels of germ cell gene VASA and GDF9. Manually collected H9 Oct4-EGFP[+] cells expressed significantly higher levels of VASA (P = 0.005) and GDF9 (P = 0.001). H9 Oct4-EGFP[+] cells developed to ovarian follicle-like structures after culture for 28 days but with low efficiency. Unlike SCF and BMP4, retinoic acid co-treatment enhanced VASA, GDF9 and SCP3 expression. A protocol is recommended to enrich differentiated HES cells with germ-cell potential by culture with granulosa cells, conditioned medium or retinoic acid, manual selection of Oct4-EGFP[+] cells, and analysis of VASA, GDF9 expression, or both. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  16. Three-dimensional bioprinting of embryonic stem cells directs highly uniform embryoid body formation

    International Nuclear Information System (INIS)

    Ouyang, Liliang; Yao, Rui; Mao, Shuangshuang; Sun, Wei; Chen, Xi; Na, Jie

    2015-01-01

    With the ability to manipulate cells temporarily and spatially into three-dimensional (3D) tissue-like construct, 3D bioprinting technology was used in many studies to facilitate the recreation of complex cell niche and/or to better understand the regulation of stem cell proliferation and differentiation by cellular microenvironment factors. Embryonic stem cells (ESCs) have the capacity to differentiate into any specialized cell type of the animal body, generally via the formation of embryoid body (EB), which mimics the early stages of embryogenesis. In this study, extrusion-based 3D bioprinting technology was utilized for biofabricating ESCs into 3D cell-laden construct. The influence of 3D printing parameters on ESC viability, proliferation, maintenance of pluripotency and the rule of EB formation was systematically studied in this work. Results demonstrated that ESCs were successfully printed with hydrogel into 3D macroporous construct. Upon process optimization, about 90% ESCs remained alive after the process of bioprinting and cell-laden construct formation. ESCs continued proliferating into spheroid EBs in the hydrogel construct, while retaining the protein expression and gene expression of pluripotent markers, like octamer binding transcription factor 4, stage specific embryonic antigen 1 and Nanog. In this novel technology, EBs were formed through cell proliferation instead of aggregation, and the quantity of EBs was tuned by the initial cell density in the 3D bioprinting process. This study introduces the 3D bioprinting of ESCs into a 3D cell-laden hydrogel construct for the first time and showed the production of uniform, pluripotent, high-throughput and size-controllable EBs, which indicated strong potential in ESC large scale expansion, stem cell regulation and fabrication of tissue-like structure and drug screening studies. (paper)

  17. 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.

  18. "Life Stage-Specific" Variations in Performance in Response to Age Stereotypes

    Science.gov (United States)

    Hehman, Jessica A.; Bugental, Daphne Blunt

    2013-01-01

    In a test of life stage-specific responses to age-based stigma, older (n = 54, ages 62-92) and younger (n = 81, ages 17-22) adults were told that a task (Weschler Adult Intelligence Scale-III block design) required either (a) speed/contemporary knowledge (YA; "youth advantage") or (b) life experience/wisdom (OA; "age…

  19. Assessing species and stage specific effects of preservation on fish oocytes over different temporal scales

    Directory of Open Access Journals (Sweden)

    M. RAKKA

    2015-09-01

    Full Text Available This study assessed the effect of 10% neutral buffered formalin and of three ethanol solutions of different concentration on Mediterranean sardine and European anchovy oocytes over several temporal scales (days, weeks, months. The two species exhibit differences both in the elemental composition and the shape of their oocytes which further allowed an appraisal of oocyte shrinkage dynamics in relation to oocyte shape, developmental stage and composition. We showed that the effect of the preservative on oocyte size is stage specific while different preservation periods of ovarian material might lead to discrepancies among studies.

  20. Mutations in wheat exhibiting growth-stage-specific resistance to biotrophic fungal pathogens.

    Science.gov (United States)

    Smith, Phil H; Howie, John A; Worland, Anthony J; Stratford, Rebecca; Boyd, Lesley A

    2004-11-01

    Two mutants were isolated in wheat that showed enhanced resistance towards Puccinia striiformis f. sp. tritici, the fungal causal agent of yellow rust. The altered phenotype of I3-48 is due to a minimum of two mutation events, each showing a partial, additive effect, with one mutation segregating with a deletion on the long arm of chromosome 4D. In the case of I3-54, the enhanced resistance is due to a single, dominant mutation. In both mutants, the expression of the enhanced resistance is growth-stage specific. With I3-54, the full resistance phenotype is apparent from the third seedling leaf onwards, while with I3-48, a full resistance phenotype is only seen on the tenth and subsequent leaves. In addition to the enhanced resistance towards yellow rust, I3-48 also shows enhanced resistance towards brown rust, and I3-54 shows enhanced resistance to powdery mildew.

  1. First genetic quantification of sex- and stage-specific feeding in the ubiquitous copepod Acartia tonsa

    DEFF Research Database (Denmark)

    Ismar, Stefanie M.H.; Kottmann, Johanna Sarah; Sommer, Ulrich

    2018-01-01

    can complement classic diet quantification methods, such as stable isotope or fatty acid analyses tools. Here, we present first results of feeding experiments assessing sex- and stage-specific food intake by the ubiquitous calanoid copepod Acartia tonsa by 18S targeted qPCR and microscopic grazing......Marine copepods provide the major food-web link between primary producers and higher trophic levels, and their feeding ecology is of acute interest in light of global change impacts on food-web functioning. Recently, quantitative polymerase chain reaction (qPCR) protocols have been developed, which...... assessment. In triplicated mixed-diet feeding treatments, three suitable A. tonsa diets, the cryptophyte Rhodomonas balthica, the haptophyte Isochrysis galbana, and the diatom Thalassiosira weissflogii, were offered in equal biomass proportions under constant conditions. Prey uptake substantially varied...

  2. Identification of differentiation-stage specific molecular markers for the osteoblastic phenotype

    DEFF Research Database (Denmark)

    Twine, Natalie; Chen, Li; Wilkins, Marc

    to age-matched control (n=4). Using RNA-seq and cluster analysis, we identified a set of stage-specific molecular markers that define the progression of OB phenotype during ex vivo culture of hMSC, predict in vivo bone formation capacity of hMSC and can be employed to study the mechanisms of impaired......The phenotype of osteoblastic (OB) cells in culture is currently defined using a limited number of markers of low sensitivity and specificity which belong mostly to extracellular matrix proteins. Also, for clinical use of human skeletal (mesenchymal) stem cells (hMSC) in bone regeneration......, there is a need to identify predictive markers for in vivo bone forming capacity. Thus, we employed Illumina RNA sequencing (RNASeq) to examine changes in gene expression across 8 time points between 0-12 days of ex vivo OB differentiation of hMSC. We identified a subset of expressed genes as potentially...

  3. A proposed staging system and stage-specific interventions for familial adenomatous polyposis

    DEFF Research Database (Denmark)

    Lynch, Patrick M; Morris, Jeffrey S; Wen, Sijin

    2016-01-01

    BACKGROUND: It is not possible to accurately count adenomas in many patients with familial adenomatous polyposis (FAP). Nevertheless, polyp counts are critical in evaluating each patient's response to interventions. However, the U.S. Food and Drug Administration no longer recognizes the decrease...... classification scheme for lower GI tract polyposis. METHODS: Twenty-four colonoscopy or sigmoidoscopy videos were reviewed by 26 clinicians familiar with diagnosis and treatment of FAP. The reviewers independently assigned a stage to a case using the proposed system and chose a stage-specific intervention...... in the review of individual cases of polyposis. Therefore, reliable and clinically relevant means for measuring trial outcomes can be developed. Outlier cases showing wide scatter in stage assignment call for individualized attention and may be inappropriate for enrollment in clinical trials for this reason....

  4. Tomato Yield and Water Use Efficiency - Coupling Effects between Growth Stage Specific Soil Water Deficits

    DEFF Research Database (Denmark)

    Chen, Si; Zhenjiang, Zhou; Andersen, Mathias Neumann

    2015-01-01

    To investigate the sensitivity of tomato yield and water use efficiency (WUE) to soil water content at different growth stages, the central composite rotatable design (CCRD) was employed in a five-factor-five-level pot experiment under regulated deficit irrigation. Two regression models concerning...... the effects of stage-specific soil water content on tomato yield and WUE were established. The results showed that the lowest available soil water (ASW) content (around 28%) during vegetative growth stage (here denoted θ1) resulted in high yield and WUE. Moderate (around 69% ASW) during blooming and fruit...... effects of ASW in two growth stages were between θ2 and θ5, θ3. In both cases a moderate θ2 was a precondition for maximum yield response to increasing θ5 and θ3. Sensitivity analysis revealed that yield was most sensitive to soil water content at fruit maturity (θ5). Numerical inspection...

  5. NF-Y recruits both transcription activator and repressor to modulate tissue- and developmental stage-specific expression of human γ-globin gene.

    Directory of Open Access Journals (Sweden)

    Xingguo Zhu

    Full Text Available The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.

  6. Electrophoretic analysis of the "cross-class" interaction between novel inhibitory serpin, squamous cell carcinoma antigen-1 and cysteine proteinases.

    Science.gov (United States)

    Nawata, S; Nakamura, K; Tanaka, T; Numa, F; Suminami, Y; Tsunaga, N; Kakegawa, H; Katunuma, N; Kato, H

    1997-05-01

    We investigated the "cross-class" interaction between cysteine proteinases and a novel inhibitory serpin, recombinant squamous cell carcinoma (rSCC) antigen-1, which inhibits a serine proteinase, chymotrypsin. rSCC antigen-1 inhibited the cysteine proteinases, papain, papaya proteinase IV and cathepsin L. Interestingly, although rSCC antigen-1 formed sodium dodecyl sulfate (SDS)- and heat-stable complexes with chymotrypsin, rSCC antigen-1 gave the 40 kDa fragment and small molecular mass peptide by incubation with papain without forming an SDS- and heat-stable complex. The cleavage was observed between the Gly353-Ser354 bond, indicating that rSCC antigen-1 interacts with cysteine proteinases not at the predicted reactive site P1-P1' portion (Ser354-Ser355), but at the Gly353-Ser354 of the P2-P1 portion. These findings promote understanding of the "suicide inhibition" mechanism of SCC antigen-1 against cysteine proteinases.

  7. Fetal antigen 1 (FA1), a circulating member of the epidermal growth factor (EGF) superfamily

    DEFF Research Database (Denmark)

    Jensen, Charlotte Harken; Krogh, T N; Støving, René Klinkby

    1997-01-01

    We describe an ELISA technique for quantification of fetal antigen 1 (FA1), a glycoprotein belonging to the EGF-superfamily. The ELISA is based on immunospecifically purified polyclonal antibodies and has a dynamic range of 0.7-5.3 ng/ml, intra- and inter-assay C.V.s of less than 3.2% and an aver...

  8. Stage-specific distribution of oxidative radicals and antioxidant enzymes in the midgut of Leptinotarsa decemlineata.

    Science.gov (United States)

    Krishnan, Natraj; Kodrík, Dalibor; Turanli, Ferit; Sehnal, Frantisek

    2007-01-01

    The titers of reactive oxygen species (ROS) represented by superoxide anion and general peroxides, and the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), are regulated in the midgut of the Colorado potato beetle (CPB) relative to the gut compartment, developmental stage, and food intake. ROS concentration is low in the potato leaves but it is very high in their digest in insect's anterior midgut. It is proposed that intensive ROS production in this gut region is linked to the processing of allelochemicals. SOD and CAT activities, low oxygen tension, and unidentified redox systems that maintain a slightly reducing milieu in the midgut lumen (pe+pH=6.95 declining to 5.36), obviously contribute to the decrease of ROS concentration along the gut length to a minimum in the wall of posterior midgut region. SOD and CAT activities are higher in the potato leaves than in the midgut tissues but the role of plant enzymes in ROS elimination within the gut lumen remains to be shown. A lower level of ROS and a higher antioxidant potential in the adult than in the larval midgut indicate stage specificity in the management of oxidative stress. The antioxidant defense is high in the diapausing adults that contain no detectable superoxide and about ten times less peroxides than the reproducing adults.

  9. Analysis of Stage-Specific Gene Expression Profiles in the Uterine Endometrium during Pregnancy in Pigs.

    Science.gov (United States)

    Kim, Mingoo; Seo, Heewon; Choi, Yohan; Yoo, Inkyu; Seo, Minseok; Lee, Chang-Kyu; Kim, Heebal; Ka, Hakhyun

    2015-01-01

    The uterine endometrium plays a critical role in regulating the estrous cycle and the establishment and maintenance of pregnancy in mammalian species. Many studies have investigated the expression and function of genes in the uterine endometrium, but the global expression pattern of genes and relationships among genes differentially expressed in the uterine endometrium during gestation in pigs remain unclear. Thus, this study investigated global gene expression profiles using microarray in pigs. Diverse transcriptome analyses including clustering, network, and differentially expressed gene (DEG) analyses were performed to detect endometrial gene expression changes during the different gestation stages. In total, 6,991 genes were found to be differentially expressed by comparing genes expressed on day (D) 12 of pregnancy with those on D15, D30, D60, D90 and D114 of pregnancy, and clustering analysis of detected DEGs distinguished 8 clusters. Furthermore, several pregnancy-related hub genes such as ALPPL2, RANBP17, NF1B, SPP1, and CST6 were discovered through network analysis. Finally, detected hub genes were technically validated by quantitative RT-PCR. These results suggest the complex network characteristics involved in uterine endometrial gene expression during pregnancy and indicate that diverse patterns of stage-specific gene expression and network connections may play a critical role in endometrial remodeling and in placental and fetal development to establish and maintenance of pregnancy in pigs.

  10. Analysis of Stage-Specific Gene Expression Profiles in the Uterine Endometrium during Pregnancy in Pigs.

    Directory of Open Access Journals (Sweden)

    Mingoo Kim

    Full Text Available The uterine endometrium plays a critical role in regulating the estrous cycle and the establishment and maintenance of pregnancy in mammalian species. Many studies have investigated the expression and function of genes in the uterine endometrium, but the global expression pattern of genes and relationships among genes differentially expressed in the uterine endometrium during gestation in pigs remain unclear. Thus, this study investigated global gene expression profiles using microarray in pigs. Diverse transcriptome analyses including clustering, network, and differentially expressed gene (DEG analyses were performed to detect endometrial gene expression changes during the different gestation stages. In total, 6,991 genes were found to be differentially expressed by comparing genes expressed on day (D 12 of pregnancy with those on D15, D30, D60, D90 and D114 of pregnancy, and clustering analysis of detected DEGs distinguished 8 clusters. Furthermore, several pregnancy-related hub genes such as ALPPL2, RANBP17, NF1B, SPP1, and CST6 were discovered through network analysis. Finally, detected hub genes were technically validated by quantitative RT-PCR. These results suggest the complex network characteristics involved in uterine endometrial gene expression during pregnancy and indicate that diverse patterns of stage-specific gene expression and network connections may play a critical role in endometrial remodeling and in placental and fetal development to establish and maintenance of pregnancy in pigs.

  11. Drosophila Chk2 and p53 proteins induce stage -specific cell death independently during oogenesis

    Science.gov (United States)

    Bakhrat, Anna; Pritchett, Tracy; Peretz, Gabriella; McCall, Kimberly; Abdu, Uri

    2011-01-01

    In Drosophila, the checkpoint protein-2 kinase (DmChk2) and its downstream effector protein, Dmp53, are required for DNA damage-mediated cell cycle arrest, DNA repair and apoptosis. In this study we focus on understanding the function of these two apoptosis inducing factors during ovarian development. We found that expression of Dmp53, but not DmChk2, led to loss of ovarian stem cells. We demonstrate that expression of DmChk2, but not Dmp53, induced mid-oogenesis cell death. DmChk2 induced cell death was not suppressed by Dmp53 mutant, revealing for the first time that in Drosophila, overexpression of DmChk2 can induce cell death which is independent of Dmp53. We found that over-expression of caspase inhibitors such as DIAP1, p35 and p49 did not suppress DmChk2- and Dmp53-induced cell death. Thus, our study reveals stage -specific effects of Dmp53 and DmChk2 in oogenesis. Moreover, our results demonstrate that although DmChk2 and Dmp53 affect different stages of ovarian development, loss of ovarian stem cells by p53 expression and mid-oogenesis cell death induced by DmChk2 do not require caspase activity. PMID:20838898

  12. The NIMA Kinase Is Required To Execute Stage-Specific Mitotic Functions after Initiation of Mitosis

    Science.gov (United States)

    Govindaraghavan, Meera; Lad, Alisha A.

    2014-01-01

    The G2-M transition in Aspergillus nidulans requires the NIMA kinase, the founding member of the Nek kinase family. Inactivation of NIMA results in a late G2 arrest, while overexpression of NIMA is sufficient to promote mitotic events independently of cell cycle phase. Endogenously tagged NIMA-GFP has dynamic mitotic localizations appearing first at the spindle pole body and then at nuclear pore complexes before transitioning to within nuclei and the mitotic spindle and back at the spindle pole bodies at mitotic exit, suggesting that it functions sequentially at these locations. Since NIMA is indispensable for mitotic entry, it has been difficult to determine the requirement of NIMA for subaspects of mitosis. We show here that when NIMA is partially inactivated, although mitosis can be initiated, a proportion of cells fail to successfully generate two daughter nuclei. We further define the mitotic defects to show that normal NIMA function is required for the formation of a bipolar spindle, nuclear pore complex disassembly, completion of chromatin segregation, and the normal structural rearrangements of the nuclear envelope required to generate two nuclei from one. In the remaining population of cells that enter mitosis with inadequate NIMA, two daughter nuclei are generated in a manner dependent on the spindle assembly checkpoint, indicating highly penetrant defects in mitotic progression without sufficient NIMA activity. This study shows that NIMA is required not only for mitotic entry but also sequentially for successful completion of stage-specific mitotic events. PMID:24186954

  13. Stage-specific effects of FGF2 on the differentiation of dental pulp cells

    Science.gov (United States)

    Sagomonyants, Karen; Mina, Mina

    2015-01-01

    Dentinogenesis is a complex and multistep process, which is regulated by various growth factors, including members of the Fibroblast Growth Factor (FGF) family. Both positive and negative effects of FGFs on dentinogenesis have been reported but the underlying mechanisms of these conflicting results are still unclear. To gain better insight into the role of FGF2 in dentinogenesis, we used dental pulp cells from various transgenic mice, in which fluorescent protein expression identifies cells at different stages of odontoblast differentiation. Our results showed that continuous exposure of pulp cells to FGF2 inhibited mineralization and revealed both stimulatory and inhibitory effects of FGF2 on expression of markers of dentinogenesis and various transgenes. During the proliferation phase of in vitro growth FGF2 increased expression of markers of dentinogenesis and the percentages of DMP1-GFP+ functional odontoblasts and DSPP-Cerulean+ odontoblasts. Additional exposure to FGF2 during the differentiation/mineralization phase of in vitro growth decreased the extent of mineralization, expression of markers of dentinogenesis, and expression of DMP1-GFP and DSPP-Cerulean transgenes. Recovery experiments showed that the inhibitory effects of FGF2 on dentinogenesis were related to the blocking of differentiation of cells into mature odontoblasts. These observations together showed stage-specific effects of FGF2 on dentinogenesis by dental pulp cells and provide critical information for the development of improved treatments for vital pulp therapy and dentin regeneration. PMID:25823776

  14. Stage-Specific Changes in Physiological and Life-History Responses to Elevated Temperature and Pco2 during the Larval Development of the European Lobster Homarus gammarus (L.).

    Science.gov (United States)

    Small, Daniel P; Calosi, Piero; Boothroyd, Dominic; Widdicombe, Steve; Spicer, John I

    2015-01-01

    An organism's physiological processes form the link between its life-history traits and the prevailing environmental conditions, especially in species with complex life cycles. Understanding how these processes respond to changing environmental conditions, thereby affecting organismal development, is critical if we are to predict the biological implications of current and future global climate change. However, much of our knowledge is derived from adults or single developmental stages. Consequently, we investigated the metabolic rate, organic content, carapace mineralization, growth, and survival across each larval stage of the European lobster Homarus gammarus, reared under current and predicted future ocean warming and acidification scenarios. Larvae exhibited stage-specific changes in the temperature sensitivity of their metabolic rate. Elevated Pco2 increased C∶N ratios and interacted with elevated temperature to affect carapace mineralization. These changes were linked to concomitant changes in survivorship and growth, from which it was concluded that bottlenecks were evident during H. gammarus larval development in stages I and IV, the transition phases between the embryonic and pelagic larval stages and between the larval and megalopa stages, respectively. We therefore suggest that natural changes in optimum temperature during ontogeny will be key to larvae survival in a future warmer ocean. The interactions of these natural changes with elevated temperature and Pco2 significantly alter physiological condition and body size of the last larval stage before the transition from a planktonic to a benthic life style. Thus, living and growing in warm, hypercapnic waters could compromise larval lobster growth, development, and recruitment.

  15. Tissue and stage-specific distribution of Wolbachia in Brugia malayi.

    Directory of Open Access Journals (Sweden)

    Kerstin Fischer

    2011-05-01

    Full Text Available BACKGROUND: Most filarial parasite species contain Wolbachia, obligatory bacterial endosymbionts that are crucial for filarial development and reproduction. They are targets for alternative chemotherapy, but their role in the biology of filarial nematodes is not well understood. Light microscopy provides important information on morphology, localization and potential function of these bacteria. Surprisingly, immunohistology and in situ hybridization techniques have not been widely used to monitor Wolbachia distribution during the filarial life cycle. METHODS/PRINCIPAL FINDINGS: A monoclonal antibody directed against Wolbachia surface protein and in situ hybridization targeting Wolbachia 16S rRNA were used to monitor Wolbachia during the life cycle of B. malayi. In microfilariae and vector stage larvae only a few cells contain Wolbachia. In contrast, large numbers of Wolbachia were detected in the lateral chords of L4 larvae, but no endobacteria were detected in the genital primordium. In young adult worms (5 weeks p.i., a massive expansion of Wolbachia was observed in the lateral chords adjacent to ovaries or testis, but no endobacteria were detected in the growth zone of the ovaries, uterus, the growth zone of the testis or the vas deferens. Confocal laser scanning and transmission electron microscopy showed that numerous Wolbachia are aligned towards the developing ovaries and single endobacteria were detected in the germline. In inseminated females (8 weeks p.i. Wolbachia were observed in the ovaries, embryos and in decreasing numbers in the lateral chords. In young males Wolbachia were found in distinct zones of the testis and in large numbers in the lateral chords in the vicinity of testicular tissue but never in mature spermatids or spermatozoa. CONCLUSIONS: Immunohistology and in situ hybridization show distinct tissue and stage specific distribution patterns for Wolbachia in B. malayi. Extensive multiplication of Wolbachia occurs in the

  16. Developmental Stage-Specific Manifestations of Absent TPO/c-MPL Signalling in Newborn Mice.

    Science.gov (United States)

    Lorenz, Viola; Ramsey, Haley; Liu, Zhi-Jian; Italiano, Joseph; Hoffmeister, Karin; Bihorel, Sihem; Mager, Donald; Hu, Zhongbo; Slayton, William B; Kile, Benjamin T; Sola-Visner, Martha; Ferrer-Marin, Francisca

    2017-12-01

    Congenital amegakaryocytic thrombocytopaenia (CAMT) is a disorder caused by c-MPL mutations that impair thrombopoietin (TPO) signalling, resulting in a near absence of megakaryocytes (MKs). While this phenotype is consistent in adults, neonates with CAMT can present with severe thrombocytopaenia despite normal MK numbers. To investigate this, we characterized MKs and platelets in newborn c-MPL –/– mice. Liver MKs in c-MPL –/– neonates were reduced in number and size compared with wild-type (WT) age-matched MKs, and exhibited ultrastructural abnormalities not found in adult c-MPL –/– MKs. Platelet counts were lower in c-MPL –/– compared with WT mice at birth and did not increase over the first 2 weeks of life. In vivo biotinylation revealed a significant reduction in the platelet half-life of c-MPL –/– newborn mice (P2) compared with age-matched WT pups, which was not associated with ultrastructural abnormalities. Genetic deletion of the pro-apoptotic Bak did not rescue the severely reduced platelet half-life of c-MPL –/– newborn mice, suggesting that it was due to factors other than platelets entering apoptosis early. Indeed, adult GFP+ (green fluorescent protein transgenic) platelets transfused into thrombocytopenic c-MPL –/– P2 pups also had a shortened lifespan, indicating the importance of cell-extrinsic factors. In addition, neonatal platelets from WT and c-MPL –/– mice exhibited reduced P-selectin surface expression following stimulation compared with adult platelets of either genotype, and platelets from c-MPL –/– neonates exhibited reduced glycoprotein IIb/IIIa (GPIIb/IIIa) activation in response to thrombin compared with age-matched WT platelets. Taken together, our findings indicate that c-MPL deficiency is associated with abnormal maturation of neonatal MKs and developmental stage-specific defects in platelet function.

  17. The Case for Stage-Specific Frailty Interventions Spanning Community Aging to Cognitive Impairment.

    Science.gov (United States)

    Chong, Mei Sian; Tay, Laura; Ismail, Noor Hafizah; Tan, Chay Hoon; Yew, Suzanne; Yeo, Audrey; Ye, Ruijing; Leung, Bernard; Ding, Yew Yoong

    2015-11-01

    To explore factors associated with frailty across the continuum of healthy aging to cognitive impairment (mild cognitive impairment [MCI], mild and moderate Alzheimer disease [AD]). Cross-sectional study. Senior activity centers and the outpatient memory clinic of a tertiary hospital. Community-dwelling and functionally independent adults aged 50 years and older and older adults attending the memory clinic with MCI, and mild and moderate AD diagnoses. We recruited 299 participants comprising 200 cognitively healthy individuals, 16 with MCI, 68 with mild AD, and 15 with moderate AD. We collected measures of comorbidities, cognitive and functional performance, physical activity level, and anthropometric and nutritional status. Frailty was defined using Buchmann criteria, and sarcopenic obesity (SO) was defined using the Asian Working Group for Sarcopenia criteria and the revised National Cholesterol and Education Panel-obesity definition of waist circumference. Multiple logistic regression was performed to identify factors associated with frailty as a whole group and separately based on cognitive subgroups. There were 16.7% of patients who met frailty criteria. Frailty prevalence was lowest in the well elderly (3.5%) and subsequently followed a U-shaped prevalence from MCI to mild and moderate AD, respectively. Specific univariate differences were noted in age, hypertension, ischemic heart disease, depressive symptoms, social differences, and functional scores. Multivariable logistic regression showed age, cognitive status, and SO to be significantly associated with frailty status. Subgroup analysis showed only SO to be significant (odds ratio [OR] 15.55, 95% confidence interval [CI] 1.63-148.42) in well elderly and only cognition to be associated with frailty (OR 0.89, 95% CI 0.80-0.99) among the cognitively impaired. Our findings lend initial support to the case for stage-specific interventions for physical frailty with the focus on SO in healthy community

  18. Life-stage-specific physiology defines invasion extent of a riverine fish

    Science.gov (United States)

    Lawrence, David J.; Beauchamp, David A.; Olden, Julian D.

    2015-01-01

    -history stage that likely sets the distributional limits of all other life-history stages. We anticipate the framework developed here could be employed to identify how similar stage-specific environmental sensitivity determines distribution in many other ectothermic species.

  19. Humanin protects against chemotherapy-induced stage-specific male germ cell apoptosis in rats.

    Science.gov (United States)

    Surampudi, P; Chang, I; Lue, Y; Doumit, T; Jia, Y; Atienza, V; Liu, P Y; Swerdloff, R S; Wang, C

    2015-05-01

    Humanin (HN) has cytoprotective action on male germ cells after testicular stress induced by heat and hormonal deprivation. To examine whether HN has protective effects on chemotherapy-induced male germ cell apoptosis, we treated four groups of adult rats with (i) vehicle (control), (ii) HN, (iii) cyclophosphamide (CP); or (iv) HN+CP. To investigate whether the protective effects of HN on germ cells require the presence of Leydig cells, another four groups of rats were pre-treated with ethane dimethanesulfonate (EDS), a Leydig cell toxicant, to eliminate Leydig cells. After 3 days, when Leydig cells were depleted by EDS, we administered: (i) vehicle, (ii) HN, (iii) CP; or (iv) HN+CP to rats. All rats were killed 12 h after the injection of HN and/or CP. Germ cell apoptosis was detected by TUNEL assay and quantified by numerical count. Compared with control and HN (alone), CP significantly increased germ cell apoptosis; HN +CP significantly reduced CP-induced apoptosis at early (I-VI) and late stages (IX-XIV) but not at middle stages (VII-VIII) of the seminiferous epithelial cycle. Pre-treatment with EDS markedly suppressed serum and intratesticular testosterone (T) levels, and significantly increased germ cell apoptosis at the middle (VII-VIII) stages. CP did not further increase germ cell apoptosis in the EDS-pre-treated rats. HN significantly attenuated germ cell apoptosis at the middle stages in EDS pre-treated rats. To investigate whether HN has any direct effects on Leydig cell function, adult Leydig cells were isolated and treated with ketoconazole (KTZ) to block testosterone synthesis. HN was not effective in preventing the reduction of T production by KTZ in vitro. We conclude that HN decreases CP and/or EDS-induced germ cell apoptosis in a stage-specific fashion. HN acts directly on germ cells to protect against EDS-induced apoptosis in the absence of Leydig cells and intratesticular testosterone levels are very low. © 2015 American Society of Andrology

  20. Humanin protects against chemotherapy-induced stage-specific male germ cell apoptosis in rats*

    Science.gov (United States)

    Lue, Y.; Doumit, T.; Jia, Y.; Atienza, V.; Liu, P. Y.; Swerdloff, R. S.; Wang, C.

    2016-01-01

    SUMMARY Humanin (HN) has cytoprotective action on male germ cells after testicular stress induced by heat and hormonal deprivation. To examine whether HN has protective effects on chemotherapy-induced male germ cell apoptosis, we treated four groups of adult rats with (i) vehicle (control), (ii) HN, (iii) cyclophosphamide (CP); or (iv) HN+CP. To investigate whether the protective effects of HN on germ cells require the presence of Leydig cells, another four groups of rats were pre-treated with ethane dimethanesulfonate (EDS), a Leydig cell toxicant, to eliminate Leydig cells. After 3 days, when Leydig cells were depleted by EDS, we administered: (i) vehicle, (ii) HN, (iii) CP; or (iv) HN+CP to rats. All rats were killed 12 h after the injection of HN and/or CP. Germ cell apoptosis was detected by TUNEL assay and quantified by numerical count. Compared with control and HN (alone), CP significantly increased germ cell apoptosis; HN +CP significantly reduced CP-induced apoptosis at early (I–VI) and late stages (IX–XIV) but not at middle stages (VII–VIII) of the seminiferous epithelial cycle. Pre-treatment with EDS markedly suppressed serum and intratesticular testosterone (T) levels, and significantly increased germ cell apoptosis at the middle (VII–VIII) stages. CP did not further increase germ cell apoptosis in the EDS-pre-treated rats. HN significantly attenuated germ cell apoptosis at the middle stages in EDS pre-treated rats. To investigate whether HN has any direct effects on Leydig cell function, adult Leydig cells were isolated and treated with ketoconazole (KTZ) to block testosterone synthesis. HN was not effective in preventing the reduction of T production by KTZ in vitro. We conclude that HN decreases CP and/or EDS-induced germ cell apoptosis in a stage-specific fashion. HN acts directly on germ cells to protect against EDS-induced apoptosis in the absence of Leydig cells and intratesticular testosterone levels are very low. PMID:25891800

  1. Transcription factor Fos-related antigen 1 is an effective target for a breast cancer vaccine

    Science.gov (United States)

    Luo, Yunping; Zhou, He; Mizutani, Masato; Mizutani, Noriko; Reisfeld, Ralph A.; Xiang, Rong

    2003-07-01

    Protection against breast cancer was achieved with a DNA vaccine against murine transcription factor Fos-related antigen 1, which is overexpressed in aggressively proliferating D2F2 murine breast carcinoma. Growth of primary s.c. tumor and dissemination of pulmonary metastases was markedly suppressed by this oral DNA vaccine, carried by attenuated Salmonella typhimurium, encoding murine Fos-related antigen 1, fused with mutant polyubiquitin, and cotransformed with secretory murine IL-18. The life span of 60% of vaccinated mice was tripled in the absence of detectable tumor growth after lethal tumor cell challenge. Immunological mechanisms involved activation of T, natural killer, and dendritic cells, as indicated by up-regulation of their activation markers and costimulatory molecules. Markedly increased specific target cell lysis was mediated by both MHC class I-restricted CD8+ T cells and natural killer cells isolated from splenocytes of vaccinated mice, including a significant release of proinflammatory cytokines IFN- and IL-2. Importantly, fluorescence analysis of fibroblast growth factor 2 and tumor cell-induced vessel growth in Matrigel plugs demonstrated marked suppression of angiogenesis only in vaccinated animals. Taken together, this multifunctional DNA vaccine proved effective in protecting against growth and metastases of breast cancer by combining the action of immune effector cells with suppression of tumor angiogenesis. vaccine | tumor | metastases | antiangiogenesis

  2. TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors.

    Science.gov (United States)

    Cebola, Inês; Rodríguez-Seguí, Santiago A; Cho, Candy H-H; Bessa, José; Rovira, Meritxell; Luengo, Mario; Chhatriwala, Mariya; Berry, Andrew; Ponsa-Cobas, Joan; Maestro, Miguel Angel; Jennings, Rachel E; Pasquali, Lorenzo; Morán, Ignasi; Castro, Natalia; Hanley, Neil A; Gomez-Skarmeta, Jose Luis; Vallier, Ludovic; Ferrer, Jorge

    2015-05-01

    The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.

  3. Does Fetal antigen 1 (FA1) identify cells with regenerative, endocrine and neuroendocrine potentials?

    DEFF Research Database (Denmark)

    Floridon, C.; Jensen, C.H.; Thorsen, P.

    2000-01-01

    -embryonic tissues from normal and pathological pregnancies revealed FA1 in stromal cells surrounding the blood islands of the yolk sac as well as in placental fibroblasts where the expression was most pronounced in diploid, androgenic complete hydatidiform moles. However, as measured by ELISA, the circulating...

  4. Toxicity of silver nanoparticles in mouse embryonic stem cells and chemical based reprogramming of somatic cells to sphere cells

    Science.gov (United States)

    Rajanahalli Krishnamurthy, Pavan

    successfully manipulated by ectopic expression of defined factors. We demonstrate that mouse fibroblasts can be converted into sphere cells by detaching fibroblast cells by proteases and then using AlbuMAX I-containing culture medium without genetic alteration. AlbuMAX I is a lipid-rich albumin. Albumin-associated lipids arachidonic acid (AA) and pluronic F-68 were responsible for this effect. The converted colonies were positive for both alkaline phosphatase and stage specific embryonic antigen-1 (SSEA-1) staining. Global gene expression analysis indicated that the sphere cells were in an intermediate state compared with MES cells and MEF cells. The sphere cells were able to differentiate into tissues representing all three embryonic germ layers following retinoic acid treatment, and also differentiated into smooth muscle cells following treatment with vascular endothelial growth factor (VEGF). The study presented a potential novel approach to transdifferentiate mouse fibroblast cells into other cell lineages mediated by AlbuMAX I-containing culture medium.

  5. Role of very late antigen-1 in T-cell-mediated immunity to systemic viral infection

    DEFF Research Database (Denmark)

    Ørding Kauffmann, Susanne; Thomsen, Allan Randrup; Christensen, Jan Pravsgaard

    2006-01-01

    The T-cell response to lymphocytic choriomeningitis virus was studied in mice lacking very late antigen-1 (VLA-1). The generation of virus-specific effector T cells was unimpaired in VLA-1(-/-) mice. In the memory phase, VLA-1 deficiency did not influence the number of memory CD8(+) T cells or th......, the current findings indicate that the expression of VLA-1 is not pivotal for T-cell-mediated antiviral immunity to a systemic infection....... or their distribution between lymphoid and nonlymphoid organs. Regarding a functional role of VLA-1, we found that intracerebral infection of both VLA-1(-/-) and wild-type (wt) mice resulted in lethal T-cell-mediated meningitis, and quantitative and qualitative analyses of the cellular exudate did not reveal any...

  6. Stage-specific control of neural crest stem cell proliferation by the small rho GTPases Cdc42 and Rac1

    DEFF Research Database (Denmark)

    Fuchs, Sebastian; Herzog, Dominik; Sumara, Grzegorz

    2009-01-01

    -renewal and proliferation of later stage, but not early migratory NCSCs. This stage-specific requirement for small Rho GTPases is due to changes in NCSCs that, during development, acquire responsiveness to mitogenic EGF acting upstream of both Cdc42 and Rac1. Thus, our data reveal distinct mechanisms for growth control......The neural crest (NC) generates a variety of neural and non-neural tissues during vertebrate development. Both migratory NC cells and their target structures contain cells with stem cell features. Here we show that these populations of neural crest-derived stem cells (NCSCs) are differentially...

  7. Life-stage-specific differences in exploitation of food mixtures: diet mixing enhances copepod egg production but not juvenile development

    DEFF Research Database (Denmark)

    Koski, Marja; Breteler, W.K.; Schogt, N.

    2006-01-01

    mortality were, however, independent of either nitrogen or HUFAs in the diet. Our results show that adult copepods are effective in combining their nutrition from several food sources, whereas juveniles are not. We suggest that there are species- and life-stage-specific differences in nutritional......, the copepods failed to moult past the first copepodite stage, and the mortality was high. In sharp contrast, mixing two nutritionally poor food species often resulted in egg production which was not significantly different from nutritionally high quality food, although hatching success in many mixtures was low...

  8. Embryonic epithelial membrane transporters.

    Science.gov (United States)

    Horster, M

    2000-12-01

    Embryonic epithelial membrane transporters are organized into transporter families that are functional in several epithelial organs, namely, in kidney, lung, pancreas, intestine, and salivary gland. Family members (subtypes) are developmentally expressed in plasma membranes in temporospatial patterns that are 1) similar for one subtype within different organs, like aquaporin-1 (AQP1) in lung and kidney; 2) different between subtypes within the same organ, like the amiloride-sensitive epithelial sodium channel (ENaC) in lung; and 3) apparently matched among members of different transporter families, as alpha-ENaC with AQP1 and -4 in lung and with AQP2 in kidney. Finally, comparison of temporal expression patterns in early embryonic development of transporters from different families [e.g., cystic fibrosis transmembrane conductance regulator (CFTR), ENaC, and outer medullary potassium channel] suggests regulatory activating or inactivating interactions in defined morphogenic periods. This review focuses on embryonic patterns, at the mRNA and immunoprotein level, of the following transporter entities expressed in epithelial cell plasma membranes: ENaC; the chloride transporters CFTR, ClC-2, bumetanide-sensitive Na-K-Cl cotransporter, Cl/OH, and Cl/HCO(3); the sodium glucose transporter-glucose transporter; the sodium/hydrogen exchanger; the sodium-phosphate cotransporter; the ATPases; and AQP. The purpose of this article is to relate temporal and spatial expression patterns in embryonic and in early postnatal epithelia to developmental changes in organ structure and function.

  9. 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...

  10. Development of physiological resistance and its stage specificity in Culex quinquefasciatus after selection with deltamethrin in Assam, India

    Directory of Open Access Journals (Sweden)

    Manas Sarkar

    2009-08-01

    Full Text Available The study investigated the development and stage specificity of physiological resistance to insecticides in a colony of Culex quinquefasciatus Say (Diptera: Culicidae mosquitoes, which are vectors of bancroftian filariasis in India, after selection with deltamethrin. Resistance was selected by exposing the larvae to the concentration of deltamethrin that caused 50% mortality in the tested population (i.e., LC50. Under continuous selection pressure, the LC50 increased steadily in subsequent generations. The estimated LC50 for the F0 generation was 0.409 μg/L; the LC50 first displayed a substantial increase in the F5 generation (5.616 μg/L and reached 121.902 μg/L in the F10 generation. The objective of this study was to establish a deltamethrin-resistant colony to develop a research programme that will study the evolution of physiological resistance patterns and stage-specific resistance responses in Cx. quinquefasciatus larvae and adults under laboratory conditions. An approximately 298-fold increase in resistance was recorded after 10 generations, as evidenced by the resistance ratio (RR50. The progress and effect of the selection pressure in the adult stage was monitored with the World Health Organisation (WHO diagnostic test. The mortality, as observed using the WHO diagnostic test, declined significantly from the F5 generation (85% onwards and the highest rate of survival (65% was observed in the F10 generation.

  11. Identification and quantitative analysis of stage-specific carbohydrates in loblolly pine (Pinus taeda) zygotic embryo and female gametophyte tissues.

    Science.gov (United States)

    Pullman, Gerald S; Buchanan, Mike

    2008-07-01

    Stage-specific analyses of starch and 18 sugars, including pentoses, hexoses, disaccharides, trisaccharides, oligosaccharides and sugar alcohols, were made throughout seed development for zygotic embryo and female gametophyte (FG) tissues of loblolly pine (Pinus taeda L.). Tissue was most often analyzed in triplicate from two open-pollinated families grown in different locations and sampled in different years. Carbohydrates were analyzed by enzymatic assay, high performance liquid chromatography or gas chromatography/mass spectrometry. For all carbohydrates quantified, peak concentrations were higher in embryo tissue than in FG tissue. Significant changes in starch and sugar concentrations occurred over time, with both seed collections showing similar trends in temporal changes. Although concentrations were not always similar, embryo and FG tissues generally showed similar patterns of change in starch and sugar concentrations over time. Total starch concentration was highest during early seed development and decreased as development progressed. The major sugars contributing to osmotic potential during early seed development were D-pinitol, sucrose, fructose and glucose. During mid-seed development, D-pinitol, sucrose, fructose, glucose, melibiose and raffinose provided major contributions to the osmotic environment. During late seed development, sucrose, raffinose, melibiose, stachyose and fructose were the major contributors to osmotic potential. These data suggest stage-specific media composition for each step in the somatic embryogenesis protocol.

  12. [Incidence of melanoma and changes in stage-specific incidence after implementation of skin cancer screening in Schleswig-Holstein].

    Science.gov (United States)

    Eisemann, N; Waldmann, A; Katalinic, A

    2014-01-01

    A pilot project in skin cancer screening (SCREEN) was conducted in Schleswig-Holstein from July 2003 to June 2004. Although the impact of this screening on the stage-specific incidence of melanoma is of great importance for screening evaluation, it remains unknown. In theory, an effective skin cancer screening program should result in a medium-term incidence decrease of melanomas with a prognostically unfavorable stage. This is studied on a population-based level by using cancer registry data. Based on data from the Cancer Registry of Schleswig-Holstein for 1999-2009, stage-specific (T-category of the TNM-classification system) age-standardized incidence rates were calculated. After implementation of the SCREEN project, the incidence of prognostically favorable melanomas (in situ and T1) was higher than before, while the incidence of advanced melanomas (T2, T3, and for women also T4) decreased considerably. The classification of tumor stages changed during the project period, which may have contributed to an artificial decrease of the stages with a poor prognosis. Nevertheless, the results are in agreement with the observed decrease of melanoma mortality in the screening region.

  13. Cloning and stage-specific expression of CK-M1 gene during metamorphosis of Japanese flounder, Paralichthys olivaceus

    Science.gov (United States)

    Chen, Yanjie; Zhang, Quanqi; Qi, Jie; Wang, Zhigang; Wang, Xubo; Sun, Yeying; Zhong, Qiwang; Li, Shuo; Li, Chunmei

    2010-05-01

    The symmetrical body of flatfish larvae changes dramatically into an asymmetrical form after metamorphosis. The molecular mechanisms responsible for this change are poorly understood. As an initial step to clarify these mechanisms, we used representational difference analysis of cDNA for the identification of genes active during metamorphosis in the Japanese flounder, Paralichthys olicaceus. One of the up-regulated genes was identified as creatine kinase muscle type 1 (CK-M1). Sequence analysis of CK-M1 revealed that it spanned 1 708 bp and encoded a protein of 382 amino acids. The overall amino acid sequence of the CK-M1 was highly conserved with those of other organisms. CK-M1 was expressed in adult fish tissues, including skeletal muscle, intestine and gill. Whole mount in-situ hybridization showed that the enhanced expression of CK-M1 expanded from the head to the whole body of larvae as metamorphosis progressed. Quantitative analysis revealed stage-specific high expression of CK-M1 during metamorphosis. The expression level of CK-M1 increased initially and peaked at metamorphosis, decreased afterward, and finally returned to the pre-metamorphosis level. This stage-specific expression pattern suggested strongly that CK-M1 was related to metamorphosis in the Japanese flounder. Its specific role in metamorphosis requires further study.

  14. Hormesis and stage specific toxicity induced by cadmium in an insect model, the queen blowfly, Phormia regina Meig

    International Nuclear Information System (INIS)

    Nascarella, Marc A.; Stoffolano, John G.; Stanek, Edward J.; Kostecki, Paul T.; Calabrese, Edward J.

    2003-01-01

    This is the first report of a heavy metal displaying a hormetic-like biphasic response for early developmental success, while at the same time displaying stage-specific toxicity at a later developmental stage. - Hormesis is an adaptive response, commonly characterized by a biphasic dose-response that can be either directly induced, or the result of compensatory biological processes following an initial disruption in homeostasis [Calabrese and Baldwin, Hum. Exp. Toxicol., 21 (2002), 91]. Low and environmentally relevant levels of dietary cadmium significantly enhanced the pupation rate of blowfly larvae, while higher doses inhibited pupation success. However, dietary cadmium at all exposure levels adversely affected the emergence of the adult fly from the pupal case. Such findings represent the first report of a heavy metal displaying a hormetic-like biphasic response for pupation success, while at the same time displaying stage-specific toxicity at a later developmental period. These conclusions are based on substantial experimentation of over 1750 blowflies, in seven replicate experiments, involving 10 concentrations per experiment. These findings indicate the need to assess the impact of environmental stressors over a broad range of potential exposures as well as throughout the entire life cycle

  15. High environmental ammonia exposure has developmental-stage specific and long-term consequences on the cortisol stress response in zebrafish.

    Science.gov (United States)

    Williams, Tegan A; Bonham, Luke A; Bernier, Nicholas J

    2017-12-01

    The capacity for early life environmental stressors to induce programming effects on the endocrine stress response in fish is largely unknown. In this study we determined the effects of high environmental ammonia (HEA) exposure on the stress response in larval zebrafish, assessed the tolerance of embryonic and larval stages to HEA, and evaluated whether early life HEA exposure has long-term consequences on the cortisol response to a novel stressor. Exposure to 500-2000μM NH 4 Cl for 16h did not affect the gene expression of corticotropin-releasing factor (CRF) system components in 1day post-fertilization (dpf) embryos, but differentially increased crfa, crfb and CRF binding protein (crfbp) expression and stimulated both dose- and time-dependent increases in the whole body cortisol of 5dpf larvae. Pre-acclimation to HEA at 1dpf did not affect the cortisol response to a subsequent NH 4 Cl exposure at 5dpf. In contrast, pre-acclimation to HEA at 5dpf caused a small but significant reduction in the cortisol response to a second NH 4 Cl exposure at 10dpf. While continuous exposure to 500-2000μM NH 4 Cl between 0 and 5dpf had a modest effect on mean survival time, exposure to 400-1000μM NH 4 Cl between 10 and 14dpf decreased mean survival time in a dose-dependent manner. Moreover, pre-acclimation to HEA at 5dpf significantly decreased the risk of mortality to continuous NH 4 Cl exposure between 10 and 14dpf. Finally, while HEA at 1dpf did not affect the cortisol stress response to a novel vortex stressor at 5dpf, the same HEA treatment at 5dpf abolished vortex stressor-induced increases in whole body cortisol at 10 and 60dpf. Together these results show that the impact of HEA on the cortisol stress response during development is life-stage specific and closely linked to ammonia tolerance. Further, we demonstrate that HEA exposure at the larval stage can have persistent effects on the capacity to respond to stressors in later life. Copyright © 2017 Elsevier Inc. All

  16. Gravity and embryonic development

    Science.gov (United States)

    Young, R. S.

    1976-01-01

    The relationship between the developing embryo (both plant and animal) and a gravitational field has long been contemplated. The difficulty in designing critical experiments on the surface of the earth because of its background of 1 g, has been an obstacle to a resolution of the problem. Biological responses to gravity (particularly in plants) are obvious in many cases; however, the influence of gravity as an environmental input to the developing embryo is not as obvious and has proven to be extremely difficult to define. In spite of this, over the years numerous attempts have been made using a variety of embryonic materials to come to grips with the role of gravity in development. Three research tools are available: the centrifuge, the clinostat, and the orbiting spacecraft. Experimental results are now available from all three sources. Some tenuous conclusions are drawn, and an attempt at a unifying theory of gravitational influence on embryonic development is made.

  17. Estimation of age- and stage-specific Catalan breast cancer survival functions using US and Catalan survival data

    Directory of Open Access Journals (Sweden)

    Marcos-Gragera Rafael

    2009-03-01

    Full Text Available Abstract Background During the last part of the 1990s the chance of surviving breast cancer increased. Changes in survival functions reflect a mixture of effects. Both, the introduction of adjuvant treatments and early screening with mammography played a role in the decline in mortality. Evaluating the contribution of these interventions using mathematical models requires survival functions before and after their introduction. Furthermore, required survival functions may be different by age groups and are related to disease stage at diagnosis. Sometimes detailed information is not available, as was the case for the region of Catalonia (Spain. Then one may derive the functions using information from other geographical areas. This work presents the methodology used to estimate age- and stage-specific Catalan breast cancer survival functions from scarce Catalan survival data by adapting the age- and stage-specific US functions. Methods Cubic splines were used to smooth data and obtain continuous hazard rate functions. After, we fitted a Poisson model to derive hazard ratios. The model included time as a covariate. Then the hazard ratios were applied to US survival functions detailed by age and stage to obtain Catalan estimations. Results We started estimating the hazard ratios for Catalonia versus the USA before and after the introduction of screening. The hazard ratios were then multiplied by the age- and stage-specific breast cancer hazard rates from the USA to obtain the Catalan hazard rates. We also compared breast cancer survival in Catalonia and the USA in two time periods, before cancer control interventions (USA 1975–79, Catalonia 1980–89 and after (USA and Catalonia 1990–2001. Survival in Catalonia in the 1980–89 period was worse than in the USA during 1975–79, but the differences disappeared in 1990–2001. Conclusion Our results suggest that access to better treatments and quality of care contributed to large improvements in

  18. Regulatory elements involved in the post-transcriptional control of stage-specific gene expression in Trypanosoma cruzi: a review

    Directory of Open Access Journals (Sweden)

    Patricia R Araújo

    2011-05-01

    Full Text Available Trypanosoma cruzi, a protozoan parasite that causes Chagas disease, exhibits unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes, RNA editing and trans-splicing. In the absence of mechanism controlling transcription initiation, organized subsets of T. cruzi genes must be post-transcriptionally co-regulated in response to extracellular signals. The mechanisms that regulate stage-specific gene expression in this parasite have become much clearer through sequencing its whole genome as well as performing various proteomic and microarray analyses, which have demonstrated that at least half of the T. cruzi genes are differentially regulated during its life cycle. In this review, we attempt to highlight the recent advances in characterising cis and trans-acting elements in the T. cruzi genome that are involved in its post-transcriptional regulatory machinery.

  19. Stem cell antigen 1-positive mesenchymal cells are the origin of follicular cells during thyroid regeneration.

    Science.gov (United States)

    Okamoto, Minoru; Hayase, Suguru; Miyakoshi, Masaaki; Murata, Tsubasa; Kimura, Shioko

    2013-01-01

    Many tissues are thought to contain adult stem/progenitor cells that are responsible for repair of the tissue where they reside upon damage and/or carcinogenesis, conditions when cellular homeostasis becomes uncontrolled. While the presence of stem/progenitor cells of the thyroid has been suggested, how these cells contribute to thyroid regeneration remains unclear. Here we show the origin of thyroid follicular cells and the process of their maturation to become follicular cells during regeneration. By using β-galactosidase (β-gal) reporter mice in conjunction with partial thyroidectomy as a model for thyroid regeneration, and bromodeoxyuridine (BrdU) long label-retaining cell analysis, we demonstrated that stem cell antigen 1 (Sca1) and BrdU-positive, but β-gal and NKX2-1 negative cells were found in the non-follicular mesenchymal area 7 days after partial thyroidectomy. They temporarily co-expressed cytokeratin 14, and were observed in part of follicles by day 35 post-partial thyroidectomy. Sca1, BrdU, β-gal, and NKX2-1-positive cells were found 120 days post-partial thyroidectomy. These results suggested that Sca1 and BrdU positive cells may participate in the formation of new thyroid follicles after partial thyroidectomy. The process of thyroid follicular cell regeneration was recapitulated in ex vivo thyroid slice collagen gel culture studies. These studies will facilitate research on thyroid stem/progenitor cells and their roles in thyroid diseases, particularly thyroid carcinomas.

  20. Identification and characterization of Eimeria tenella apical membrane antigen-1 (AMA1.

    Directory of Open Access Journals (Sweden)

    Lianlian Jiang

    Full Text Available Apical membrane antigen-1 (AMA1 is a micronemal protein of apicomplexan parasites that appears to be essential during the invasion of host cells. In this study, a full-length cDNA of AMA1 was identified from Eimeria tenella (Et using expressed sequence tag and the rapid amplification of cDNA ends technique. EtAMA1 had an open reading frame of 1608 bp encoding a protein of 535 amino acids. Quantitative real-time PCR analysis revealed that EtAMA1 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second-generation merozoites. The ectodomain sequence was expressed as recombinant EtAMA1 (rEtAMA1 and rabbit polyclonal antibodies raised against the rEtAMA1 recognized a 58-kDa native parasite protein by Western Blotting and had a potent inhibitory effect on parasite invasion, decreasing it by approximately 70%. Immunofluorescence analysis and immunohistochemistry analysis showed EtAMA1 might play an important role in sporozoite invasion and development.

  1. Effects of Pulsed Electromagnetic Field on Differentiation of HUES-17 Human Embryonic Stem Cell Line

    Directory of Open Access Journals (Sweden)

    Yi-Lin Wu

    2014-08-01

    Full Text Available Electromagnetic fields are considered to potentially affect embryonic development, but the mechanism is still unknown. In this study, human embryonic stem cell (hESC line HUES-17 was applied to explore the mechanism of exposure on embryonic development to pulsed electromagnetic field (PEMF for 400 pulses at different electric field intensities and the differentiation of HUES-17 cells was observed after PEMF exposure. The expression of alkaline phosphatase (AP, stage-specific embryonic antigen-3 (SSEA-3, SSEA-4 and the mRNA level and protein level of Oct4, Sox2 and Nanog in HUES-17 cells remained unchanged after PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m. Four hundred pulses PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m did not affect the differentiation of HUES-17 cells. The reason why electromagnetic fields affect embryonic development may be due to other mechanisms rather than affecting the differentiation of embryonic stem cells.

  2. Zinc coordination is required for and regulates transcription activation by Epstein-Barr nuclear antigen 1.

    Directory of Open Access Journals (Sweden)

    Siddhesh Aras

    2009-06-01

    Full Text Available Epstein-Barr Nuclear Antigen 1 (EBNA1 is essential for Epstein-Barr virus to immortalize naïve B-cells. Upon binding a cluster of 20 cognate binding-sites termed the family of repeats, EBNA1 transactivates promoters for EBV genes that are required for immortalization. A small domain, termed UR1, that is 25 amino-acids in length, has been identified previously as essential for EBNA1 to activate transcription. In this study, we have elucidated how UR1 contributes to EBNA1's ability to transactivate. We show that zinc is necessary for EBNA1 to activate transcription, and that UR1 coordinates zinc through a pair of essential cysteines contained within it. UR1 dimerizes upon coordinating zinc, indicating that EBNA1 contains a second dimerization interface in its amino-terminus. There is a strong correlation between UR1-mediated dimerization and EBNA1's ability to transactivate cooperatively. Point mutants of EBNA1 that disrupt zinc coordination also prevent self-association, and do not activate transcription cooperatively. Further, we demonstrate that UR1 acts as a molecular sensor that regulates the ability of EBNA1 to activate transcription in response to changes in redox and oxygen partial pressure (pO(2. Mild oxidative stress mimicking such environmental changes decreases EBNA1-dependent transcription in a lymphoblastoid cell-line. Coincident with a reduction in EBNA1-dependent transcription, reductions are observed in EBNA2 and LMP1 protein levels. Although these changes do not affect LCL survival, treated cells accumulate in G0/G1. These findings are discussed in the context of EBV latency in body compartments that differ strikingly in their pO(2 and redox potential.

  3. Hypoxia Inducible Factors have distinct and stage-specific roles during reprogramming of human cells to pluripotency

    Science.gov (United States)

    Mathieu, Julie; Zhou, Wenyu; Xing, Yalan; Sperber, Henrik; Ferreccio, Amy; Agoston, Zsuzsa; Kuppusamy, Kavitha T; Moon, Randall T; Ruohola-Baker, Hannele

    2014-01-01

    SUMMARY Pluripotent stem cells have distinct metabolic requirements, and reprogramming cells to pluripotency requires a shift from oxidative to glycolytic metabolism. Here, we show that this shift occurs early during reprogramming of human cells and requires Hypoxia Inducible Factors in a stage-specific manner. HIF1α and HIF2α are both necessary to initiate this metabolic switch and for acquisition of pluripotency, and stabilization of either protein during early phases of reprogramming is sufficient to induce the switch to glycolytic metabolism. In contrast, stabilization of HIF2α during later stages represses reprogramming, due at least in part to up-regulation of TNF-related apoptosis-inducing ligand (TRAIL). TRAIL inhibits iPSC generation by repressing apoptotic caspase 3 activity specifically in cells undergoing reprogramming, but not hESCs, and inhibiting TRAIL activity enhances hiPSC generation. These results shed light on the mechanisms underlying the metabolic shifts associated with acquisition of a pluripotent identity during reprogramming. PMID:24656769

  4. Stage-Specific Fatty Acid Fluxes Play a Regulatory Role in Glycerolipid Metabolism during Seed Development in Jatropha curcas L.

    Science.gov (United States)

    Chaitanya, Bharatula Sri Krishna; Kumar, Sumit; Kaki, Shiva Shanker; Balakrishna, Marrapu; Karuna, Mallampalli Sri Lakshmi; Prasad, Rachapudi Badari Narayana; Sastry, Pidaparty Seshadri; Reddy, Attipalli Ramachandra

    2015-12-23

    The present study describes the changes in lipid profile as well as fatty acid fluxes during seed development in Jatropha curcas L. Endosperm from 34, 37, and 40 days after anthesis (DAA), incubated with [(14)C]acetate, showed significant synthesis of phosphatidylcholine (PC) at seed maturation. The fatty acid methyl ester profile showed PC from 34 DAA was rich in palmitic acid (16:0), whereas PC from 37 and 40 DAA was rich in oleic acid (18:1n-9). Molecular species analysis of diacylglycerol (DAG) indicated DAG (16:0/18:2n-6) was in abundance at 34 DAA, whereas DAG (18:1n-9/18:2n-6) was significantly high at 40 DAA. Triacylglycerol (TAG) analysis revealed TAG (16:0/18:2n-6/16:0) was abundant at 34 DAA, whereas TAG (18:1n-9/18:2n-6/18:1n-9) formed the majority at 40 DAA. Expression of two types of diacylglycerol acyltransferases varied with seed maturation. These data demonstrate stage-specific distinct pools of PC and DAG synthesis during storage TAG accumulation in Jatropha seed.

  5. Stage-Specific Changes in Plasmodium Metabolism Required for Differentiation and Adaptation to Different Host and Vector Environments.

    Directory of Open Access Journals (Sweden)

    Anubhav Srivastava

    2016-12-01

    Full Text Available Malaria parasites (Plasmodium spp. encounter markedly different (nutritional environments during their complex life cycles in the mosquito and human hosts. Adaptation to these different host niches is associated with a dramatic rewiring of metabolism, from a highly glycolytic metabolism in the asexual blood stages to increased dependence on tricarboxylic acid (TCA metabolism in mosquito stages. Here we have used stable isotope labelling, targeted metabolomics and reverse genetics to map stage-specific changes in Plasmodium berghei carbon metabolism and determine the functional significance of these changes on parasite survival in the blood and mosquito stages. We show that glutamine serves as the predominant input into TCA metabolism in both asexual and sexual blood stages and is important for complete male gametogenesis. Glutamine catabolism, as well as key reactions in intermediary metabolism and CoA synthesis are also essential for ookinete to oocyst transition in the mosquito. These data extend our knowledge of Plasmodium metabolism and point towards possible targets for transmission-blocking intervention strategies. Furthermore, they highlight significant metabolic differences between Plasmodium species which are not easily anticipated based on genomics or transcriptomics studies and underline the importance of integration of metabolomics data with other platforms in order to better inform drug discovery and design.

  6. Proteome profiling in murine models of multiple sclerosis: identification of stage specific markers and culprits for tissue damage.

    Directory of Open Access Journals (Sweden)

    Ralf A Linker

    Full Text Available The identification of new biomarkers is of high interest for the prediction of the disease course and also for the identification of pathomechanisms in multiple sclerosis (MS. To specify markers of the chronic disease phase, we performed proteome profiling during the later phase of myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis (MOG-EAE, day 35 after immunization as a model disease mimicking many aspects of secondary progressive MS. In comparison to healthy controls, high resolution 2 dimensional gel electrophoresis revealed a number of regulated proteins, among them glial fibrilary acidic protein (GFAP. Phase specific up-regulation of GFAP in chronic EAE was confirmed by western blotting and immunohistochemistry. Protein levels of GFAP were also increased in the cerebrospinal fluid of MS patients with specificity for the secondary progressive disease phase. In a next step, proteome profiling of an EAE model with enhanced degenerative mechanisms revealed regulation of alpha-internexin, syntaxin binding protein 1, annexin V and glutamate decarboxylase in the ciliary neurotrophic factor (CNTF knockout mouse. The identification of these proteins implicate an increased apoptosis and enhanced axonal disintegration and correlate well the described pattern of tissue injury in CNTF -/- mice which involve oligodendrocyte (OL apoptosis and axonal injury.In summary, our findings underscore the value of proteome analyses as screening method for stage specific biomarkers and for the identification of new culprits for tissue damage in chronic autoimmune demyelination.

  7. Functional interrogation of Plasmodium genus metabolism identifies species- and stage-specific differences in nutrient essentiality and drug targeting.

    Directory of Open Access Journals (Sweden)

    Alyaa M Abdel-Haleem

    2018-01-01

    Full Text Available Several antimalarial drugs exist, but differences between life cycle stages among malaria species pose challenges for developing more effective therapies. To understand the diversity among stages and species, we reconstructed genome-scale metabolic models (GeMMs of metabolism for five life cycle stages and five species of Plasmodium spanning the blood, transmission, and mosquito stages. The stage-specific models of Plasmodium falciparum uncovered stage-dependent changes in central carbon metabolism and predicted potential targets that could affect several life cycle stages. The species-specific models further highlight differences between experimental animal models and the human-infecting species. Comparisons between human- and rodent-infecting species revealed differences in thiamine (vitamin B1, choline, and pantothenate (vitamin B5 metabolism. Thus, we show that genome-scale analysis of multiple stages and species of Plasmodium can prioritize potential drug targets that could be both anti-malarials and transmission blocking agents, in addition to guiding translation from non-human experimental disease models.

  8. BMP signaling regulates the fate of chondro-osteoprogenitor cells in facial mesenchyme in a stage-specific manner.

    Science.gov (United States)

    Celá, Petra; Buchtová, Marcela; Veselá, Iva; Fu, Kathy; Bogardi, Jean-Philippe; Song, Yiping; Barlow, Amanda; Buxton, Paul; Medalová, Jirina; Francis-West, Philippa; Richman, Joy M

    2016-09-01

    Lineage tracing has shown that most of the facial skeleton is derived from cranial neural crest cells. However, the local signals that influence postmigratory, neural crest-derived mesenchyme also play a major role in patterning the skeleton. Here, we study the role of BMP signaling in regulating the fate of chondro-osteoprogenitor cells in the face. A single Noggin-soaked bead inserted into stage 15 chicken embryos induced an ectopic cartilage resembling the interorbital septum within the palate and other midline structures. In contrast, the same treatment in stage 20 embryos caused a loss of bones. The molecular basis for the stage-specific response to Noggin lay in the simultaneous up-regulation of SOX9 and downregulation of RUNX2 in the maxillary mesenchyme, increased cell adhesiveness as shown by N-cadherin induction around the beads and increased RA pathway gene expression. None of these changes were observed in stage 20 embryos. These experiments demonstrate how slight changes in expression of growth factors such as BMPs could lead to gain or loss of cartilage in the upper jaw during vertebrate evolution. In addition, BMPs have at least two roles: one in patterning the skull and another in regulating the skeletogenic fates of neural crest-derived mesenchyme. Developmental Dynamics 245:947-962, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Stage-specific appearance of cytoplasmic microtubules around the surviving nuclei during the third prezygotic division of Paramecium.

    Science.gov (United States)

    Wang, Yi-Wen; Yuan, Jin-Qiang; Gao, Xin; Yang, Xian-Yu

    2012-12-01

    There are six micronuclear divisions during conjugation of Paramecium caudatum: three prezygotic and three postzygotic divisions. Four haploid nuclei are formed during the first two meiotic prezygotic divisions. Usually only one meiotic product is located in the paroral cone (PC) region at the completion of meiosis, which survives and divides mitotically to complete the third prezygotic division to yield a stationary and a migratory pronucleus. The remaining three located outside of the PC degenerate. The migratory pronuclei are then exchanged between two conjugants and fuse with the stationary pronuclei to form synkarya, which undergo three successive divisions (postzygotic divisions). However, little is known about the surviving mechanism of the PC nuclei. In the current study, stage-specific appearance of cytoplasmic microtubules (cMTs) was indicated during the third prezygotic division by immunofluorescence labeling with anti-alpha tubulin antibodies surrounding the surviving nuclei, including the PC nuclei and the two types of prospective pronuclei. This suggested that cMTs were involved in the formation of a physical barrier, whose function may relate to sequestering and protecting the surviving nuclei from the major cytoplasm, where degeneration of extra-meiotic products occurs, another important nuclear event during the third prezygotic division.

  10. Stage-specific heat effects: timing and duration of heat waves alter demographic rates of a global insect pest.

    Science.gov (United States)

    Zhang, Wei; Rudolf, Volker H W; Ma, Chun-Sen

    2015-12-01

    The frequency and duration of periods with high temperatures are expected to increase under global warming. Thus, even short-lived organisms are increasingly likely to experience periods of hot temperatures at some point of their life-cycle. Despite recent progress, it remains unclear how various temperature experiences during the life-cycle of organisms affect demographic traits. We simulated hot days (daily mean temperature of 30 °C) increasingly experienced under field conditions and investigated how the timing and duration of such hot days during the life cycle of Plutella xylostella affects adult traits. We show that hot days experienced during some life stages (but not all) altered adult lifespan, fecundity, and oviposition patterns. Importantly, the effects of hot days were contingent on which stage was affected, and these stage-specific effects were not always additive. Thus, adults that experience different temporal patterns of hot periods (i.e., changes in timing and duration) during their life-cycle often had different demographic rates and reproductive patterns. These results indicate that we cannot predict the effects of current and future climate on natural populations by simply focusing on changes in the mean temperature. Instead, we need to incorporate the temporal patterns of heat events relative to the life-cycle of organisms to describe population dynamics and how they will respond to future climate change.

  11. Functional interrogation of Plasmodium genus metabolism identifies species- and stage-specific differences in nutrient essentiality and drug targeting

    KAUST Repository

    Abdel-Haleem, Alyaa M.

    2018-01-04

    Several antimalarial drugs exist, but differences between life cycle stages among malaria species pose challenges for developing more effective therapies. To understand the diversity among stages and species, we reconstructed genome-scale models (GEMs) of metabolism for five life cycle stages and five species of Plasmodium spanning the blood, transmission, and mosquito stages. The stage-specific models of Plasmodium falciparum uncovered stage-dependent changes in central carbon metabolism and predicted potential targets that could affect several life cycle stages. The species-specific models further highlight differences between experimental animal models and the human-infecting species. Comparisons between human- and rodent-infecting species revealed differences in thiamine (vitamin B1), choline, and pantothenate (vitamin B5) metabolism. Thus, we show that genome-scale analysis of multiple stages and species of Plasmodium can prioritize potential drug targets that could be both anti-malarials and transmission blocking agents, in addition to guiding translation from non-human experimental disease models.

  12. Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

    International Nuclear Information System (INIS)

    Kim, Sun Young; Song, Kyung-A; Kieff, Elliott; Kang, Myung-Soo

    2012-01-01

    Highlights: ► Evidence that targeting EBNA1 dimer, an EBV onco-antigen, can be achievable. ► A small molecule and a peptide as EBNA1 dimerization inhibitors identified. ► Both inhibitors associated with EBNA1 and blocked EBNA1 DNA binding activity. ► Also, prevented its dimerization, and repressed viral gene transcription. -- Abstract: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)’s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459–607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-Jκ binding to the Jκ site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560–574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated with EBNA1 in vitro, and repressed EBNA1-dependent transcription in vivo. Collectively, this study describes two

  13. Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun Young; Song, Kyung-A [Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Samsung Biomedical Research Institute (SBRI), Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Kieff, Elliott [Department of Medicine, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States); Kang, Myung-Soo, E-mail: mkang@skku.edu [Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Samsung Biomedical Research Institute (SBRI), Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Department of Medicine, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States)

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer Evidence that targeting EBNA1 dimer, an EBV onco-antigen, can be achievable. Black-Right-Pointing-Pointer A small molecule and a peptide as EBNA1 dimerization inhibitors identified. Black-Right-Pointing-Pointer Both inhibitors associated with EBNA1 and blocked EBNA1 DNA binding activity. Black-Right-Pointing-Pointer Also, prevented its dimerization, and repressed viral gene transcription. -- Abstract: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)'s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459-607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-J{kappa} binding to the J{kappa} site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560-574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated

  14. Population genetic structure and natural selection of apical membrane antigen-1 in Plasmodium vivax Korean isolates.

    Science.gov (United States)

    Kang, Jung-Mi; Lee, Jinyoung; Cho, Pyo-Yun; Moon, Sung-Ung; Ju, Hye-Lim; Ahn, Seong Kyu; Sohn, Woon-Mok; Lee, Hyeong-Woo; Kim, Tong-Soo; Na, Byoung-Kuk

    2015-11-16

    Plasmodium vivax apical membrane antigen-1 (PvAMA-1) is a leading candidate antigen for blood stage malaria vaccine. However, antigenic variation is a major obstacle in the development of an effective vaccine based on this antigen. In this study, the genetic structure and the effect of natural selection of PvAMA-1 among Korean P. vivax isolates were analysed. Blood samples were collected from 66 Korean patients with vivax malaria. The entire PvAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. The PvAMA-1 sequence of each isolate was sequenced and the polymorphic characteristics and effect of natural selection were analysed using the DNASTAR, MEGA4, and DnaSP programs. Thirty haplotypes of PvAMA-1, which were further classified into seven different clusters, were identified in the 66 Korean P. vivax isolates. Domain II was highly conserved among the sequences, but substantial nucleotide diversity was observed in domains I and III. The difference between the rates of non-synonymous and synonymous mutations suggested that the gene has evolved under natural selection. No strong evidence indicating balancing or positive selection on PvAMA-1 was identified. Recombination may also play a role in the resulting genetic diversity of PvAMA-1. This study is the first comprehensive analysis of nucleotide diversity across the entire PvAMA-1 gene using a single population sample from Korea. Korean PvAMA-1 had limited genetic diversity compared to PvAMA-1 in global isolates. The overall pattern of genetic polymorphism of Korean PvAMA-1 differed from other global isolates and novel amino acid changes were also identified in Korean PvAMA-1. Evidences for natural selection and recombination event were observed, which is likely to play an important role in generating genetic diversity across the PvAMA-1. These results provide useful information for the understanding the population structure of P. vivax circulating in Korea and have important

  15. Population genetic structure and natural selection of Plasmodium falciparum apical membrane antigen-1 in Myanmar isolates.

    Science.gov (United States)

    Kang, Jung-Mi; Lee, Jinyoung; Moe, Mya; Jun, Hojong; Lê, Hương Giang; Kim, Tae Im; Thái, Thị Lam; Sohn, Woon-Mok; Myint, Moe Kyaw; Lin, Khin; Shin, Ho-Joon; Kim, Tong-Soo; Na, Byoung-Kuk

    2018-02-07

    Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is one of leading blood stage malaria vaccine candidates. However, genetic variation and antigenic diversity identified in global PfAMA-1 are major hurdles in the development of an effective vaccine based on this antigen. In this study, genetic structure and the effect of natural selection of PfAMA-1 among Myanmar P. falciparum isolates were analysed. Blood samples were collected from 58 Myanmar patients with falciparum malaria. Full-length PfAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. PfAMA-1 sequence of each isolate was sequenced. Polymorphic characteristics and effect of natural selection were analysed with using DNASTAR, MEGA4, and DnaSP programs. Polymorphic nature and natural selection in 459 global PfAMA-1 were also analysed. Thirty-seven different haplotypes of PfAMA-1 were identified in 58 Myanmar P. falciparum isolates. Most amino acid changes identified in Myanmar PfAMA-1 were found in domains I and III. Overall patterns of amino acid changes in Myanmar PfAMA-1 were similar to those in global PfAMA-1. However, frequencies of amino acid changes differed by country. Novel amino acid changes in Myanmar PfAMA-1 were also identified. Evidences for natural selection and recombination event were observed in global PfAMA-1. Among 51 commonly identified amino acid changes in global PfAMA-1 sequences, 43 were found in predicted RBC-binding sites, B-cell epitopes, or IUR regions. Myanmar PfAMA-1 showed similar patterns of nucleotide diversity and amino acid polymorphisms compared to those of global PfAMA-1. Balancing natural selection and intragenic recombination across PfAMA-1 are likely to play major roles in generating genetic diversity in global PfAMA-1. Most common amino acid changes in global PfAMA-1 were located in predicted B-cell epitopes where high levels of nucleotide diversity and balancing natural selection were found. These results highlight the

  16. Embryonic effects of radiation on ICR mice depending developmental stages

    International Nuclear Information System (INIS)

    Gu, Yeun Hwa; Kusama, Tomoko; Kai, Michiaki

    1995-01-01

    The ICR pregnant mice were irradiated at 1.5Gy in every 6 hours in the period of organogenesis in order to classify the stage specificity of the embryonic effects of radiation and the stage of development differentiation of the primordium of each major organ. Intrauterine death, fetal body weight and external malformation in live fetuses were observed on day 18 of gestation. There was no statistically significant difference in the intrauterine mortality at any stage organogenesis. The fetal body weight of the mice irradiated in the intermediate stage of organogenesis showed significantly lower. There were specific highly sensitive stages in the incidences of each external malformation, that is exencephalia, open eyelid, cleft palate, anomalies of extremities and anomalies of the tail. At these stage, the primordial of the major organs are established in ICR mice

  17. Inhibition of Host Cell Lysosome Spreading by Trypanosoma cruzi Metacyclic Stage-Specific Surface Molecule gp90 Downregulates Parasite Invasion.

    Science.gov (United States)

    Rodrigues, João Paulo Ferreira; Sant'ana, Guilherme Hideki Takahashi; Juliano, Maria Aparecida; Yoshida, Nobuko

    2017-09-01

    Successful infection by Trypanosoma cruzi , the agent of Chagas' disease, is critically dependent on host cell invasion by metacyclic trypomastigote (MT) forms. Two main metacyclic stage-specific surface molecules, gp82 and gp90, play determinant roles in target cell invasion in vitro and in oral T. cruzi infection in mice. The structure and properties of gp82, which is highly conserved among T. cruzi strains, are well known. Information on gp90 is still rather sparse. Here, we attempted to fill that gap. gp90, purified from poorly invasive G strain MT and expressing gp90 at high levels, inhibited HeLa cell lysosome spreading and the gp82-mediated internalization of a highly invasive CL strain MT expressing low levels of a diverse gp90 molecule. A recombinant protein containing the conserved C-terminal domain of gp90 exhibited the same properties as the native G strain gp90: it counteracted the host cell lysosome spreading induced by recombinant gp82 and exhibited an inhibitory effect on HeLa cell invasion by CL strain MT. Assays to identify the gp90 sequence associated with the property of downregulating MT invasion, using synthetic peptides spanning the gp90 C-terminal domain, revealed the sequence GVLYTADKEW. These data, plus the findings that lysosome spreading was induced upon HeLa cell interaction with CL strain MT, but not with G strain MT, and that in mixed infection CL strain MT internalization was inhibited by G strain MT, suggest that the inhibition of target cell lysosome spreading is the mechanism by which the gp90 molecule exerts its downregulatory role. Copyright © 2017 Rodrigues et al.

  18. A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes

    Directory of Open Access Journals (Sweden)

    Pilar T. V. Florentino

    2018-04-01

    Full Text Available Trypanosoma cruzi is the etiologic agent of Chagas’ disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels, it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas’ disease.

  19. Genome wide analysis reveals Zic3 interaction with distal regulatory elements of stage specific developmental genes in zebrafish.

    Directory of Open Access Journals (Sweden)

    Cecilia L Winata

    2013-10-01

    Full Text Available Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.

  20. Inducing hair follicle neogenesis with secreted proteins enriched in embryonic skin.

    Science.gov (United States)

    Fan, Sabrina Mai-Yi; Tsai, Chia-Feng; Yen, Chien-Mei; Lin, Miao-Hsia; Wang, Wei-Hung; Chan, Chih-Chieh; Chen, Chih-Lung; Phua, Kyle K L; Pan, Szu-Hua; Plikus, Maksim V; Yu, Sung-Liang; Chen, Yu-Ju; Lin, Sung-Jan

    2018-03-13

    Organ development is a sophisticated process of self-organization. However, despite growing understanding of the developmental mechanisms, little is known about how to reactivate them postnatally for regeneration. We found that treatment of adult non-hair fibroblasts with cell-free extract from embryonic skin conferred upon them the competency to regenerate hair follicles. Proteomics analysis identified three secreted proteins enriched in the embryonic skin, apolipoprotein-A1, galectin-1 and lumican that together were essential and sufficient to induce new hair follicles. These 3 proteins show a stage-specific co-enrichment in the perifolliculogenetic embryonic dermis. Mechanistically, exposure to embryonic skin extract or to the combination of the 3 proteins altered the gene expression to an inductive hair follicle dermal papilla fibroblast-like profile and activated Igf and Wnt signaling, which are crucial for the regeneration process. Therefore, a cocktail of organ-specific extracellular proteins from the embryonic environment can render adult cells competent to re-engage in developmental interactions for organ neogenesis. Identification of factors that recreate the extracellular context of respective developing tissues can become an important strategy to promote regeneration in adult organs. Copyright © 2018 Elsevier Ltd. All rights reserved.

  1. Interaction between cysteine synthase and serine O-acetyltransferase proteins and their stage specific expression in Leishmania donovani.

    Science.gov (United States)

    Singh, Kuljit; Singh, Krishn Pratap; Equbal, Asif; Suman, Shashi S; Zaidi, Amir; Garg, Gaurav; Pandey, Krishna; Das, Pradeep; Ali, Vahab

    2016-12-01

    Leishmania possess a unique trypanothione redox metabolism with undebated roles in protection from oxidative damage and drug resistance. The biosynthesis of trypanothione depends on l-cysteine bioavailability which is regulated by cysteine biosynthesis pathway. The de novo cysteine biosynthesis pathway is comprised of serine O-acetyltransferase (SAT) and cysteine synthase (CS) enzymes which sequentially mediate two consecutive steps of cysteine biosynthesis, and is absent in mammalian host. However, despite the apparent dependency of redox metabolism on cysteine biosynthesis pathway, the role of SAT and CS in redox homeostasis has been unexplored in Leishmania parasites. Herein, we have characterized CS and SAT to investigate their interaction and relative abundance of these proteins in promastigote vs. amastigote growth stages of L. donovani. CS and SAT genes of L. donovani (LdCS and LdSAT) were cloned, expressed, and fusion proteins purified to homogeneity with affinity column chromatography. Purified LdCS contains PLP as cofactor and showed optimum enzymatic activity at pH 7.5. Enzyme kinetics showed that LdCS catalyses the synthesis of cysteine using O-acetylserine and sulfide with a K m of 15.86 mM and 0.17 mM, respectively. Digitonin fractionation and indirect immunofluorescence microscopy showed that LdCS and LdSAT are localized in the cytoplasm of promastigotes. Size exclusion chromatography, co-purification, pull down and immuno-precipitation assays demonstrated a stable complex formation between LdCS and LdSAT proteins. Furthermore, LdCS and LdSAT proteins expression/activity was upregulated in amastigote growth stage of the parasite. Thus, the stage specific differential expression of LdCS and LdSAT suggests that it may have a role in the redox homeostasis of Leishmania. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  2. Tissue- and stage-specific Wnt target gene expression is controlled subsequent to beta-catenin recruitment to cis-regulatory modules

    NARCIS (Netherlands)

    Nakamura, Y.; de Paiva Alves, E.; Veenstra, G.J.C.; Hoppler, S.

    2016-01-01

    Key signalling pathways, such as canonical Wnt/beta-catenin signalling, operate repeatedly to regulate tissue- and stage-specific transcriptional responses during development. Although recruitment of nuclear beta-catenin to target genomic loci serves as the hallmark of canonical Wnt signalling,

  3. Adenosine-uridine-rich element is one of the required cis-elements for epimastigote form stage-specific gene expression of the congolense epimastigote specific protein.

    Science.gov (United States)

    Suganuma, Keisuke; Mochabo, Kennedy Miyoro; Hakimi, Hassan; Yamasaki, Shino; Yamagishi, Junya; Asada, Masahito; Kawazu, Shin-Ichiro; Inoue, Noboru

    2013-09-01

    It is known that gene expression in kinetoplastida is regulated post-transcriptionally. Several previous studies have shown that stage-specific gene expression in trypanosomes is regulated by cis-elements located in the 3' untranslated region (UTR) of each mRNA and also by RNA binding proteins. Our previous study revealed that gene expression of congolense epimastigote specific protein (cesp) was regulated by cis-elements located in the 3'UTR. In the present study, we identified the adenosine and uridine rich region in the cesp 3'UTR. Using transgenic trypanosome cell lines with different egfp expression cassettes, we showed that this adenosine and uridine rich region is one of the regulatory elements for epimastigote form (EMF) stage-specific gene expression via the regulatory cis-element of the eukaryotic AU rich element (ARE). Therefore this required element within the cesp 3'UTR was designated as T. congolense ARE. This required cis-element might selectively stabilize mRNA in the EMF stage and destabilize mRNA in other stages. By RNA electro mobility shift assay, unknown stage-specific RNA binding proteins (RBPs) whose sequences specifically interacted with the required cis-element were found. These results indicate that EMF stage specific cis-element and RBP complexes might specifically stabilize cesp mRNA in EMF. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. RETINOIC ACID INDUCTION OF CLEFT PALATE IN EGF AND TGF-ALPHA KNOCKOUT MICE: STAGE SPECIFIC INFLUENCES OF GROWTH FACTOR EXPRESSION

    Science.gov (United States)

    ABBOTT, B. D., LEFFLER, K.E. AND BUCKALEW, A.R, Reproductive Toxicology Division, NHEERL, ORD, US EPA, Research Triangle Park, North Carolina. Retinoic acid induction of cleft palate (CP) in EGF and TGF knockout mice: Stage specific influences of growth factor expression.<...

  5. Involvement of lymphocyte function-associated antigen-1 (LFA-1) in HIV infection: inhibition by monoclonal antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Mathiesen, Lars Reinhardt

    1991-01-01

    Monoclonal antibodies (MAbs) against the alpha- and beta-chain of lymphocyte-associated antigen-1 (LFA-1) were examined for inhibition of HIV-1 infection in vitro. Infection of the T cell line MT4 and the monocytic cell line U937 by isolates HTLVIIIB and SSI-002, respectively was inhibited...... in a concentration dependent manner by MAb against the beta-chain but not against the alpha-chain. No cross-reactivity was found between MAb against LFA-1 and against the CD4 receptor (MAb Leu3a). MAbs against the beta-chain and the CD4 receptor were found to act synergistically in inhibiting HIV infection...

  6. 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.

  7. Efficient femtosecond driven SOX 17 delivery into mouse embryonic stem cells: differentiation studies

    Science.gov (United States)

    Thobakgale, Lebogang; Manoto, Sello Lebohang; Lemboumba, Satuurnin Ombinda; Maaza, Malik; Mthunzi-Kufa, Patience

    2017-02-01

    Embryonic stem cells have great promise in regenerative medicine because of their ability to self-renew and differentiate into various cell types. Delivery of therapeutic genes into cells has already been achieved using of chemical agents and viral vectors with high transfection efficiencies. However, these methods have also been documented as toxic and in the latter case they can cause latent cell infections. In this study we use femtosecond laser pulses to optically deliver genetic material in mouse embryonic stem cells. Femtosecond laser pulses in contrast to the conventional approach, minimises the risk of unwanted side effects because photons are used to create transient pores on the membrane which allow free entry of molecules with no need for delivery agents. Using an Olympus microscope, fluorescence imaging of the samples post irradiation was performed and decreased expression of stage specific embryonic antigen one (SSEA-1) consistent with on-going cellular differentiation was observed. Our results also show that femtosecond laser pulses were effective in delivering SOX 17 plasmid DNA (pSOX17) which resulted in the differentiation of mouse embryonic stem cells into endoderm cells. We thus concluded that laser transfection of stem cells for the purpose of differentiation, holds potential for applications in tissue engineering as a method of generating new cell lines.

  8. Identification of Protective B-Cell Epitopes within the Novel Malaria Vaccine Candidate Plasmodium falciparum Schizont Egress Antigen 1.

    Science.gov (United States)

    Nixon, Christina E; Park, Sangshin; Pond-Tor, Sunthorn; Raj, Dipak; Lambert, Lynn E; Orr-Gonzalez, Sachy; Barnafo, Emma K; Rausch, Kelly M; Friedman, Jennifer F; Fried, Michal; Duffy, Patrick E; Kurtis, Jonathan D

    2017-07-01

    Naturally acquired antibodies to Plasmodium falciparum schizont egress antigen 1 (PfSEA-1A) are associated with protection against severe malaria in children. Vaccination of mice with SEA-1A from Plasmodium berghei (PbSEA-1A) decreases parasitemia and prolongs survival following P. berghei ANKA challenge. To enhance the immunogenicity of PfSEA-1A, we identified five linear B-cell epitopes using peptide microarrays probed with antisera from nonhuman primates vaccinated with recombinant PfSEA-1A (rPfSEA-1A). We evaluated the relationship between epitope-specific antibody levels and protection from parasitemia in a longitudinal treatment-reinfection cohort in western Kenya. Antibodies to three epitopes were associated with 16 to 17% decreased parasitemia over an 18-week high transmission season. We are currently designing immunogens to enhance antibody responses to these three epitopes. Copyright © 2017 American Society for Microbiology.

  9. Design and synthesis of prostate cancer antigen-1 (PCA-1/ALKBH3) inhibitors as anti-prostate cancer drugs.

    Science.gov (United States)

    Nakao, Syuhei; Mabuchi, Miyuki; Shimizu, Tadashi; Itoh, Yoshihiro; Takeuchi, Yuko; Ueda, Masahiro; Mizuno, Hiroaki; Shigi, Naoko; Ohshio, Ikumi; Jinguji, Kentaro; Ueda, Yuko; Yamamoto, Masatatsu; Furukawa, Tatsuhiko; Aoki, Shunji; Tsujikawa, Kazutake; Tanaka, Akito

    2014-02-15

    A series of 1-aryl-3,4-substituted-1H-pyrazol-5-ol derivatives was synthesized and evaluated as prostate cancer antigen-1 (PCA-1/ALKBH3) inhibitors to obtain a novel anti-prostate cancer drug. After modifying 1-(1H-benzimidazol-2-yl)-3,4-dimethyl-1H-pyrazol-5-ol (1), a hit compound found during random screening using a recombinant PCA-1/ALKBH3, 1-(1H-5-methylbenzimidazol-2-yl)-4-benzyl-3-methyl-1H-pyrazol-5-ol (35, HUHS015), was obtained as a potent PCA-1/ALKBH3 inhibitor both in vitro and in vivo. The bioavailability (BA) of 35 was 7.2% in rats after oral administration. As expected, continuously administering 35 significantly suppressed the growth of DU145 cells, which are human hormone-independent prostate cancer cells, in a mouse xenograft model without untoward effects. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. [Cytotoxic effects of etoposide at different stages of differentiation of embryoid bodies formed by mouse embryonic stem cells].

    Science.gov (United States)

    Gordeeva, O F

    2013-01-01

    The initial stages of in vitro differentiation of embryonic stem cells are considered as unique three-dimensional models of early development of mammals for basic, pharmacological, and toxicological studies. It has been previously shown (Gordeeva, 2012) that the assessment of embryotoxicity in the model of undifferentiated embryonic stem cells can be insufficiently accurate in predicting toxic effects on mammalian embryos. In view of this, we performed a comparative study of the damaging effects of the cytostatic etoposide in undifferentiated embryonic stem cells and embryoid bodiesof different stages of differentiation that have similar three-dimensional structures with early embryos. The analysis of growth, cell death, and dynamics of differentiation of embryonic stem cells and embryoid bodies exposed to etoposide showed that the cytostatic and cytotoxic effects of etoposide are stage-specific. The damaging effects of etoposide were maximum in the undifferentiated embryonic stem cells and decreased with growth and differentiation of embryoid bodies. We assume that the increase in the cell volume of embryoid bodies and the development of the hypertrophic we suggest that the increase of embryoid body volume and overgrowth of extraembryonic endoderm layer lead to a decrease in the diffusion, transport, and metabolism of chemical and bioactive substances and prevent the damaging effects.

  11. Regulation of gene expression in the protozoan parasite Entamoeba invadens identification of core promoter elements and promoters with stage-specific expression patterns

    Science.gov (United States)

    Manna, Dipak; Ehrenkaufer, Gretchen M.; Singh, Upinder

    2014-01-01

    Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. Entamoeba histolytica is an important human pathogen and is a leading parasitic cause of death globally. During its life cycle, Entamoeba converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Despite being central to its biology, the triggers that are involved in the developmental pathways of this parasite are not well understood. In order to define the transcriptional network associated with stage conversion we used Entamoeba invadens which serves as a model system for Entamoeba developmental biology, and performed RNA sequencing at different developmental time points . In this study RNA-Seq data was utilized to define basal transcriptional control elements as well as to identify promoters which regulate stage-specific gene expression patterns. We discovered that the 5’ and 3’ untranslated regions of E. invadens genes are short, a median of 20 nucleotides (nt) and 26 nt respectively. Bioinformatics analysis of DNA sequences proximate to the start and stop codons identified two conserved motifs: (i) E. invadens Core Promoter Motif - GAAC-Like (EiCPM-GL) (GAACTACAAA), and (ii) E. invadens 3’- U-Rich Motif (Ei3’-URM) (TTTGTT) in the 5’ and 3’ flanking regions, respectively. Electrophoretic mobility shift assays demonstrated that both motifs specifically bind nuclear protein(s) from E. invadens trophozoites. Additionally, we identified select genes with stage-specific expression patterns and analyzed the ability of each gene promoter to drive a luciferase reporter gene during the developmental cycle. This approach confirmed three trophozoite-specific, four encystation-specific and two excystation-specific promoters. This work lays the framework for use of stage-specific promoters to express proteins of interest in a particular life-cycle stage, adding to the molecular toolbox for genetic

  12. Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns.

    Science.gov (United States)

    Manna, Dipak; Ehrenkaufer, Gretchen M; Singh, Upinder

    2014-10-01

    Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. Entamoeba histolytica is an important human pathogen and is a leading parasitic cause of death globally. During its life cycle, Entamoeba converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Despite being central to its biology, the triggers that are involved in the developmental pathways of this parasite are not well understood. In order to define the transcriptional network associated with stage conversion we used Entamoeba invadens which serves as a model system for Entamoeba developmental biology, and performed RNA sequencing at different developmental time points. In this study RNA-Seq data was utilised to define basal transcriptional control elements as well as to identify promoters which regulate stage-specific gene expression patterns. We discovered that the 5' and 3' untranslated regions of E. invadens genes are short, a median of 20 nucleotides (nt) and 26 nt respectively. Bioinformatics analysis of DNA sequences proximate to the start and stop codons identified two conserved motifs: (i) E. invadens Core Promoter Motif - GAAC-Like (EiCPM-GL) (GAACTACAAA), and (ii) E. invadens 3'-U-Rich Motif (Ei3'-URM) (TTTGTT) in the 5' and 3' flanking regions, respectively. Electrophoretic mobility shift assays demonstrated that both motifs specifically bind nuclear protein(s) from E. invadens trophozoites. Additionally, we identified select genes with stage-specific expression patterns and analysed the ability of each gene promoter to drive a luciferase reporter gene during the developmental cycle. This approach confirmed three trophozoite-specific, four encystation-specific and two excystation-specific promoters. This work lays the framework for use of stage-specific promoters to express proteins of interest in a particular life-cycle stage, adding to the molecular toolbox for genetic manipulation of E

  13. Embryonic development during chronic acceleration

    Science.gov (United States)

    Smith, A. H.; Abbott, U. K.

    1982-01-01

    Experiments carried out on chicken eggs indicate that the embryo is affected during very early development, especially over the first four days, and during hatching. In the first four days, the brain develops as well as the anlage for all other organs. In addition, the heart commences to function and the extraembryonic membranes that compartmentalize the egg contents form. The latter require an appreciable extension and folding of tissue which may be disrupted by the mechanical load. Observations of embryonic abnormalities that occur during chronic acceleration suggest an inhibition of development of the axial skeleton, which is rarely seen otherwise, a general retardation of embryonic growth, and circulatory problems. The final stages of development (after 18 days) involve the uptake of fluids, the transition to aerial respiration, and the reorientation of the embryo into a normal hatching position. At 4 G mortality is very high during this period, with a majority of embryos failing to reorient into the normal hatching position.

  14. Electroporation of Embryonic Kidney Explants

    Science.gov (United States)

    Haddad, Nicholas; Houle, Daniel; Gupta, Indra R.

    Metanephric kidney development in the mouse begins at embryonic day (E) 10.5, when the ureteric bud (UB), an outgrowth of the epithelial nephric duct, invades the neighboring metanephric mesenchyme (MM). The ureteric bud then undergoes a series of branching events to form the collecting duct network of the adult kidney (Fig. 19.1). As each ureteric bud tip forms, the adjacent undifferentiated mesenchyme is induced to epithelialize and form a nephron, the functional unit of the adult kidney that filters waste. Rodent embryonic kidneys can be dissected and cultured as explants such that branching morphogenesis and nephrogenesis can be observed ex vivo (Rothenpieler and Dressler, 1993; Vega et al., 1996; Piscione et al., 1997; Gupta et al., 2003).

  15. Constitutive transgene expression of Stem Cell Antigen-1 in the hair follicle alters the sensitivity to tumor formation and progression

    Directory of Open Access Journals (Sweden)

    Rikke Christensen

    2017-08-01

    Full Text Available The cell surface protein Stem Cell Antigen-1 (Sca-1 marks stem or progenitor cells in several murine tissues and is normally upregulated during cancer development. Although the specific function of Sca-1 remains unknown, Sca-1 seems to play a role in proliferation, differentiation and cell migration in a number of tissues. In the skin epithelium, Sca-1 is highly expressed in the interfollicular epidermis but is absent in most compartments of the hair follicle; however, the function of Sca-1 in the skin has not been investigated. To explore the role of Sca-1 in normal and malignant skin development we generated transgenic mice that express Sca-1 in the hair follicle stem cells that are normally Sca-1 negative. Development of hair follicles and interfollicular epidermis appeared normal in Sca-1 mutant mice; however, follicular induction of Sca-1 expression in bulge region and isthmus stem cells reduced the overall yield of papillomas in a chemical carcinogenesis protocol. Despite that fewer papillomas developed in transgenic mice a higher proportion of the papillomas underwent malignant conversion. These findings suggest that overexpression of Sca-1 in the hair follicle stem cells contributes at different stages of tumour development. In early stages, overexpression of Sca-1 decreases tumour formation while at later stages overexpression of Sca-1 seems to drive tumours towards malignant progression.

  16. Molecular cloning, characterization and antigenicity of Babesia sp. BQ1 (Lintan) (Babesia cf. motasi) apical membrane antigen-1 (AMA-1).

    Science.gov (United States)

    Niu, Qingli; Liu, Zhijie; Yang, Jifei; Guan, Guiquan; Pan, Yuping; Luo, Jianxun; Yin, Hong

    2017-04-01

    Apical membrane antigen-1 (AMA-1) has been described as a potential vaccine candidate in apicomplexan parasites. Here we characterize the ama-1 gene. The full-length ama-1 gene of Babesia sp. BQ1 (Lintan) (BLTAMA-1) is 1785 bp, which contains an open reading frame (ORF) encoding a 65-kDa protein of 594 amino acid residues; by definition, the 5' UTR precedes the first methionine of the ORF. Phylogenetic analysis based on AMA-1 amino acid sequences clearly separated Piroplasmida from other Apicomplexa parasites. The Babesia sp. BQ1 (Lintan) AMA-1 sequence is most closely associated with that of B. ovata and B. bigemina, with high bootstrap value. A recombinant protein encoding a conserved region and containing ectodomains I and II of BLTAMA-1 was constructed. BLTrAMA-1-DI/DII proteins were tested for reactivity with sera from sheep infected by Babesia sp. BQ1 (Lintan). In Western-blot analysis, native Babesia sp. BQ1 (Lintan) AMA-1 proteins were recognized by antibodies raised in rabbits against BLTrAMA-1 in vitro. The results of this study are discussed in terms of gene characterization, taxonomy and antigenicity.

  17. Specific T-cell recognition of the merozoite proteins rhoptry-associated protein 1 and erythrocyte-binding antigen 1 of Plasmodium falciparum

    DEFF Research Database (Denmark)

    Jakobsen, P H; Hviid, L; Theander, T G

    1993-01-01

    The merozoite proteins merozoite surface protein 1 (MSP-1) and rhoptry-associated protein 1 (RAP-1) and synthetic peptides containing sequences of MSP-1, RAP-1, and erythrocyte-binding antigen 1, induced in vitro proliferative responses of lymphocytes collected from Ghanaian blood donors living i...

  18. Fetal antigen 1, an EGF multidomain protein in the sex hormone-producing cells of the gonads and the microenvironment of germ cells

    DEFF Research Database (Denmark)

    Jensen, Charlotte Harken; Erb, K; Westergaard, L G

    1999-01-01

    Fetal antigen 1 (FA1), an epidermal growth factor (EGF) multidomain glycoprotein, was investigated in the human reproductive system. Immunohistochemical analysis of the male reproductive system revealed staining for FA1 in the Leydig cells only. Concentrations of FA1 in seminal plasma and serum w...

  19. Sequence diversity and natural selection at domain I of the apical membrane antigen 1 among Indian Plasmodium falciparum populations

    Directory of Open Access Journals (Sweden)

    Kumar Ashwani

    2007-11-01

    Full Text Available Abstract Background The Plasmodium falciparum apical membrane antigen 1 (AMA1 is a leading malaria vaccine candidate antigen. The complete AMA1 protein is comprised of three domains where domain I exhibits high sequence polymorphism and is thus named as the hyper-variable region (HVR. The present study describes the extent of genetic polymorphism and natural selection at domain I of the ama1 gene among Indian P. falciparum isolates. Methods The part of the ama1 gene covering domain I was PCR amplified and sequenced from 157 P. falciparum isolates collected from five different geographical regions of India. Statistical and phylogenetic analyses of the sequences were done using DnaSP ver. 4. 10. 9 and MEGA version 3.0 packages. Results A total of 57 AMA1 haplotypes were observed among 157 isolates sequenced. Forty-six of these 57 haplotypes are being reported here for the first time. The parasites collected from the high malaria transmission areas (Assam, Orissa, and Andaman and Nicobar Islands showed more haplotypes (H and nucleotide diversity π as compared to low malaria transmission areas (Uttar Pradesh and Goa. The comparison of all five Indian P. falciparum subpopulations indicated moderate level of genetic differentiation and limited gene flow (Fixation index ranging from 0.048 to 0.13 between populations. The difference between rates of non-synonymous and synonymous mutations, Tajima's D and McDonald-Kreitman test statistics suggested that the diversity at domain I of the AMA1 antigen is due to positive natural selection. The minimum recombination events were also high indicating the possible role of recombination in generating AMA1 allelic diversity. Conclusion The level of genetic diversity and diversifying selection were higher in Assam, Orissa, and Andaman and Nicobar Islands populations as compared to Uttar Pradesh and Goa. The amounts of gene flow among these populations were moderate. The data reported here will be valuable for the

  20. Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)

    Science.gov (United States)

    Bueno, Lilian Lacerda; Lobo, Francisco Pereira; Morais, Cristiane Guimarães; Mourão, Luíza Carvalho; de Ávila, Ricardo Andrez Machado; Soares, Irene Silva; Fontes, Cor Jesus; Lacerda, Marcus Vinícius; Olórtegui, Carlos Chavez; Bartholomeu, Daniella Castanheira; Fujiwara, Ricardo Toshio; Braga, Érika Martins

    2011-01-01

    Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290–307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies. PMID:21713006

  1. Correcting bias due to missing stage data in the non-parametric estimation of stage-specific net survival for colorectal cancer using multiple imputation.

    Science.gov (United States)

    Falcaro, Milena; Carpenter, James R

    2017-06-01

    Population-based net survival by tumour stage at diagnosis is a key measure in cancer surveillance. Unfortunately, data on tumour stage are often missing for a non-negligible proportion of patients and the mechanism giving rise to the missingness is usually anything but completely at random. In this setting, restricting analysis to the subset of complete records gives typically biased results. Multiple imputation is a promising practical approach to the issues raised by the missing data, but its use in conjunction with the Pohar-Perme method for estimating net survival has not been formally evaluated. We performed a resampling study using colorectal cancer population-based registry data to evaluate the ability of multiple imputation, used along with the Pohar-Perme method, to deliver unbiased estimates of stage-specific net survival and recover missing stage information. We created 1000 independent data sets, each containing 5000 patients. Stage data were then made missing at random under two scenarios (30% and 50% missingness). Complete records analysis showed substantial bias and poor confidence interval coverage. Across both scenarios our multiple imputation strategy virtually eliminated the bias and greatly improved confidence interval coverage. In the presence of missing stage data complete records analysis often gives severely biased results. We showed that combining multiple imputation with the Pohar-Perme estimator provides a valid practical approach for the estimation of stage-specific colorectal cancer net survival. As usual, when the percentage of missing data is high the results should be interpreted cautiously and sensitivity analyses are recommended. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. 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.

  3. Cryopreservation of embryonic axes of groundnut ( Arachis ...

    African Journals Online (AJOL)

    An efficient cryopreservation protocol was developed for groundnut embryonic axes using vitrification technique. Embryonic axes obtained from seeds of four groundnut genotypes were dehydrated in Plant Vitrification Solution (PVS2) solution for different durations (0, 1, 2, 3, 4 and 5 h) before plunged into liquid nitrogen ...

  4. the production of mouse embryonic stem cells

    Indian Academy of Sciences (India)

    MADU

    result of the anarchic development of early embryonic cells left behind during development. However, their localization in the genital organs supported an alternative hypothesis: they were the result of an anarchic multiplication of the. Series. What history tells us. VII. Twenty-five years ago: the production of mouse embryonic ...

  5. Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1

    Directory of Open Access Journals (Sweden)

    Wang Pu

    2010-10-01

    Full Text Available Abstract The Epstein-Barr Virus (EBV Nuclear Antigen 1 (EBNA1 protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP, regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP combined with massively parallel deep-sequencing (ChIP-Seq was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA

  6. Temporal genetic changes in Plasmodium vivax apical membrane antigen 1 over 19 years of transmission in southern Mexico.

    Science.gov (United States)

    Flores-Alanis, Alejandro; González-Cerón, Lilia; Santillán, Frida; Ximenez, Cecilia; Sandoval, Marco A; Cerritos, René

    2017-05-02

    Mexico advanced to the pre-elimination phase in 2009 due to a significant reduction in malaria cases, and since 2000, Plasmodium vivax is the only species transmitted. During the last two decades, malaria transmission has been mostly local and isolated to a few regions. It is important to gain further insights into the impact of control measures on the parasite population structure. Hence, the aim of the current study was to determine detailed changes in P. vivax genetic diversity and population structure based on analysing the gene that encodes the apical membrane antigen 1 (pvama1). This analysis covered from control to pre-elimination (1993-2011) in a hypo-endemic region in southern Mexico. The 213 pvama1 I-II sequences presently analysed were grouped into six periods of three years each. They showed low genetic diversity, with 15 haplotypes resolved. Among the DNA sequences, there was a gradual decrease in genetic diversity, the number of mixed genotype infections and the intensity of positive selection, in agreement with the parallel decline in malaria cases. At the same time, linkage disequilibrium (R 2 ) increased. The three-dimensional haplotype network revealed that pvama1 I-II haplotypes were separated by 1-11 mutational steps, and between one another by 0-3 unsampled haplotypes. In the temporal network, seven haplotypes were detected in at least two of the six-time layers, and only four distinct haplotypes were evidenced in the pre-elimination phase. Structure analysis indicated that three subpopulations fluctuated over time. Only 8.5% of the samples had mixed ancestry. In the pre-elimination phase, subpopulation P1 was drastically reduced, and the admixture was absent. The results suggest that P. vivax in southern Mexico evolved based on local adaptation into three "pseudoclonal" subpopulations that diversified at the regional level and persisted over time, although with varying frequency. Control measures and climate events influenced the number of

  7. High antibody titer against apical membrane antigen-1 is required to protect against malaria in the Aotus model.

    Directory of Open Access Journals (Sweden)

    Sheetij Dutta

    Full Text Available A Plasmodium falciparum 3D7 strain Apical Membrane Antigen-1 (AMA1 vaccine, formulated with AS02(A adjuvant, slowed parasite growth in a recent Phase 1/2a trial, however sterile protection was not observed. We tested this AS02(A, and a Montanide ISA720 (ISA formulation of 3D7 AMA1 in Aotus monkeys. The 3D7 parasite does not invade Aotus erythrocytes, hence two heterologous strains, FCH/4 and FVO, were used for challenge, FCH/4 AMA1 being more homologous to 3D7 than FVO AMA1. Following three vaccinations, the monkeys were challenged with 50,000 FCH/4 or 10,000 FVO parasites. Three of the six animals in the AMA+ISA group were protected against FCH/4 challenge. One monkey did not become parasitemic, another showed only a short period of low level parasitemia that self-cured, and a third animal showed a delay before exhibiting its parasitemic phase. This is the first protection shown in primates with a recombinant P. falciparum AMA1 without formulation in Freund's complete adjuvant. No animals in the AMA+AS02(A group were protected, but this group exhibited a trend towards reduced growth rate. A second group of monkeys vaccinated with AMA+ISA vaccine was not protected against FVO challenge, suggesting strain-specificity of AMA1-based protection. Protection against FCH/4 strain correlated with the quantity of induced antibodies, as the protected animals were the only ones to have in vitro parasite growth inhibitory activity of >70% at 1:10 serum dilution; immuno-fluorescence titers >8,000; ELISA titers against full-length AMA1 >300,000 and ELISA titer against AMA1 domains1+2 >100,000. A negative correlation between log ELISA titer and day 11 cumulative parasitemia (Spearman rank r = -0.780, p value = 0.0001, further confirmed the relationship between antibody titer and protection. High titers of cross-strain inhibitory antibodies against AMA1 are therefore critical to confer solid protection, and the Aotus model can be used to down-select future AMA1

  8. Mechanisms of embryonic stomach development.

    Science.gov (United States)

    McCracken, Kyle W; Wells, James M

    2017-06-01

    The stomach is a digestive organ that has important roles in human physiology and pathophysiology. The developmental origin of the stomach is the embryonic foregut, which also gives rise a number of other structures. There are several signaling pathways and transcription factors that are known to regulate stomach development at different stages, including foregut patterning, stomach specification, and gastric regionalization. These developmental events have important implications in later homeostasis and disease in the adult stomach. Here we will review the literature that has shaped our current understanding of the molecular mechanisms that coordinate gastric organogenesis. Further we will discuss how developmental paradigms have guided recent efforts to differentiate stomach tissue from pluripotent stem cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Human embryonic stem cells handbook

    Directory of Open Access Journals (Sweden)

    Carlo Alberto Redi

    2013-03-01

    Full Text Available After the Nobel prize in physiology or medicine was awarded jointly to Sir John Gurdon and Shinya Yamanaka for the discovery that mature cells can be reprogrammed to become pluripotent it became imperative to write down the review for a book entirely devoted to human embryonic stem cells (hES, those cells that are a urgent need for researchers, those cells that rekindle the ethical debates and finally, last but not least, those cells whose study paved the way to obtain induced pluripotent stem cells by the OSKC’s Yamanaka method (the OSKC acronim refers, for those not familiar with the topic, to the four stemness genes used to transfect somatic fibroblasts: Oct4, Sox2, Klf4 and c-Myc....

  10. Undifferentiated embryonal sarcoma of liver

    Directory of Open Access Journals (Sweden)

    Avyakta Kallam

    2015-12-01

    Full Text Available Undifferentiated embryonal sarcoma of the liver (UESL is a rare malignant hepatic tumor. A 47 year old male presented with symptoms of sour taste in his mouth, occasional nausea, indigestion and 15-pound weight loss over two months. He had an unremarkable upper gastrointestinal endoscopy. Imaging showed a large liver mass in the left hepatic lobe that was resected and then reported as UESL. He went on to develop lung metastases and was initially treated with doxorubicin and ifosfamide followed by switching of therapy to gemcitabine and docetaxel due to progression of disease. He had a good response after two cycles and went on to receive four more cycles, achieving stable disease. We can therefore conclude that the combination of gemcitabine and docetaxel is a potential therapeutic option for patients with UESL.

  11. Undifferentiated Embryonal Sarcoma of Liver.

    Science.gov (United States)

    Kallam, Avyakta; Krishnamurthy, Jairam; Kozel, Jessica; Shonka, Nicole

    2015-12-29

    Undifferentiated embryonal sarcoma of the liver (UESL) is a rare malignant hepatic tumor. A 47 year old male presented with symptoms of sour taste in his mouth, occasional nausea, indigestion and 15-pound weight loss over two months. He had an unremarkable upper gastrointestinal endoscopy. Imaging showed a large liver mass in the left hepatic lobe that was resected and then reported as UESL. He went on to develop lung metastases and was initially treated with doxorubicin and ifosfamide followed by switching of therapy to gemcitabine and docetaxel due to progression of disease. He had a good response after two cycles and went on to receive four more cycles, achieving stable disease. We can therefore conclude that the combination of gemcitabine and docetaxel is a potential therapeutic option for patients with UESL.

  12. Variable Extent of Lineage-Specificity and Developmental Stage-Specificity of Cohesin and CCCTC-Binding Factor Binding Within the Immunoglobulin and T Cell Receptor Loci

    Directory of Open Access Journals (Sweden)

    Salvatore Loguercio

    2018-03-01

    Full Text Available CCCTC-binding factor (CTCF is largely responsible for the 3D architecture of the genome, in concert with the action of cohesin, through the creation of long-range chromatin loops. Cohesin is hypothesized to be the main driver of these long-range chromatin interactions by the process of loop extrusion. Here, we performed ChIP-seq for CTCF and cohesin in two stages each of T and B cell differentiation and examined the binding pattern in all six antigen receptor (AgR loci in these lymphocyte progenitors and in mature T and B cells, ES cells, and fibroblasts. The four large AgR loci have many bound CTCF sites, most of which are only occupied in lymphocytes, while only the CTCF sites at the end of each locus near the enhancers or J genes tend to be bound in non-lymphoid cells also. However, despite the generalized lymphocyte restriction of CTCF binding in AgR loci, the Igκ locus is the only locus that also shows significant lineage-specificity (T vs. B cells and developmental stage-specificity (pre-B vs. pro-B in CTCF binding. We show that cohesin binding shows greater lineage- and stage-specificity than CTCF at most AgR loci, providing more specificity to the loops. We also show that the culture of pro-B cells in IL7, a common practice to expand the number of cells before ChIP-seq, results in a CTCF-binding pattern resembling pre-B cells, as well as other epigenetic and transcriptional characteristics of pre-B cells. Analysis of the orientation of the CTCF sites show that all sites within the large V portions of the Igh and TCRβ loci have the same orientation. This suggests either a lack of requirement for convergent CTCF sites creating loops, or indicates an absence of any loops between CTCF sites within the V region portion of those loci but only loops to the convergent sites at the D-J-enhancer end of each locus. The V region portions of the Igκ and TCRα/δ loci, by contrast, have CTCF sites in both orientations, providing many options for

  13. Bioelectric patterning during oogenesis: stage-specific distribution of membrane potentials, intracellular pH and ion-transport mechanisms in Drosophila ovarian follicles.

    Science.gov (United States)

    Krüger, Julia; Bohrmann, Johannes

    2015-01-16

    Bioelectric phenomena have been found to exert influence on various developmental and regenerative processes. Little is known about their possible functions and the cellular mechanisms by which they might act during Drosophila oogenesis. In developing follicles, characteristic extracellular current patterns and membrane-potential changes in oocyte and nurse cells have been observed that partly depend on the exchange of protons, potassium ions and sodium ions. These bioelectric properties have been supposed to be related to various processes during oogenesis, e. g. pH-regulation, osmoregulation, cell communication, cell migration, cell proliferation, cell death, vitellogenesis and follicle growth. Analysing in detail the spatial distribution and activity of the relevant ion-transport mechanisms is expected to elucidate the roles that bioelectric phenomena play during oogenesis. To obtain an overview of bioelectric patterning along the longitudinal and transversal axes of the developing follicle, the spatial distributions of membrane potentials (Vmem), intracellular pH (pHi) and various membrane-channel proteins were studied systematically using fluorescent indicators, fluorescent inhibitors and antisera. During mid-vitellogenic stages 9 to 10B, characteristic, stage-specific Vmem-patterns in the follicle-cell epithelium as well as anteroposterior pHi-gradients in follicle cells and nurse cells were observed. Corresponding distribution patterns of proton pumps (V-ATPases), voltage-dependent L-type Ca(2+)-channels, amiloride-sensitive Na(+)-channels and Na(+),H(+)-exchangers (NHE) and gap-junction proteins (innexin 3) were detected. In particular, six morphologically distinguishable follicle-cell types are characterized on the bioelectric level by differences concerning Vmem and pHi as well as specific compositions of ion channels and carriers. Striking similarities between Vmem-patterns and activity patterns of voltage-dependent Ca(2+)-channels were found, suggesting

  14. Inhibition of Antigen Presentation by the Glycine/Alanine Repeat Domain Is Not Conserved in Simian Homologues of Epstein-Barr Virus Nuclear Antigen 1

    OpenAIRE

    Blake, Neil W.; Moghaddam, Amir; Rao, Pasupuleti; Kaur, Amitinder; Glickman, Rhona; Cho, Young-gyu; Marchini, Andrew; Haigh, Tracey; Johnson, R. Paul; Rickinson, Alan B.; Wang, Fred

    1999-01-01

    Most humans and Old World nonhuman primates are infected for life with Epstein-Barr virus (EBV) or closely related gammaherpesviruses in the same lymphocryptovirus (LCV) subgroup. Several potential strategies for immune evasion and persistence have been proposed based on studies of EBV infection in humans, but it has been difficult to test their actual contribution experimentally. Interest has focused on the EBV nuclear antigen 1 (EBNA1) because of its essential role in the maintenance and re...

  15. lncRNA Functional Networks in Oligodendrocytes Reveal Stage-Specific Myelination Control by an lncOL1/Suz12 Complex in the CNS.

    Science.gov (United States)

    He, Danyang; Wang, Jincheng; Lu, Yulan; Deng, Yaqi; Zhao, Chuntao; Xu, Lingli; Chen, Yinhuai; Hu, Yueh-Chiang; Zhou, Wenhao; Lu, Q Richard

    2017-01-18

    Long noncoding RNAs (lncRNAs) are emerging as important regulators of cellular functions, but their roles in oligodendrocyte myelination remain undefined. Through de novo transcriptome reconstruction, we establish dynamic expression profiles of lncRNAs at different stages of oligodendrocyte development and uncover a cohort of stage-specific oligodendrocyte-restricted lncRNAs, including a conserved chromatin-associated lncOL1. Co-expression network analyses further define the association of distinct oligodendrocyte-expressing lncRNA clusters with protein-coding genes and predict lncRNA functions in oligodendrocyte myelination. Overexpression of lncOL1 promotes precocious oligodendrocyte differentiation in the developing brain, whereas genetic inactivation of lncOL1 causes defects in CNS myelination and remyelination following injury. Functional analyses illustrate that lncOL1 interacts with Suz12, a component of polycomb repressive complex 2, to promote oligodendrocyte maturation, in part, through Suz12-mediated repression of a differentiation inhibitory network that maintains the precursor state. Together, our findings reveal a key lncRNA epigenetic circuitry through interaction with chromatin-modifying complexes in control of CNS myelination and myelin repair. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Proteomic Analysis of the Excretory and Secretory Proteins of Haemonchus contortus (HcESP Binding to Goat PBMCs In Vivo Revealed Stage-Specific Binding Profiles.

    Directory of Open Access Journals (Sweden)

    Javaid Ali Gadahi

    Full Text Available Haemonchus contortus is a parasitic gastrointestinal nematode, and its excretory and secretory products (HcESPs interact extensively with the host cells. In this study, we report the interaction of proteins from HcESPs at different developmental stages to goat peripheral blood mononuclear cells (PBMCs in vivo using liquid chromatography-tandem mass spectrometry. A total of 407 HcESPs that interacted with goat PBMCs at different time points were identified from a H. contortus protein database using SEQUEST searches. The L4 and L5 stages of H. contortus represented a higher proportion of the identified proteins compared with the early and late adult stages. Both stage-specific interacting proteins and proteins that were common to multiple stages were identified. Forty-seven interacting proteins were shared among all stages. The gene ontology (GO distributions of the identified goat PBMC-interacting proteins were nearly identical among all developmental stages, with high representation of binding and catalytic activity. Cellular, metabolic and single-organism processes were also annotated as major biological processes, but interestingly, more proteins were annotated as localization processes at the L5 stage than at the L4 and adult stages. Based on the clustering of homologous proteins, we improved the functional annotations of un-annotated proteins identified at different developmental stages. Some unnamed H. contortus ATP-binding cassette proteins, including ADP-ribosylation factor and P-glycoprotein-9, were identified by STRING protein clustering analysis.

  17. Multiplex zymography captures stage-specific activity profiles of cathepsins K, L, and S in human breast, lung, and cervical cancer.

    Science.gov (United States)

    Chen, Binbin; Platt, Manu O

    2011-07-14

    Cathepsins K, L, and S are cysteine proteases upregulated in cancer and proteolyze extracellular matrix to facilitate metastasis, but difficulty distinguishing specific cathepsin activity in complex tissue extracts confounds scientific studies and employing them for use in clinical diagnoses. Here, we have developed multiplex cathepsin zymography to profile cathepsins K, L, and S activity in 10 μg human breast, lung, and cervical tumors by exploiting unique electrophoretic mobility and renaturation properties. Frozen breast, lung, and cervix cancer tissue lysates and normal organ tissue lysates from the same human patients were obtained (28 breast tissues, 23 lung tissues, and 23 cervix tissues), minced and homogenized prior to loading for cathepsin gelatin zymography to determine enzymatic activity. Cleared bands of cathepsin activity were identified and validated in tumor extracts and detected organ- and stage-specific differences in activity. Cathepsin K was unique compared to cathepsins L and S. It was significantly higher for all cancers even at the earliest stage tested (stage I for lung and cervix (n = 6, p zymography, yielded 100% sensitivity and specificity for 20 breast tissue samples tested (10 normal; 10 tumor) in part due to the consistent absence of cathepsin K in normal breast tissue across all patients. To summarize, this sensitive assay provides quantitative outputs of cathepsins K, L, and S activities from mere micrograms of tissue and has potential use as a supplement to histological methods of clinical diagnoses of biopsied human tissue.

  18. Nbn and atm cooperate in a tissue and developmental stage-specific manner to prevent double strand breaks and apoptosis in developing brain and eye.

    Science.gov (United States)

    Rodrigues, Paulo M G; Grigaravicius, Paulius; Remus, Martina; Cavalheiro, Gabriel R; Gomes, Anielle L; Rocha-Martins, Maurício; Martins, Mauricio R; Frappart, Lucien; Reuss, David; McKinnon, Peter J; von Deimling, Andreas; Martins, Rodrigo A P; Frappart, Pierre-Olivier

    2013-01-01

    Nibrin (NBN or NBS1) and ATM are key factors for DNA Double Strand Break (DSB) signaling and repair. Mutations in NBN or ATM result in Nijmegen Breakage Syndrome and Ataxia telangiectasia. These syndromes share common features such as radiosensitivity, neurological developmental defects and cancer predisposition. However, the functional synergy of Nbn and Atm in different tissues and developmental stages is not yet understood. Here, we show in vivo consequences of conditional inactivation of both genes in neural stem/progenitor cells using Nestin-Cre mice. Genetic inactivation of Atm in the central nervous system of Nbn-deficient mice led to reduced life span and increased DSBs, resulting in increased apoptosis during neural development. Surprisingly, the increase of DSBs and apoptosis was found only in few tissues including cerebellum, ganglionic eminences and lens. In sharp contrast, we showed that apoptosis associated with Nbn deletion was prevented by simultaneous inactivation of Atm in developing retina. Therefore, we propose that Nbn and Atm collaborate to prevent DSB accumulation and apoptosis during development in a tissue- and developmental stage-specific manner.

  19. Stage-specific binding profiles of cohesin in resting and activated B lymphocytes suggest a role for cohesin in immunoglobulin class switching and maturation.

    Directory of Open Access Journals (Sweden)

    Gamze Günal-Sadık

    Full Text Available The immunoglobulin heavy chain locus (Igh features higher-order chromosomal interactions to facilitate stage-specific assembly of the Ig molecule. Cohesin, a ring-like protein complex required for sister chromatid cohesion, shapes chromosome architecture and chromatin interactions important for transcriptional regulation and often acts together with CTCF. Cohesin is likely involved in B cell activation and Ig class switch recombination. Hence, binding profiles of cohesin in resting mature murine splenic B lymphocytes and at two stages after cell activation were elucidated by chromatin immunoprecipitation and deep sequencing. Comparative genomic analysis revealed cohesin extensively changes its binding to transcriptional control elements after 48 h of stimulation with LPS/IL-4. Cohesin was clearly underrepresented at switch regions regardless of their activation status, suggesting that switch regions need to be cohesin-poor. Specific binding changes of cohesin at B-cell specific gene loci Pax5 and Blimp-1 indicate new cohesin-dependent regulatory pathways. Together with conserved cohesin/CTCF sites at the Igh 3'RR, a prominent cohesin/CTCF binding site was revealed near the 3' end of Cα where PolII localizes to 3' enhancers. Our study shows that cohesin likely regulates B cell activation and maturation, including Ig class switching.

  20. Foxc2CreERT2knock-in mice mark stage-specific Foxc2-expressing cells during mouse organogenesis.

    Science.gov (United States)

    Amin, Mohammed Badrul; Miura, Naoyuki; Uddin, Mohammad Khaja Mafij; Islam, Mohammod Johirul; Yoshida, Nobuaki; Iseki, Sachiko; Kume, Tsutomu; Trainor, Paul A; Saitsu, Hirotomo; Aoto, Kazushi

    2017-01-01

    Foxc2, a member of the winged helix transcription factor family, is essential for eye, calvarial bone, cardiovascular and kidney development in mice. Nevertheless, how Foxc2-expressing cells and their descendent cells contribute to the development of these tissues and organs has not been elucidated. Here, we generated a Foxc2 knock-in (Foxc2 CreERT2 ) mouse, in which administration of estrogen receptor antagonist tamoxifen induces nuclear translocation of Cre recombinase in Foxc2-expressing cells. By crossing with ROSA-LacZ reporter mice (Foxc2 CreERT2 ; R26R), the fate of Foxc2 positive (Foxc2 + ) cells was analyzed through LacZ staining at various embryonic stages. We found Foxc2 + cell descendants in the supraoccipital and exoccipital bone in E18.5 embryos, when tamoxifen was administered at embryonic day (E) 8.5. Furthermore, Foxc2 + descendant cranial neural crest cells at E8-10 were restricted to the corneal mesenchyme, while Foxc2 + cell derived cardiac neural crest cells at E6-12 were found in the aorta, pulmonary trunk and valves, and endocardial cushions. Foxc2 + cell descendant contributions to the glomerular podocytes in the kidney were also observed following E6.5 tamoxifen treatment. Our results are consistent with previous reports of Foxc2 expression during early embryogenesis and the Foxc2 CreERT2 mouse provides a tool to investigate spatiotemporal roles of Foxc2 and contributions of Foxc2 + expressing cells during mouse embryogenesis. © 2016 Japanese Teratology Society.

  1. Childhood Central Nervous System Embryonal Tumors Treatment

    Science.gov (United States)

    ... Reporting & Auditing Grant Transfer Grant Closeout Contracts & Small Business Training Cancer Training at NCI (Intramural) Funding for ... are open only to patients who have not started treatment. Children who have CNS embryonal tumors should ...

  2. Life Cycle Stage-resolved Proteomic Analysis of the Excretome/Secretome from Strongyloides ratti—Identification of Stage-specific Proteases*

    Science.gov (United States)

    Soblik, Hanns; Younis, Abuelhassan Elshazly; Mitreva, Makedonka; Renard, Bernhard Y.; Kirchner, Marc; Geisinger, Frank; Steen, Hanno; Brattig, Norbert W.

    2011-01-01

    A wide range of biomolecules, including proteins, are excreted and secreted from helminths and contribute to the parasite's successful establishment, survival, and reproduction in an adverse habitat. Excretory and secretory proteins (ESP) are active at the interface between parasite and host and comprise potential targets for intervention. The intestinal nematode Strongyloides spp. exhibits an exceptional developmental plasticity in its life cycle characterized by parasitic and free-living generations. We investigated ESP from infective larvae, parasitic females, and free-living stages of the rat parasite Strongyloides ratti, which is genetically very similar to the human pathogen, Strongyloides stercoralis. Proteomic analysis of ESP revealed 586 proteins, with the largest number of stage-specific ESP found in infective larvae (196), followed by parasitic females (79) and free-living stages (35). One hundred and forty proteins were identified in all studied stages, including anti-oxidative enzymes, heat shock proteins, and carbohydrate-binding proteins. The stage-selective ESP of (1) infective larvae included an astacin metalloproteinase, the L3 Nie antigen, and a fatty acid retinoid-binding protein; (2) parasitic females included a prolyl oligopeptidase (prolyl serine carboxypeptidase), small heat shock proteins, and a secreted acidic protein; (3) free-living stages included a lysozyme family member, a carbohydrate-hydrolyzing enzyme, and saponin-like protein. We verified the differential expression of selected genes encoding ESP by qRT-PCR. ELISA analysis revealed the recognition of ESP by antibodies of S. ratti-infected rats. A prolyl oligopeptidase was identified as abundant parasitic female-specific ESP, and the effect of pyrrolidine-based prolyl oligopeptidase inhibitors showed concentration- and time-dependent inhibitory effects on female motility. The characterization of stage-related ESP from Strongyloides will help to further understand the interaction of

  3. MKP1-dependent PTH modulation of bone matrix mineralization in female mice is osteoblast maturation stage specific and involves P-ERK and P-p38 MAPKs.

    Science.gov (United States)

    Mahalingam, Chandrika D; Sampathi, Bharat Reddy; Sharma, Sonali; Datta, Tanuka; Das, Varsha; Abou-Samra, Abdul B; Datta, Nabanita S

    2013-03-01

    Limited information is available on the role of MAPK phosphatase 1 (MKP1) signaling in osteoblasts. We have recently reported distinct roles for MKP1 during osteoblast proliferation, differentiation, and skeletal responsiveness to parathyroid hormone (PTH). As MKP1 regulates the phosphorylation status of MAPKs, we investigated the involvement of P-ERK and P-p38 MAPKs in MKP1 knockout (KO) early and mature osteoblasts with respect to mineralization and PTH response. Calvarial osteoblasts from 9-14-week-old WT and MKP1 KO male and female mice were examined. Western blot analysis revealed downregulation and sustained expressions of P-ERK and P-p38 with PTH treatment in differentiated osteoblasts derived from KO males and females respectively. Exposure of early osteoblasts to p38 inhibitor, SB203580 (S), markedly inhibited mineralization in WT and KO osteoblasts from both genders as determined by von Kossa assay. In osteoblasts from males, ERK inhibitor U0126 (U), not p38 inhibitor (S), prevented the inhibitory effects of PTH on mineralization in early or mature osteoblasts. In osteoblasts from KO females, PTH sustained mineralization in early osteoblasts and decreased mineralization in mature cells. This effect of PTH was attenuated by S in early osteoblasts and by U in mature KO cells. Changes in matrix Gla protein expression with PTH in KO osteoblasts did not correlate with mineralization, indicative of MKP1-dependent additional mechanisms essential for PTH action on osteoblast mineralization. We conclude that PTH regulation of osteoblast mineralization in female mice is maturation stage specific and involves MKP1 modulation of P-ERK and P-p38 MAPKs.

  4. Cytological and transcriptional dynamics analysis of host plant revealed stage-specific biological processes related to compatible rice-Ustilaginoidea virens interaction.

    Directory of Open Access Journals (Sweden)

    Jinquan Chao

    Full Text Available Rice false smut, a fungal disease caused by Ustilaginoidea virens is becoming a severe detriment to rice production worldwide. However, little is known about the molecular response of rice to attacks by the smut pathogen. In this article, we define the initial infection process as having three stages: initial colonization on the pistil (stage 1, S1, amplification on the anther (stage 2, S2 and sporulation in the anther chambers (stage 3, S3. Based on the transcriptome of rice hosts in response to U. virens in two separate years, we identified 126, 204, and 580 specific regulated genes in their respective stages S1, S2, and S3, respectively, by excluding common expression patterns in other openly biotic/abiotic databases using bioinformatics. As the disease progresses, several stage-specific biological processes (BP terms were distinctively enriched: "Phosphorylation" in stage S1, "PCD" in S2, and "Cell wall biogenesis" in S3, implying a concise signal cascade indicative of the tactics that smut pathogens use to control host rice cells during infection. 113 regulated genes were coexpressed among the three stages. They shared highly conserved promoter cis-element in the promoters in response to the regulation of WRKY and Myb for up-regulation, and ABA and Ca2+ for down regulation, indicating their potentially critical roles in signal transduction during rice-U. virens interaction. We further analyzed seven highly regulated unique genes; four were specific to pollen development, implying that pollen-related genes play critical roles in the establishment of rice susceptibility to U. virens. To my knowledge, this is the first report about probing of molecular response of rice to smut pathogen infection, which will greatly expand our understanding of the molecular events surrounding infection by rice false smut.

  5. Racial disparities in stage-specific gastric cancer: analysis of results from the Surveillance Epidemiology and End Results (SEER) program database.

    Science.gov (United States)

    Zhang, Gang; Zhao, Xing; Li, Jie; Yuan, Yu; Wen, Ming; Hao, Xin; Li, Ping; Zhang, Aimin

    2017-08-01

    The incidence of gastric cancer is declining in western countries but continues to represent a serious health problem worldwide, especially in Asia and among Asian Americans. This study aimed to investigate ethnic disparities in stage-specific gastric cancer, including differences in incidence, treatment and survival. The cohort study was analyzed using the data set of patients with gastric cancer registered in the Surveillance, Epidemiology, and End Results (SEER) program from 2004 to 2013. Among 54,165 patients with gastric cancer, 38,308 were whites (70.7%), 7546 were blacks (13.9%), 494 were American Indian/Alaskan Natives (0.9%) and 7817 were Asians/Pacific Islanders (14.4%). Variables were patient demographics, disease characteristics, surgery/radiation treatment, overall survival (OS) and cause specific survival (CSS). Asians/Pacific Islanders demonstrated the highest incidence rates for gastric cancer compared with other groups and had the greatest decline in incidence during the study period (13.03 to 9.28 per 100,000/year), as well as the highest percentage of patients with American Joint Committee on Cancer (AJCC) early stage gastric cancer. There were significant differences between groups in treatment across stages I-IV (all p<0.001); Asians/Pacific Islanders had the highest rate of surgery plus radiation (45.1%). Significant differences were found in OS and CSS between groups (p<0.001); OS was highest among Asians/Pacific Islanders. Multivariate analysis revealed that age, race, grade, stage, location, and second primary cancer were valid prognostic factors for survival. Marked ethnic disparities exist in age-adjusted incidence of primary gastric cancer, with significant differences between races in age, gender, histological type, grade, AJCC stage, location, second cancer, treatment and survival. Copyright © 2017 American Federation for Medical Research.

  6. Overexpression and cosuppression of xylem-related genes in an early xylem differentiation stage-specific manner by the AtTED4 promoter.

    Science.gov (United States)

    Endo, Satoshi; Iwamoto, Kuninori; Fukuda, Hiroo

    2018-02-01

    Tissue-specific overexpression of useful genes, which we can design according to their cause-and-effect relationships, often gives valuable gain-of-function phenotypes. To develop genetic tools in woody biomass engineering, we produced a collection of Arabidopsis lines that possess chimeric genes of a promoter of an early xylem differentiation stage-specific gene, Arabidopsis Tracheary Element Differentiation-related 4 (AtTED4) and late xylem development-associated genes, many of which are uncharacterized. The AtTED4 promoter directed the expected expression of transgenes in developing vascular tissues from young to mature stage. Of T2 lines examined, 42%, 49% and 9% were judged as lines with the nonrepeat type insertion, the simple repeat type insertion and the other repeat type insertion of transgenes. In 174 T3 lines, overexpression lines were confirmed for 37 genes, whereas only cosuppression lines were produced for eight genes. The AtTED4 promoter activity was high enough to overexpress a wide range of genes over wild-type expression levels, even though the wild-type expression is much higher than AtTED4 expression for several genes. As a typical example, we investigated phenotypes of pAtTED4::At5g60490 plants, in which both overexpression and cosuppression lines were included. Overexpression but not cosuppression lines showed accelerated xylem development, suggesting the positive role of At5g60490 in xylem development. Taken together, this study provides valuable results about behaviours of various genes expressed under an early xylem-specific promoter and about usefulness of their lines as genetic tools in woody biomass engineering. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  7. Differential expression of genes in soybean in response to the causal agent of Asian soybean rust (Phakopsora pachyrhizi Sydow) is soybean growth stage-specific.

    Science.gov (United States)

    Panthee, Dilip R; Marois, James J; Wright, David L; Narváez, Dario; Yuan, Joshua S; Stewart, C Neal

    2009-01-01

    Understanding plant host response to a pathogen such as Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), under different environmental conditions and growth stages is crucial for developing a resistant plant variety. The main objective of this study was to perform global transcriptome profiling of P. pachyrhizi-exposed soybean (Glycine max) with susceptible reaction to the pathogen from two distinct developmental growth stages using whole genome Affymetrix microarrays of soybean followed by confirmation using a resistant genotype. Soybean cv. 5601T (susceptible to ASR) at the V(4) and R(1) growth stages and Glycine tomentella (resistant to ASR) plants were inoculated with P. pachyrhizi and leaf samples were collected after 72 h of inoculation for microarray analysis. Upon analyzing the data using Array Assist software at 5% false discovery rate (FDR), a total of 5,056 genes were found significantly differentially expressed at V(4) growth stage, of which 2,401 were up-regulated, whereas 579 were found differentially expressed at R(1) growth stage, of which 264 were up-regulated. There were 333 differentially expressed common genes between the V(4) and R(1) growth stages, of which 125 were up-regulated. A large difference in number of differentially expressed genes between the two growth stages indicates that the gene expression is growth-stage-specific. We performed real-time RT-PCR analysis on nine of these genes from both growth stages and both plant species and found results to be congruent with those from the microarray analysis.

  8. A two- and three-dimensional approach for visualizing human embryonic stem cell differentiation

    DEFF Research Database (Denmark)

    Brøchner, Christian Beltoft; Vestentoft, Peter S; Lynnerup, Niels

    2010-01-01

    Undifferentiated human embryonic stem cells are characterized by expression of specific cell markers like the transcription factors OCT4, SOX2, and NANOG, the stage-specific embryonic antigen SSEA4, and the tumor-related antigens TRA-1-60 and TRA-1-81 and by their ability to differentiate under...... the expected markers. We describe a technique allowing paraffin embedding an entire hESC colony (e.g., 150 microm thick) and prepare 2-microm thick serial sections. Different staining procedures applied to individual sections produce a 2D survey of the developing hESC colony. Furthermore, a new and useful...... visualization of this 2D-expression pattern can be created by developing a 3D-model of the culture, based on serial paraffin sections. Individual sections are stained using individual markers. Using 3D image processing software such as Mimics or 3D-Doctor, the actual 3D-rendering of an entire colony can...

  9. The Cross-talk Between TGF-β1 and Dlk1 Mediates Early Chondrogenesis During Embryonic Endochondral Ossification

    DEFF Research Database (Denmark)

    Taipaleenmaki, Hanna; M, Linda; Chen, Li

    2012-01-01

    Dlkl/Pref-1/FA1 (delta like-1/preadipocyte factor-1/Fetal Antigen-1) is a novel surface marker for embryonic chondroprogenitor cells undergoing lineage progression from proliferation to prehypertrophic stages. However, mechanisms mediating control of its expression during chondrogenesis...... during mesenchymal condensation and chondrocyte proliferation, in parallel with expression of Sox9 and Col2a1, and was down-regulated upon the expression of Col10a1 by hypertrophic chondrocytes. Among a number of molecules that affected chondrogenesis, TGF-β1-induced proliferation of chondroprogenitors...... was associated with decreased Dlk1 expression. This effect was abolished by TGF-β signalling inhibitor SB431542, suggesting regulation of Dlk1/FA1 by TGF-β1 signalling in chondrogenesis. TGF-β1-induced Smad phosphorylation and chondrogenesis were significantly increased in Dlk1 (-/-) MEF, while they were blocked...

  10. Specific T-cell recognition of the merozoite proteins rhoptry-associated protein 1 and erythrocyte-binding antigen 1 of Plasmodium falciparum

    DEFF Research Database (Denmark)

    Jakobsen, P H; Hviid, L; Theander, T G

    1993-01-01

    The merozoite proteins merozoite surface protein 1 (MSP-1) and rhoptry-associated protein 1 (RAP-1) and synthetic peptides containing sequences of MSP-1, RAP-1, and erythrocyte-binding antigen 1, induced in vitro proliferative responses of lymphocytes collected from Ghanaian blood donors living...... by individuals living in an area with a high transmission rate of malaria. Most of the donor plasma samples tested contained immunoglobulin G (IgG) and IgM antibodies recognizing the merozoite proteins, while only a minority showed high IgG reactivity to the synthetic peptides....

  11. 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

  12. Stage-specific inhibition of MHC class I presentation by the Epstein-Barr virus BNLF2a protein during virus lytic cycle.

    Directory of Open Access Journals (Sweden)

    Nathan P Croft

    2009-06-01

    Full Text Available The gamma-herpesvirus Epstein-Barr virus (EBV persists for life in infected individuals despite the presence of a strong immune response. During the lytic cycle of EBV many viral proteins are expressed, potentially allowing virally infected cells to be recognized and eliminated by CD8+ T cells. We have recently identified an immune evasion protein encoded by EBV, BNLF2a, which is expressed in early phase lytic replication and inhibits peptide- and ATP-binding functions of the transporter associated with antigen processing. Ectopic expression of BNLF2a causes decreased surface MHC class I expression and inhibits the presentation of indicator antigens to CD8+ T cells. Here we sought to examine the influence of BNLF2a when expressed naturally during EBV lytic replication. We generated a BNLF2a-deleted recombinant EBV (DeltaBNLF2a and compared the ability of DeltaBNLF2a and wild-type EBV-transformed B cell lines to be recognized by CD8+ T cell clones specific for EBV-encoded immediate early, early and late lytic antigens. Epitopes derived from immediate early and early expressed proteins were better recognized when presented by DeltaBNLF2a transformed cells compared to wild-type virus transformants. However, recognition of late antigens by CD8+ T cells remained equally poor when presented by both wild-type and DeltaBNLF2a cell targets. Analysis of BNLF2a and target protein expression kinetics showed that although BNLF2a is expressed during early phase replication, it is expressed at a time when there is an upregulation of immediate early proteins and initiation of early protein synthesis. Interestingly, BNLF2a protein expression was found to be lost by late lytic cycle yet DeltaBNLF2a-transformed cells in late stage replication downregulated surface MHC class I to a similar extent as wild-type EBV-transformed cells. These data show that BNLF2a-mediated expression is stage-specific, affecting presentation of immediate early and early proteins, and

  13. Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Séverine A Degrelle

    Full Text Available Somatic cell nuclear transfer (SCNT is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each; one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular "uncoupling". Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538, we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity and subsequent pregnancy loss. Finally

  14. Embryonal sarcoma of the liver: case report

    International Nuclear Information System (INIS)

    Moreno, Luz A; Garzon C, Julian G; Montoya Ruben D; Murcia Susana

    2009-01-01

    Embryonal hepatic sarcoma occupies the third place in frequency among hepatic tumors in the pediatric population. Its clinical symptoms are usually non specific, and its appearance in diagnostic images is of a solid mass with a cystic component. Even though a histological confirmation is necessary, the initial management is oriented based on the imaging findings. This article presents a case report of a 9 year old girl with the diagnosis of an embryonal hepatic sarcoma; a description is done of the main clinical, histological and imaging characteristics.

  15. Autophagy in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Thien Tra

    Full Text Available Autophagy (macroautophagy is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC.

  16. Pitx2 in Embryonic and Adult Myogenesis

    Directory of Open Access Journals (Sweden)

    Amelia E. Aranega

    2017-05-01

    Full Text Available Skeletal muscle is a heterogeneous tissue that represents between 30 and 38% of the human body mass and has important functions in the organism, such as maintaining posture, locomotor impulse, or pulmonary ventilation. The genesis of skeletal muscle during embryonic development is a process controlled by an elaborate regulatory network combining the interplay of extrinsic and intrinsic regulatory mechanisms that transform myogenic precursor cells into functional muscle fibers through a finely tuned differentiation program. However, the capacity of generating muscle still remains once these fibers have matured. Adult myogenesis resembles many of the embryonic morphogenetic episodes and depends on the activation of satellite cells that have the potential to differentiate into new muscle fibers. Pitx2 is a member of the bicoid family of homeodomain transcription factors that play an important role in morphogenesis. In the last decade, Pitx2 has emerged as a key element involved in the fine-tuning mechanism that regulates skeletal-muscle development as well as the differentiation and cell fate of satellite cells in adult muscle. Here we present an integrative view of all aspects of embryonic and adult myogenesis in which Pitx2 is involved, from embryonic development to satellite-cell proliferation, fate specification, and differentiation. Those new Pitx2 functions on satellite-cell biology might open new perspectives to develop therapeutic strategies for muscular disorders.

  17. Pitx2 in Embryonic and Adult Myogenesis.

    Science.gov (United States)

    Hernandez-Torres, Francisco; Rodríguez-Outeiriño, Lara; Franco, Diego; Aranega, Amelia E

    2017-01-01

    Skeletal muscle is a heterogeneous tissue that represents between 30 and 38% of the human body mass and has important functions in the organism, such as maintaining posture, locomotor impulse, or pulmonary ventilation. The genesis of skeletal muscle during embryonic development is a process controlled by an elaborate regulatory network combining the interplay of extrinsic and intrinsic regulatory mechanisms that transform myogenic precursor cells into functional muscle fibers through a finely tuned differentiation program. However, the capacity of generating muscle still remains once these fibers have matured. Adult myogenesis resembles many of the embryonic morphogenetic episodes and depends on the activation of satellite cells that have the potential to differentiate into new muscle fibers. Pitx2 is a member of the bicoid family of homeodomain transcription factors that play an important role in morphogenesis. In the last decade, Pitx2 has emerged as a key element involved in the fine-tuning mechanism that regulates skeletal-muscle development as well as the differentiation and cell fate of satellite cells in adult muscle. Here we present an integrative view of all aspects of embryonic and adult myogenesis in which Pitx2 is involved, from embryonic development to satellite-cell proliferation, fate specification, and differentiation. Those new Pitx2 functions on satellite-cell biology might open new perspectives to develop therapeutic strategies for muscular disorders.

  18. Embryonic Development In Clarias gariepinus (Buchell, 1822 ...

    African Journals Online (AJOL)

    The embryonic development in Clarias gariepinus was studied under laboratory conditions. The development stages of eggs starting from first cleavage to hatching were examined microscopically. The accurate timing and detailed description of each stage were recorded. Photomicrograph of important stages, segmentation ...

  19. Testicular Embryonic Rhabdomyosarcoma, Case report with brief ...

    African Journals Online (AJOL)

    Testicular Embryonic Rhabdomyosarcoma, Case report with brief literature review. AM Adam, MMAM Ibnouf, IAF Allah. Abstract. Background: Rhabdomyosarcoma (RMS) is a malignant solid tumour arising from mesenchymal tissues which normally differentiate to form striated muscle. It can occur in a wide variety of sites.

  20. How the embryonic chick brain twists

    Science.gov (United States)

    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 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. PMID:28334695

  1. Epigenetic control of embryonic stem cell fate

    DEFF Research Database (Denmark)

    Christophersen, Nicolaj Strøyer; Helin, Kristian

    2010-01-01

    Embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and are pluripotent, as they are able to differentiate into all cell types of the adult organism. Once established, the pluripotent ES cells can be maintained under defined culture conditions, but can als...

  2. Dynamics of nuclear matrix proteome during embryonic ...

    Indian Academy of Sciences (India)

    Drosophila melanogaster embryos and show that 65% of the NuMat proteome is dynamic during development. Our ... [Varma P and Mishra RK 2011 Dynamics of nuclear matrix proteome during embryonic development in Drosophila melanogaster. J. Biosci. 36 .... functional group X, D = Number of proteins unique to late.

  3. 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

  4. Dynamics of nuclear matrix proteome during embryonic ...

    Indian Academy of Sciences (India)

    Embryonic development is a complex and dynamic process that involves spatiotemporal expression of genes in a highly coordinated manner. Multiple levels of nuclear architecture maintain the fidelity of gene expression programme. One of the components of nuclear architecture, which is believed to play an important role ...

  5. the production of mouse embryonic stem cells

    Indian Academy of Sciences (India)

    MADU

    Much research and lively debate focuses on the potential value of embryonic stem (ES) cells for regenerative medicine, the ethical issues raised by the use of human embryos to generate them, and the efforts presently made to circumvent this problem. The preparation of human ES cells in 1998 (Thomson et al 1998) ...

  6. Embryonal rhabdomyosarcoma of the cervix | Ocheke | African ...

    African Journals Online (AJOL)

    Embryonal rhabdomyosarcoma (sarcoma botyroides) of the cervix, which is rare, is described in a 16-yearold. The combined use of chemotherapy, radiotherapy and surgery has markedly improved survival in those with this condition. However, our patient did not benefit from this treatment modality due to late presentation ...

  7. Embryonic Stem Cells and their Genetic Modification

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 13; Issue 2. Embryonic Stem Cells and their Genetic Modification - The Nobel Prize in Physiology or Medicine 2007. Mitradas M Panicker. General Article Volume 13 Issue 2 February 2008 pp 172-180 ...

  8. The effects of storage temperature and position on embryonic ...

    African Journals Online (AJOL)

    The present study was carried out with the aim of determining the effects of storage temperature and storage position on embryonic mortalities in ostrich eggs. A total of 229 ostrich eggs was collected from two commercial ostrich farms. The effects of storage temperature on embryonic mortality differed. Embryonic deaths ...

  9. Description of post-implantation embryonic stages in European roe deer (Capreolus capreolus) after embryonic diapause.

    Science.gov (United States)

    Beyes, M; Nause, N; Bleyer, M; Kaup, F-J; Neumann, S

    2017-12-01

    The embryonic stage of development is defined as the period between fertilization and the establishment of most of the organ systems by the end of this period. Development in this stage is rapid. In many mammalian species, particularly in humans, the interval between fertilization and implantation is exactly determined and continuous without intermission. However, European roe deer (Capreolus capreolus) embryos undergo a reversible retardation of development. This interesting reproduction strategy is called embryonic diapause (delayed implantation). After this period of embryonic arrest, development continues without further interruption. The aim of this study was to investigate embryonic development after diapause in European roe deer. Because of the embryonic diapause and the unknown date of fertilization, it was impossible to assign the embryos to a certain gestational age (days). This study describes normal stages of embryonic development mainly based on the external morphological traits of 56 well-preserved post-implantation roe deer embryos and attempts to assign the embryos to certain development stages. Carnegie stages of human embryos were used as an orientation for staging roe deer embryos. We observed a considerable range of variation of embryonic stages investigated until the end of January. We found post-implantation stages of embryonic development already at the end of December and foetuses at the end of January. Moreover, assigning the embryos to a particular stage of development allows the comparison between pairs of twins and triplets. We showed that twins and triplets were always at the same development level, despite the discrepancy in inter-twin and inter-triplet size. © 2017 Blackwell Verlag GmbH.

  10. A trade-off between embryonic development rate and immune function of avian offspring is concealed by embryonic temperature

    Science.gov (United States)

    Martin, Thomas E.; Arriero, Elena; Majewska, Ania

    2011-01-01

    Long embryonic periods are assumed to reflect slower intrinsic development that are thought to trade off to allow enhanced physiological systems, such as immune function. Yet, the relatively rare studies of this trade-off in avian offspring have not found the expected trade-off. Theory and tests have not taken into account the strong extrinsic effects of temperature on embryonic periods of birds. Here, we show that length of the embryonic period did not explain variation in two measures of immune function when temperature was ignored, based on studies of 34 Passerine species in tropical Venezuela (23 species) and north temperate Arizona (11 species). Variation in immune function was explained when embryonic periods were corrected for average embryonic temperature, in order to better estimate intrinsic rates of development. Immune function of offspring trades off with intrinsic rates of embryonic development once the extrinsic effects of embryonic temperatures are taken into account.

  11. Temporal regulation of embryonic M-phases.

    Directory of Open Access Journals (Sweden)

    Franck Chesnel

    2008-02-01

    Full Text Available Temporal regulation of M-phases of the cell cycle requires precise molecular mechanisms that differ among different cells. This variable regulation is particularly clear during embryonic divisions. The first embryonic mitosis in the mouse lasts twice as long as the second one. In other species studied so far (C. elegans, Sphaerechinus granularis, Xenopus laevis, the first mitosis is also longer than the second, yet the prolongation is less pronounced than in the mouse. We have found recently that the mechanisms prolonging the first embryonic M-phase differ in the mouse and in Xenopus embryos. In the mouse, the metaphase of the first mitosis is specifically prolonged by the unknown mechanism acting similarly to the CSF present in oocytes arrested in the second meiotic division. In Xenopus, higher levels of cyclins B participate in the M-phase prolongation, however, without any cell cycle arrest. In Xenopus embryo cell-free extracts, the inactivation of the major M-phase factor, MPF, depends directly on dissociation of cyclin B from CDK1 subunit and not on cyclin B degradation as was thought before. In search for other mitotic proteins behaving in a similar way as cyclins B we made two complementary proteomic screens dedicated to identifying proteins ubiquitinated and degraded by the proteasome upon the first embryonic mitosis in Xenopus laevis. The first screen yielded 175 proteins. To validate our strategy we are verifying now which of them are really ubiquitinated. In the second one, we identified 9 novel proteins potentially degraded via the proteasome. Among them, TCTP (Translationally Controlled Tumor Protein, a 23-kDa protein, was shown to be partially degraded during mitosis (as well as during meiotic exit. We characterized the expression and the role of this protein in Xenopus, mouse and human somatic cells, Xenopus and mouse oocytes and embryos. TCTP is a mitotic spindle protein positively regulating cellular proliferation. Analysis of

  12. Changes in glycosphingolipid composition during differentiation of human embryonic stem cells to ectodermal or endodermal lineages.

    Science.gov (United States)

    Liang, Yuh-Jin; Yang, Bei-Chia; Chen, Jin-Mei; Lin, Yu-Hsing; Huang, Chia-Lin; Cheng, Yuan-Yuan; Hsu, Chi-Yen; Khoo, Kay-Hooi; Shen, Chia-Ning; Yu, John

    2011-12-01

    Glycosphingolipids (GSLs) are ubiquitous components of cell membranes that can act as mediators of cell adhesion and signal transduction and can possibly be used as cell type-specific markers. Our previous study indicated that there was a striking switch in the core structures of GSLs during differentiation of human embryonic stem cells (hESCs) into embryoid body (EB), suggesting a close association of GSLs with cell differentiation. In this study, to further clarify if alterations in GSL patterns are correlated with lineage-specific differentiation of hESCs, we analyzed changes in GSLs as hESCs were differentiated into neural progenitors or endodermal cells by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and tandem mass spectrometry (MS/MS) analyses. During hESC differentiation into neural progenitor cells, we found that the core structures of GSLs switched from globo- and lacto- to mostly ganglio-series dominated by GD3. On the other hand, when hESCs were differentiated into endodermal cells, patterns of GSLs totally differed from those observed in EB outgrowth and neural progenitors. The most prominent GSL identified by the MALDI-MS and MS/MS analysis was Gb(4) Ceramide, with no appreciable amount of stage-specific embryonic antigens 3 or 4, or GD3, in endodermal cells. These changes in GSL profiling were accompanied by alterations in the biosynthetic pathways of expressions of key glycosyltransferases. Our findings suggest that changes in GSLs are closely associated with lineage specificity and differentiation of hESCs. Copyright © 2011 AlphaMed Press.

  13. Glycosphingolipid dynamics in human embryonic stem cell and cancer: their characterization and biomedical implications.

    Science.gov (United States)

    Ho, Ming-Yi; Yu, Alice L; Yu, John

    2017-12-01

    Glycosphingolipids (GSLs) are composed of complex glycans linked to sphingosines and various fatty acid chains. Antibodies against several GSLs designated as stage-specific embryonic antigens (SSEAs), have been widely used to characterize differentiation of embryonic stem (ES) cells. In view of the cross-reactivities of these antibodies with multiple glycans, a few laboratories have employed advanced mass spectrometry (MS) technologies to define the dynamic changes of surface GSLs upon ES differentiation. However, the amphiphilic nature and heterogeneity of GSLs make them difficult to decipher. In our studies, systematic survey of GSL expression profiles in human ES cells and differentiated derivatives was conducted, primarily with matrix-assisted laser desorption/ionization MS (MALDI-MS) and MS/MS analyses. In addition to the well-known ES-specific markers, SSEA-3 and SSEA-4, several previously undisclosed globo- and lacto-series GSLs, including Gb4Cer, Lc4Cer, fucosyl Lc4Cer, Globo H, and disialyl Gb5Cer were identified in the undifferentiated human ES and induced pluripotent stem cells. Furthermore, during differentiation to embryoid body outgrowth, the core structures of GSLs switched from globo- and lacto- to ganglio-series. Lineage-specific differentiation was also marked by alterations of specific GSLs. During differentiation into neural progenitors, core structures shifted to primarily ganglio-series dominated by GD3. GSL patterns shifted to prominent expression of Gb4Cer with little SSEA-3 and- 4 or GD3 during endodermal differentiation. Several issues relevant to MS analysis and novel GSLs in ES cells were discussed. Finally, unique GSL signatures in ES and cancer cells are exploited in glycan-targeted anti-cancer immunotherapy and their mechanistic investigations were discussed using anti-GD2 mAb and Globo H as examples.

  14. Embryonic development of Pelteobagrus fulvidraco (Richardson, 1846)

    Science.gov (United States)

    Wang, Weimin; Abbas, Khalid; Yan, Ansheng

    2006-12-01

    For production enhancement and procedure upgrade, the developmental phases of laboratory-reared eggs of catfish Pelteobagrus fulvidraco were investigated. Twenty mature females and 10 males were collected from Dadongmen wholesale fisheries market in Wuhan City on May 8, 2003. Zygotes were stripped from mature fish after hormone-induced ovulation, fertilized, and incubated through whole embryonic development. The fertilized eggs were stocked in density of 100 eggs/L in white square tanks of 10 L. Incubation water was dechlorinated tap water with continuous aeration. The tanks were lit directly with 60 W fluorescent bulbs with a 12 light: 12 dark photoperiod. Water temperature, dissolved oxygen and pH were 29.0±0.5°C, 6.7±0.4 mg/L and 7.4±2, respectively. The results showed that the eggs of P. fulvidraco were yellow, sticky and contained much yolk. The mean diameter of fertilized eggs was 2.03 mm. At the water temperature of 29.0±0.5°C, the ontogenesis spent about 33 h after fertilization. From fertilization to hatching, the embryonic development can be divided into 30 40 phases, which varies in the emphasis and direction of development. The detailed embryonic movement was also described.

  15. Embryonic and foetal mortality in buffalo species

    Directory of Open Access Journals (Sweden)

    Marco Rendina

    2010-01-01

    Full Text Available The aim of this study was to verify the incidence of late embryonic mortality (between 25 and 45 days post-insemination; LEM and foetal mortality (between 45 and 70 days postinsemination; FM in buffaloes synchronized and mated by AI during the transitional period. The trial was performed on 288 multiparous Mediterranean Buffaloes, synchronized and inseminated by AI. Trans-rectal ultrasonography was performed 25, 45, and 70 days post-insemination to assess embryonic development. Milk samples were collected on Days 10, 20, 25, 30, and 45 post-insemination to determine progesterone concentration in whey. Pregnancy rate on Day 25 after AI was 48.6% but declined to 35.4% and to 30.6% by Day 45 and 70 respectively, representing a LEM of 27.1% and a FM of 13.7%. Progesterone concentration was higher (P<0.01 in pregnant compared to LEM buffaloes after 20 days post-insemination. Differences (P<0.05 were found between FM and LEM buffaloes on Days 25 and 30. Furthermore, progesterone concentration in pregnant buffaloes was higher (P=0.09 than that of FM buffaloes on Day 30 and 45. In conclusion, the success of application of reproductive biotechnologies in the transitional period depends from the incidence of embryonic and foetal mortality.

  16. Studies on the isolation, structural analysis and tissue localization of fetal antigen 1 and its relation to a human adrenal-specific cDNA, pG2

    DEFF Research Database (Denmark)

    Jensen, Charlotte Harken; Teisner, Børge; Højrup, Peter

    1993-01-01

    sequence was back-translated into the appropriate degenerate sequence of nucleic acids, fetal antigen 1 could be partially aligned to a 'human adrenal-specific mRNA, pG2'. The indirect immunoperoxidase technique demonstrated fetal antigen 1 in fetal hepatocytes, glandular cells of fetal pancreas...... is encoded by the mRNA defined by the cDNA clone pG2, but definitive sequencing and expression studies of this mRNA have not been achieved. Udgivelsesdato: 1993-Apr...

  17. Identification of a rare point mutation at C-terminus of merozoite surface antigen-1 gene of Plasmodium falciparum in eastern Indian isolates.

    Science.gov (United States)

    Raj, Dipak Kumar; Das, Bibhu Ranjan; Dash, A P; Supakar, Prakash C

    2004-01-01

    Merozoite surface antigen-1 (MSA-1) of Plasmodium falciparum is highly immunogenic in human. Several studies suggest that MSA-1 protein is an effective target for a protective immune response. Attempt has been made to find new point mutations by analyzing 244 bp [codon 1655(R) to 1735 (I)] relatively conserved C-terminus region of MSA-1 gene in 125 isolates. This region contains two EGF like domains, which are involved in generating protective immune response in human. Point mutations in this region are very much important in view of vaccine development. Searching of mutational hot spots in MSA-1 protein by sequencing method in a representative number of isolates is quite critical and expensive. Therefore, in this study slot blot and PCR-SSCP method have been used to find out new mutations in the individual isolates showing alterations in the mobility of DNA fragment. Sequencing of the altered bands from the SSCP gel shows a rare non-synonymous point mutation in 7 (5.6%) of the 125 isolates at amino acid position 1704 of MSA-1 gene where isoleucine is replaced by valine.

  18. Effects of Intermittent Administration of Parathyroid Hormone (1-34 on Bone Differentiation in Stromal Precursor Antigen-1 Positive Human Periodontal Ligament Stem Cells

    Directory of Open Access Journals (Sweden)

    Xiaoxiao Wang

    2016-01-01

    Full Text Available Periodontitis is the most common cause of tooth loss and bone destruction in adults worldwide. Human periodontal ligament stem cells (hPDLSCs may represent promising new therapeutic biomaterials for tissue engineering applications. Stromal precursor antigen-1 (STRO-1 has been shown to have roles in adherence, proliferation, and multipotency. Parathyroid hormone (PTH has been shown to enhance proliferation in osteoblasts. Therefore, in this study, we aimed to compare the functions of STRO-1(+ and STRO-1(− hPDLSCs and to investigate the effects of PTH on the osteogenic capacity of STRO-1(+ hPDLSCs in order to evaluate their potential applications in the treatment of periodontitis. Our data showed that STRO-1(+ hPDLSCs expressed higher levels of the PTH-1 receptor (PTH1R than STRO-1(− hPDLSCs. In addition, intermittent PTH treatment enhanced the expression of PTH1R and osteogenesis-related genes in STRO-1(+ hPDLSCs. PTH-treated cells also exhibited increased alkaline phosphatase activity and mineralization ability. Therefore, STRO-1(+ hPDLSCs represented a more promising cell resource for biomaterials and tissue engineering applications. Intermittent PTH treatment improved the capacity for STRO-1(+ hPDLSCs to repair damaged tissue and ameliorate the symptoms of periodontitis.

  19. The CD11a partner in Sus scrofa lymphocyte function-associated antigen-1 (LFA-1: mRNA cloning, structure analysis and comparison with mammalian homologues

    Directory of Open Access Journals (Sweden)

    Thomas Anne VT

    2005-10-01

    Full Text Available Abstract Background Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2, the most abundant and widely expressed beta2-integrin, is required for many cellular adhesive interactions during the immune response. Many studies have shown that LFA-1 is centrally involved in the pathogenesis of several diseases caused by Repeats-in-toxin (RTX -producing bacteria. Results The porcine-LFA-1 CD11a (alpha subunit coding sequence was cloned, sequenced and compared with the available mammalian homologues in this study. Despite some focal differences, it shares all the main characteristics of these latter. Interestingly, as in sheep and humans, an allelic variant with a triplet insertion resulting in an additional Gln-744 was consistently identified, which suggests an allelic polymorphism that might be biologically relevant. Conclusion Together with the pig CD18-encoding cDNA, which has been available for a long time, the sequence data provided here will allow the successful expression of porcine CD11a, thus giving the first opportunity to express the Sus scrofa beta2-integrin LFA-1 in vitro as a tool to examine the specificities of inflammation in the porcine species.

  20. Protein kinase a dependent phosphorylation of apical membrane antigen 1 plays an important role in erythrocyte invasion by the malaria parasite.

    Directory of Open Access Journals (Sweden)

    Kerstin Leykauf

    2010-06-01

    Full Text Available Apicomplexan parasites are obligate intracellular parasites that infect a variety of hosts, causing significant diseases in livestock and humans. The invasive forms of the parasites invade their host cells by gliding motility, an active process driven by parasite adhesion proteins and molecular motors. A crucial point during host cell invasion is the formation of a ring-shaped area of intimate contact between the parasite and the host known as a tight junction. As the invasive zoite propels itself into the host-cell, the junction moves down the length of the parasite. This process must be tightly regulated and signalling is likely to play a role in this event. One crucial protein for tight-junction formation is the apical membrane antigen 1 (AMA1. Here we have investigated the phosphorylation status of this key player in the invasion process in the human malaria parasite Plasmodium falciparum. We show that the cytoplasmic tail of P. falciparum AMA1 is phosphorylated at serine 610. We provide evidence that the enzyme responsible for serine 610 phosphorylation is the cAMP regulated protein kinase A (PfPKA. Importantly, mutation of AMA1 serine 610 to alanine abrogates phosphorylation of AMA1 in vivo and dramatically impedes invasion. In addition to shedding unexpected new light on AMA1 function, this work represents the first time PKA has been implicated in merozoite invasion.

  1. Heat shock factor 1 upregulates transcription of Epstein-Barr Virus nuclear antigen 1 by binding to a heat shock element within the BamHI-Q promoter

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Feng-Wei [The State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou (China); Wu, Xian-Rui [Department of Surgery, Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou (China); Liu, Wen-Ju; Liao, Yi-Ji [The State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou (China); Lin, Sheng [Laboratory of Integrated Biosciences, School of Life Science, Sun Yat-sen University, Guangzhou (China); Zong, Yong-Sheng; Zeng, Mu-Sheng; Zeng, Yi-Xin [The State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou (China); Mai, Shi-Juan, E-mail: maishj@sysucc.org.cn [The State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou (China); Xie, Dan, E-mail: xied@mail.sysu.edu.cn [The State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou (China)

    2011-12-20

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for maintenance of the episome and establishment of latency. In this study, we observed that heat treatment effectively induced EBNA1 transcription in EBV-transformed B95-8 and human LCL cell lines. Although Cp is considered as the sole promoter used for the expression of EBNA1 transcripts in the lymphoblastoid cell lines, the RT-PCR results showed that the EBNA1 transcripts induced by heat treatment arise from Qp-initiated transcripts. Using bioinformatics, a high affinity and functional heat shock factor 1 (HSF1)-binding element within the - 17/+4 oligonucleotide of the Qp was found, and was determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Moreover, heat shock and exogenous HSF1 expression induced Qp activity in reporter assays. Further, RNA interference-mediated HSF1 gene silencing attenuated heat-induced EBNA1 expression in B95-8 cells. These results provide evidence that EBNA1 is a new target for the transcription factor HSF1.

  2. The homeobox gene Hex regulates hepatocyte differentiation from embryonic stem cell-derived endoderm.

    Science.gov (United States)

    Kubo, Atsushi; Kim, Yon Hui; Irion, Stefan; Kasuda, Shogo; Takeuchi, Mitsuaki; Ohashi, Kazuo; Iwano, Masayuki; Dohi, Yoshiko; Saito, Yoshihiko; Snodgrass, Ralph; Keller, Gordon

    2010-02-01

    We investigated the role of the hematopoietically expressed homeobox (Hex) in the differentiation and development of hepatocytes within embryonic stem cell (ESC)-derived embryoid bodies (EBs). Analyses of hepatic endoderm derived from Hex(-/-) EBs revealed a dramatic reduction in the levels of albumin (Alb) and alpha-fetoprotein (Afp) expression. In contrast, stage-specific forced expression of Hex in EBs from wild-type ESCs led to the up-regulation of Alb and Afp expression and secretion of Alb and transferrin. These inductive effects were restricted to c-kit(+) endoderm-enriched EB-derived populations, suggesting that Hex functions at the level of hepatic specification of endoderm in this model. Microarray analysis revealed that Hex regulated the expression of a broad spectrum of hepatocyte-related genes, including fibrinogens, apolipoproteins, and cytochromes. When added to the endoderm-induced EBs, bone morphogenetic protein 4 acted synergistically with Hex in the induction of expression of Alb, Afp, carbamoyl phosphate synthetase, transcription factor 1, and CCAAT/enhancer binding protein alpha. These findings indicate that Hex plays a pivotal role during induction of liver development from endoderm in this in vitro model and suggest that this strategy may provide important insight into the generation of functional hepatocytes from ESCs.

  3. Self-renewal of embryonic-stem-cell-derived progenitors by organ-matched mesenchyme.

    Science.gov (United States)

    Sneddon, Julie B; Borowiak, Malgorzata; Melton, Douglas A

    2012-11-29

    One goal of regenerative medicine, to use stem cells to replace cells lost by injury or disease, depends on producing an excess of the relevant cell for study or transplantation. To this end, the stepwise differentiation of stem cells into specialized derivatives has been successful for some cell types, but a major problem remains the inefficient conversion of cells from one stage of differentiation to the next. If specialized cells are to be produced in large numbers it will be necessary to expand progenitor cells, without differentiation, at some steps of the process. Using the pancreatic lineage as a model for embryonic-stem-cell differentiation, we demonstrate that this is a solvable problem. Co-culture with organ-matched mesenchyme permits proliferation and self-renewal of progenitors, without differentiation, and enables an expansion of more than a million-fold for human endodermal cells with full retention of their developmental potential. This effect is specific both to the mesenchymal cell and to the progenitor being amplified. Progenitors that have been serially expanded on mesenchyme give rise to glucose-sensing, insulin-secreting cells when transplanted in vivo. Theoretically, the identification of stage-specific renewal signals can be incorporated into any scheme for the efficient production of large numbers of differentiated cells from stem cells and may therefore have wide application in regenerative biology.

  4. Distinctive Roles of Canonical and Noncanonical Wnt Signaling in Human Embryonic Cardiomyocyte Development

    Directory of Open Access Journals (Sweden)

    Silvia Mazzotta

    2016-10-01

    Full Text Available Wnt signaling is a key regulator of vertebrate heart development; however, specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components, monitor pathway activity, and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A, via FZD7 and canonical signaling, regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B, via ROR2 and noncanonical signaling, regulate MESP1 expression and cardiovascular development; and that later in development WNT2, WNT5A/5B, and WNT11, via FZD4 and FZD6, regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling. Our findings confirm in human development previously proposed roles for canonical Wnt signaling in sequential stages of vertebrate cardiomyogenesis, and identify more precise roles for noncanonical signaling and for individual Wnt signal and Wnt receptor genes in human cardiomyocyte development.

  5. Morphogenesis and three-dimensional movement of the stomach during the human embryonic period.

    Science.gov (United States)

    Kaigai, N; Nako, A; Yamada, S; Uwabe, C; Kose, K; Takakuwa, T

    2014-05-01

    The stomach develops as the local widening of the foregut after Carnegie stage (CS) 13 that moves in a dramatic and dynamic manner during the embryonic period. Using the magnetic resonance images of 377 human embryos, we present the morphology, morphometry, and three-dimensional movement of the stomach during CS16 and CS23. The stomach morphology revealed stage-specific features. The angular incisura and the cardia were formed at CS18. The change in the angular incisura angle was approximately 90° during CS19 and CS20, and was stomach revealed that the stomach gradually becomes "deflected" during development. The stomach may appear to move to the left laterally and caudally due to its deflection and differential growth. The track of the reference points in the stomach may reflect the visual three-dimensional movement. The movement of point M, representing the movement of the greater curvature, was different from that of points C (cardia) and P (pyloric antrum). The P and C were located just around the midsagittal plane in all the stages observed. Point M moved in the caudal-left lateral direction until CS22. Moreover, the vector CP does not rotate around the dorsoventral axis, as widely believed, but around the transverse axis. The plane CPM rotated mainly around the longitudinal axis. The data obtained will be useful for prenatal diagnosis in the near future. Copyright © 2013 Wiley Periodicals, Inc.

  6. Phase 1/2a study of the malaria vaccine candidate apical membrane antigen-1 (AMA-l) administered in adjuvant system AS01B or AS02A

    NARCIS (Netherlands)

    M.D. Spring (Michele Donna); J.F. Cummings (James); C.F. Ockenhouse (Christian); S. Dutta (Shantanu); R. Reidler (Randall); E. Angov (Evelina); E. Bergmann-Leitner (Elke); V.A. Stewart (Ann); S. Bittner (Stacey); L. Juompan (Laure); M.G. Kortepeter (Mark); R. Nielsen (Robin); U. Krzych (Urszula); E. Tierney (Ev); L.A. Ware (Lisa); M. Dowler (Megan); C.C. Hermsen (Cornelus); R.W. Sauerwein (Robert); S.J. de Vlas (Sake); O. Ofori-Anyinam (Opokua); D.E. Lanar (David); J.L. Williams (Jack); K.E. Kester (Kent); K. Tucker (Kathryn); M. Shi (Meng); E. Malkin (Elissa); C. Long (Carole); C.L. Diggs (Carter); L. Soisson (Lorraine Amory); M.C. Dubois; W.R. Ballou (Ripley); J. Cohen (Joe); D.G. Heppner (Gray)

    2009-01-01

    textabstractBackground: This Phase 1/2a study evaluated the safety, immunogenicity, and efficacy of an experimental malaria vaccine comprised of the recombinant Plasmodium falciparum protein apical membrane antigen-1 (AMA-1) representing the 3D7 allele formulated with either the AS01B or AS02A

  7. Cytokine signalling in embryonic stem cells

    DEFF Research Database (Denmark)

    Kristensen, David Møbjerg; Kalisz, Mark; Nielsen, Jens Høiriis

    2006-01-01

    signalling pathways have been documented. In addition, gp130 activation leads to both PI3K and Src activation. The canonical Wnt pathway is sufficient to maintain self-renewal of both human ES cells and mouse ES cells. It seems quite possible that the main pathway maintaining self-renewal in ES cells...... is the Wnt pathway, while the LIF-JAK-STAT3 pathway is present in mouse cells as an adaptation for sustaining self-renewal during embryonic diapause, a condition of delayed implantation in mammals. In keeping with this scenario, the Wnt pathway has been shown to elevate the level of c-myc. Thus, the two...

  8. Synthetic strategies for studying embryonic development.

    Science.gov (United States)

    Ouyang, Xiaohu; Chen, James K

    2010-06-25

    Developmental biology has evolved from a descriptive science to one based on genetic principles and molecular mechanisms. Although molecular biology and genetic technologies have been the primary drivers of this transformation, synthetic strategies have been increasingly utilized to interrogate the mechanisms of embryonic patterning with spatial and temporal precision. In this review, we survey how chemical tools and engineered proteins have been used to perturb developmental processes at the DNA, RNA, protein, and cellular levels. We discuss the design principles, experimental capabilities, and limitations of each method, as well as future challenges for the chemical and developmental biology communities.

  9. 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

  10. Association analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope and smoking status in Brazilian patients with rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Michel Alexandre Yazbek

    2011-01-01

    Full Text Available INTRODUCTION: Epstein-Barr virus exposure appears to be an environmental trigger for rheumatoid arthritis that interacts with other risk factors. Relationships among anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status have been observed in patients with rheumatoid arthritis from different populations. OBJECTIVE: To perform an association analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status in Brazilian patients with rheumatoid arthritis. METHODS: In a case-control study, 140 rheumatoid arthritis patients and 143 healthy volunteers who were matched for age, sex, and ethnicity were recruited. Anti-Epstein-Barr nuclear antigen-1 antibodies and anti-cyclic citrullinated peptide antibodies were examined using an enzyme-linked immunosorbent assay, and shared epitope alleles were identified by genotyping. Smoking information was collected from all subjects. A comparative analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status was performed in the patient group. Logistic regression analysis models were used to analyze the risk of rheumatoid arthritis. RESULTS: Anti-Epstein-Barr nuclear antigen-1 antibodies were not associated with anti-cyclic citrullinated peptide antibodies, shared epitope alleles, or smoking status. Anti-cyclic citrullinated peptide antibody positivity was significantly higher in smoking patients with shared epitope alleles (OR = 3.82. In a multivariate logistic regression analysis using stepwise selection, only anti-cyclic citrullinated peptide antibodies were found to be independently associated with rheumatoid arthritis (OR = 247.9. CONCLUSION: Anti-Epstein-Barr nuclear antigen-1 antibodies did not increase the risk of rheumatoid arthritis and were not associated with the rheumatoid arthritis risk factors studied. Smoking

  11. Life stage-specific effects of the fungicide pyrimethanil and temperature on the snail Physella acuta (Draparnaud, 1805) disclose the pitfalls for the aquatic risk assessment under global climate change

    International Nuclear Information System (INIS)

    Seeland, Anne; Albrand, Jennifer; Oehlmann, Jörg; Müller, Ruth

    2013-01-01

    It can be suggested that the combined stress of pesticide pollution and suboptimal temperature influences the sensitivity of life stages of aquatic invertebrates differently. The embryo, juvenile, half- and full-life-cycle toxicity tests performed with the snail Physella acuta at different concentrations (0.06–0.5 or 1.0 mg L −1 ) of the model fungicide pyrimethanil at 15, 20 and 25 °C revealed, that pyrimethanil caused concentration-dependent effects at all test temperatures. Interestingly, the ecotoxicity of pyrimethanil was higher at lower (suboptimal) temperature for embryo hatching and F 1 reproduction, but its ecotoxicity for juvenile growth and F 0 reproduction increased with increasing temperature. The life-stage specific temperature-dependent ecotoxicity of pyrimethanil and the high fungicide susceptibility of the invasive snail clearly demonstrate the complexity of pesticide–temperature interactions and the challenge to draw conclusions for the risk of pesticides under the impact of global climate change. -- Highlights: ► Physella acuta reacts highly sensitively to exposure to pyrimethanil. ► The ecotoxicity of pyrimethanil is life-stage specific. ► Pyrimethanil and temperature stress influenced the development interactively. -- The aquatic risk of pesticides under climate change cannot be adequately assessed by recent strategies for the regular risk assessment of agrochemicals

  12. Inhibition of antigen presentation by the glycine/alanine repeat domain is not conserved in simian homologues of Epstein-Barr virus nuclear antigen 1.

    Science.gov (United States)

    Blake, N W; Moghaddam, A; Rao, P; Kaur, A; Glickman, R; Cho, Y G; Marchini, A; Haigh, T; Johnson, R P; Rickinson, A B; Wang, F

    1999-09-01

    Most humans and Old World nonhuman primates are infected for life with Epstein-Barr virus (EBV) or closely related gammaherpesviruses in the same lymphocryptovirus (LCV) subgroup. Several potential strategies for immune evasion and persistence have been proposed based on studies of EBV infection in humans, but it has been difficult to test their actual contribution experimentally. Interest has focused on the EBV nuclear antigen 1 (EBNA1) because of its essential role in the maintenance and replication of the episomal viral genome in latently infected cells and because EBNA1 endogenously expressed in these cells is protected from presentation to the major histocompatibility complex class-I restricted cytotoxic T-lymphocyte (CTL) response through the action of an internal glycine-alanine repeat (GAR). Given the high degree of biologic conservation among LCVs which infect humans and Old World primates, we hypothesized that strategies essential for viral persistence would be well conserved among viruses of this subgroup. We show that the rhesus LCV EBNA1 shares sequence homology with the EBV and baboon LCV EBNA1 and that the rhesus LCV EBNA1 is a functional homologue for EBV EBNA1-dependent plasmid maintenance and replication. Interestingly, all three LCVs possess a GAR domain, but the baboon and rhesus LCV EBNA1 GARs fail to inhibit antigen processing and presentation as determined by using three different in vitro CTL assays. These studies suggest that inhibition of antigen processing and presentation by the EBNA1 GAR may not be an essential mechanism for persistent infection by all LCV and that other mechanisms may be important for immune evasion during LCV infection.

  13. Proteome analysis of chick embryonic cerebrospinal fluid.

    Science.gov (United States)

    Parada, Carolina; Gato, Angel; Aparicio, Mariano; Bueno, David

    2006-01-01

    During early stages of embryo development, the brain cavity is filled with embryonic cerebrospinal fluid (E-CSF), a complex fluid containing different protein fractions that contributes to the regulation of the survival, proliferation and neurogenesis of the neuroectodermal stem cells. Using 2-DE, protein sequencing and database searches, we identified and analyzed the proteome of the E-CSF from chick embryos (Gallus gallus). We identified 26 different gene products, including proteins related to the extracellular matrix, proteins associated with the regulation of osmotic pressure and metal transport, proteins related to cell survival, MAP kinase activators, proteins involved in the transport of retinol and vitamin D, antioxidant and antimicrobial proteins, intracellular proteins and some unknown proteins. Most of these gene products are involved in the regulation of developmental processes during embryogenesis in systems other than E-CSF. Interestingly, 14 of them are also present in adult human CSF proteome, and it has been reported that they are altered in the CSF of patients suffering neurodegenerative diseases and/or neurological disorders. Understanding these molecules and the mechanisms they control during embryonic neurogenesis is a key contribution to the general understanding of CNS development, and may also contribute to greater knowledge of these human diseases.

  14. MPSS profiling of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Vasicek Tom

    2004-08-01

    Full Text Available Abstract Background Pooled human embryonic stem cells (hESC cell lines were profiled to obtain a comprehensive list of genes common to undifferentiated human embryonic stem cells. Results Pooled hESC lines were profiled to obtain a comprehensive list of genes common to human ES cells. Massively parallel signature sequencing (MPSS of approximately three million signature tags (signatures identified close to eleven thousand unique transcripts, of which approximately 25% were uncharacterised or novel genes. Expression of previously identified ES cell markers was confirmed and multiple genes not known to be expressed by ES cells were identified by comparing with public SAGE databases, EST libraries and parallel analysis by microarray and RT-PCR. Chromosomal mapping of expressed genes failed to identify major hotspots and confirmed expression of genes that map to the X and Y chromosome. Comparison with published data sets confirmed the validity of the analysis and the depth and power of MPSS. Conclusions Overall, our analysis provides a molecular signature of genes expressed by undifferentiated ES cells that can be used to monitor the state of ES cells isolated by different laboratories using independent methods and maintained under differing culture conditions

  15. MPSS profiling of human embryonic stem cells.

    Science.gov (United States)

    Brandenberger, Ralph; Khrebtukova, Irina; Thies, R Scott; Miura, Takumi; Jingli, Cai; Puri, Raj; Vasicek, Tom; Lebkowski, Jane; Rao, Mahendra

    2004-08-10

    Pooled human embryonic stem cells (hESC) cell lines were profiled to obtain a comprehensive list of genes common to undifferentiated human embryonic stem cells. Pooled hESC lines were profiled to obtain a comprehensive list of genes common to human ES cells. Massively parallel signature sequencing (MPSS) of approximately three million signature tags (signatures) identified close to eleven thousand unique transcripts, of which approximately 25% were uncharacterised or novel genes. Expression of previously identified ES cell markers was confirmed and multiple genes not known to be expressed by ES cells were identified by comparing with public SAGE databases, EST libraries and parallel analysis by microarray and RT-PCR. Chromosomal mapping of expressed genes failed to identify major hotspots and confirmed expression of genes that map to the X and Y chromosome. Comparison with published data sets confirmed the validity of the analysis and the depth and power of MPSS. Overall, our analysis provides a molecular signature of genes expressed by undifferentiated ES cells that can be used to monitor the state of ES cells isolated by different laboratories using independent methods and maintained under differing culture conditions

  16. Cryopreservation of embryonic axes of maize ( Zea mays L.) by ...

    African Journals Online (AJOL)

    A storage protocol at cryogenic temperature was established for embryonic axes of maize using a basic vitrification protocol with direct immersion in liquid nitrogen (-196ºC). The response of isolated embryonic axes of five maize genotypes to plant vitrification solution (PVS2) at different concentrations was studied. Recovery ...

  17. Embryonic adaptations and nutrition in the viviparous teleost Clinus ...

    African Journals Online (AJOL)

    Embryos of Clinus dorsalis absorb nutrients from the embiyotrophe, secreted by the follicular epithelium. Autoradiographic studies revealed that the principal areas of nutrient absorption are the embryonic gut and epidermis. A histological and electron microscopic study of embryonic structure revealed an extensively ...

  18. Transcriptome analysis of primary bovine extra-embryonic cultured cells

    Directory of Open Access Journals (Sweden)

    Séverine A. Degrelle

    2015-12-01

    Full Text Available The dataset described in this article pertains to the article by Hue et al. (2015 entitled “Primary bovine extra-embryonic cultured cells: A new resource for the study of in vivo peri-implanting phenotypes and mesoderm formation” [1]. In mammals, extra-embryonic tissues are essential to support not only embryo patterning but also embryo survival, especially in late implanting species. These tissues are composed of three cell types: trophoblast (bTCs, endoderm (bXECs and mesoderm (bXMCs. Until now, it is unclear how these cells interact. In this study, we have established primary cell cultures of extra-embryonic tissues from bovine embryos collected at day-18 after artificial insemination. We used our homemade bovine 10K array (GPL7417 to analyze the gene expression profiles of these primary extra-embryonic cultured cells compared to the corresponding cells from in vivo micro-dissected embryos. Here, we described the experimental design, the isolation of bovine extra-embryonic cell types as well as the microarray expression analysis. The dataset has been deposited in Gene Expression Omnibus (GEO (accession number GSE52967. Finally, these primary cell cultures were a powerful tool to start studying their cellular properties, and will further allow in vitro studies on cellular interactions among extra-embryonic tissues, and potentially between extra-embryonic vs embryonic tissues.

  19. 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

  20. Influx mechanisms in the embryonic and adult rat choroid plexus

    DEFF Research Database (Denmark)

    Saunders, Norman R; Dziegielewska, Katarzyna M; Møllgård, Kjeld

    2015-01-01

    The transcriptome of embryonic and adult rat lateral ventricular choroid plexus, using a combination of RNA-Sequencing and microarray data, was analyzed by functional groups of influx transporters, particularly solute carrier (SLC) transporters. RNA-Seq was performed at embryonic day (E) 15 and a...... that the choroid plexus in embryonic brain plays a major role in supplying the developing brain with essential nutrients.......The transcriptome of embryonic and adult rat lateral ventricular choroid plexus, using a combination of RNA-Sequencing and microarray data, was analyzed by functional groups of influx transporters, particularly solute carrier (SLC) transporters. RNA-Seq was performed at embryonic day (E) 15...... in the adult plexus were expressed at higher levels than in embryos. These results are compared with earlier published physiological studies of amino acid and monocarboxylate transport in developing rodents. This comparison shows correlation of high expression of some transporters in the developing brain...

  1. Development of rat embryonic spinal ganglion cells in damaged nerve.

    Science.gov (United States)

    Petrova, E S; Isaeva, E N; Korzhevskii, D E

    2014-09-01

    The development of dissociated cells from rat embryonic spinal ganglion after transplantation to damaged nerve of adult animals was studied using immunohistochemical differentiation markers of neural and glial cells. The cell suspension obtained after dissociation of rat embryonic spinal ganglia (embryonic day 15) was injected into the proximal segment of crushed sciatic nerve. The nerve was damaged by ligation for 40 sec. Progenitor cells were labeled with 5-bromo-2'-deoxyuridine (BrdU) before transplantation. BrdU-immunopositive cells were detected in the nerve trunks of recipients on days 1, 21, and 28 after transplantation. Dissociated cells of rat embryonic spinal ganglion (embryonic day 15) survived for at least 4 weeks after transplantation to the nerve and differentiate into NeuN-immunopositive neurons with morphological properties of sensory neurons and satellite cells containing S100 protein.

  2. Identifying Patient-Specific Epstein-Barr Nuclear Antigen-1 Genetic Variation and Potential Autoreactive Targets Relevant to Multiple Sclerosis Pathogenesis.

    Directory of Open Access Journals (Sweden)

    Monika Tschochner

    Full Text Available Epstein-Barr virus (EBV infection represents a major environmental risk factor for multiple sclerosis (MS, with evidence of selective expansion of Epstein-Barr Nuclear Antigen-1 (EBNA1-specific CD4+ T cells that cross-recognize MS-associated myelin antigens in MS patients. HLA-DRB1*15-restricted antigen presentation also appears to determine susceptibility given its role as a dominant risk allele. In this study, we have utilised standard and next-generation sequencing techniques to investigate EBNA-1 sequence variation and its relationship to HLA-DR15 binding affinity, as well as examining potential cross-reactive immune targets within the central nervous system proteome.Sanger sequencing was performed on DNA isolated from peripheral blood samples from 73 Western Australian MS cases, without requirement for primary culture, with additional FLX 454 Roche sequencing in 23 samples to identify low-frequency variants. Patient-derived viral sequences were used to predict HLA-DRB1*1501 epitopes (NetMHCII, NetMHCIIpan and candidates were evaluated for cross recognition with human brain proteins.EBNA-1 sequence variation was limited, with no evidence of multiple viral strains and only low levels of variation identified by FLX technology (8.3% nucleotide positions at a 1% cut-off. In silico epitope mapping revealed two known HLA-DRB1*1501-restricted epitopes ('AEG': aa 481-496 and 'MVF': aa 562-577, and two putative epitopes between positions 502-543. We identified potential cross-reactive targets involving a number of major myelin antigens including experimentally confirmed HLA-DRB1*15-restricted epitopes as well as novel candidate antigens within myelin and paranodal assembly proteins that may be relevant to MS pathogenesis.This study demonstrates the feasibility of obtaining autologous EBNA-1 sequences directly from buffy coat samples, and confirms divergence of these sequences from standard laboratory strains. This approach has identified a number of

  3. Evaluation of protective immune responses induced by DNA vaccines encoding Toxoplasma gondii surface antigen 1 (SAG1) and 14-3-3 protein in BALB/c mice.

    Science.gov (United States)

    Meng, Min; He, Shenyi; Zhao, Guanghui; Bai, Yang; Zhou, Huaiyu; Cong, Hua; Lu, Gang; Zhao, Qunli; Zhu, Xing-Quan

    2012-11-26

    Toxoplasmosis, caused by an obligate intracellular protozoan parasite Toxoplasma gondii, has been a serious clinical and veterinary problem. Effective DNA vaccines against T. gondii can prevent and control the spread of toxoplasmosis, which is important for both human health and the farming industry. The T. gondii 14-3-3 protein has been proved to be antigenic and immunogenic and was a potential vaccine candidate against toxoplasmosis. In this study, we evaluated the immune responses induced by recombinant plasmids encoding T. gondii surface antigen 1 (SAG1) and 14-3-3 protein by immunizing BALB/c mice intramuscularly. In the present study, BALB/c mice were randomly divided into five groups, including three experimental groups (pSAG1, p14-3-3 and pSAG1/14-3-3) and two control groups (PBS and pBudCE4.1), and were immunized intramuscularly three times. The levels of IgG antibodies and cytokine production in mouse sera were determined by enzyme-linked immunosorbent assays (ELISA). Two weeks after the last immunization, all mice were challenged intraperitoneally (i.p.) with 1×10(4) tachyzoites of T. gondii and the survival time of mice was observed and recorded every day. Mice vaccinated with pSAG1, p14-3-3 or pSAG1/14-3-3 developed high levels of IgG2a and gamma interferon (IFN-γ) and low levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) compared to control groups (PBS or pBudCE4.1), which suggested a modulated Th1 type immune response (Pmice in experimental groups was longer than control groups (Pmice and was a novel DNA vaccine candidate against toxoplasmosis, and the immune protective efficacy elicited by SAG1 gene was also demonstrated. Our results also showed multi-gene vaccine significantly enhanced immune responses and protective efficacy and was superior to the single-gene vaccine.

  4. Phase 1/2a study of the malaria vaccine candidate apical membrane antigen-1 (AMA-1 administered in adjuvant system AS01B or AS02A.

    Directory of Open Access Journals (Sweden)

    Michele D Spring

    Full Text Available This Phase 1/2a study evaluated the safety, immunogenicity, and efficacy of an experimental malaria vaccine comprised of the recombinant Plasmodium falciparum protein apical membrane antigen-1 (AMA-1 representing the 3D7 allele formulated with either the AS01B or AS02A Adjuvant Systems.After a preliminary safety evaluation of low dose AMA-1/AS01B (10 microg/0.5 mL in 5 adults, 30 malaria-naïve adults were randomly allocated to receive full dose (50 microg/0.5 mL of AMA-1/AS01B (n = 15 or AMA-1/AS02A (n = 15, followed by a malaria challenge. All vaccinations were administered intramuscularly on a 0-, 1-, 2-month schedule. All volunteers experienced transient injection site erythema, swelling and pain. Two weeks post-third vaccination, anti-AMA-1 Geometric Mean Antibody Concentrations (GMCs with 95% Confidence Intervals (CIs were high: low dose AMA-1/AS01B 196 microg/mL (103-371 microg/mL, full dose AMA-1/AS01B 279 microg/mL (210-369 microg/mL and full dose AMA-1/AS02A 216 microg/mL (169-276 microg/mL with no significant difference among the 3 groups. The three vaccine formulations elicited equivalent functional antibody responses, as measured by growth inhibition assay (GIA, against homologous but not against heterologous (FVO parasites as well as demonstrable interferon-gamma (IFN-gamma responses. To assess efficacy, volunteers were challenged with P. falciparum-infected mosquitoes, and all became parasitemic, with no significant difference in the prepatent period by either light microscopy or quantitative polymerase chain reaction (qPCR. However, a small but significant reduction of parasitemia in the AMA-1/AS02A group was seen with a statistical model employing qPCR measurements.All three vaccine formulations were found to be safe and highly immunogenic. These immune responses did not translate into significant vaccine efficacy in malaria-naïve adults employing a primary sporozoite challenge model, but encouragingly, estimation of parasite

  5. 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...

  6. Human embryonic stem cells and patent protection

    Directory of Open Access Journals (Sweden)

    Radovanović Sanja M.

    2015-01-01

    Full Text Available Given the importance of biotechnological research in modern diagnostics and therapeutics, on the one hand, and stimulative function of a patent, on the other hand, this work deals with the question of the possibility of pa-tent protection of human embryonic stem cells. Taking into account that this is a biotechnological invention, the key question that this paper highlights is the interpretation of the provisions of their patentability. Namely, thanks to the advanced methods of isolation, purification and preparation for implementation, modern patent systems do not exclude a priori living organisms from patent protection. Therefore, the analysis of representative administrative decisions or court rulings sought to define the criteria that would be applied in order to give patent protection to a certain biotechnological invention (stem cells while others do not.

  7. 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...... will be available over the coming years. However, in order to reach this goal further systematic research is needed. Such cell lines hold promises for developing adequate models for human ES cell therapy and they may open for new avenues for the production of genetically modified animals as the ES cells ahve...

  8. Human embryonic stem cells and microenvironment

    Directory of Open Access Journals (Sweden)

    Banu İskender

    2014-09-01

    Full Text Available Human embryonic stem cells (hESCs possess a great potential in the field of regenerative medicine by their virtue of pluripotent potential with indefinite proliferation capabilities. They can self renew themselves and differentiate into three embryonic germ layers. Although they are conventionally grown on mitotically inactivated mouse feeder cells, there are in vitro culture systems utilizing feeder cells of human origin in order to prevent cross-species contamination. Recently established in vitro culture systems suggested that direct interaction with feeder cells is not necessary but rather attachment to a substrate is required to ensure long-term, efficient hESC culture in vitro. This substrate is usually composed of a mixture of extracellular matrix components representing in vivo natural niche. In hESC biology, the mechanism of interaction of hESCs with extracellular matrix molecules remained insufficiently explored area of research due to their transient nature of interaction with the in vivo niche. However, an in vitro culture system established using extracellular matrix molecules may provide a safer alternative to culture systems with feeder cells while paving the way to Good Manufacturing Practice-GMP production of hESCs for therapeutic purposes. Therefore, it is essential to study the interaction of extracellular matrix molecules with hESCs in order to standardize in vitro culture systems for large-scale production of hESCs in a less labor-intensive way. This would not only provide valuable information regarding the mechanisms that control pluripotency but also serve to dissect the molecular signaling pathways of directed differentiation for prospective therapeutic applications in the future. J Clin Exp Invest 2014; 5 (3: 486-495

  9. Embryonic development of the cricket Gryllus bimaculatus.

    Science.gov (United States)

    Donoughe, Seth; Extavour, Cassandra G

    2016-03-01

    Extensive research into Drosophila melanogaster embryogenesis has improved our understanding of insect developmental mechanisms. However, Drosophila development is thought to be highly divergent from that of the ancestral insect and arthropod in many respects. We therefore need alternative models for arthopod development that are likely to be more representative of basally-branching clades. The cricket Gryllus bimaculatus is such a model, and currently has the most sophisticated functional genetic toolkit of any hemimetabolous insect. The existing cricket embryonic staging system is fragmentary, and it is based on morphological landmarks that are not easily visible on a live, undissected egg. To address this problem, here we present a complementary set of "egg stages" that serve as a guide for identifying the developmental progress of a cricket embryo from fertilization to hatching, based solely on the external appearance of the egg. These stages were characterized using a combination of brightfield timelapse microscopy, timed brightfield micrographs, confocal microscopy, and measurements of egg dimensions. These egg stages are particularly useful in experiments that involve egg injection (including RNA interference, targeted genome modification, and transgenesis), as injection can alter the speed of development, even in control treatments. We also use 3D reconstructions of fixed embryo preparations to provide a comprehensive description of the morphogenesis and anatomy of the cricket embryo during embryonic rudiment assembly, germ band formation, elongation, segmentation, and appendage formation. Finally, we aggregate and schematize a variety of published developmental gene expression patterns. This work will facilitate further studies on G. bimaculatus development, and serve as a useful point of reference for other studies of wild type and experimentally manipulated insect development in fields from evo-devo to disease vector and pest management. Copyright

  10. Intestinal lineage commitment of embryonic stem cells.

    Science.gov (United States)

    Cao, Li; Gibson, Jason D; Miyamoto, Shingo; Sail, Vibhavari; Verma, Rajeev; Rosenberg, Daniel W; Nelson, Craig E; Giardina, Charles

    2011-01-01

    Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue. Copyright © 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  11. Energy and nutrient utilisation by embryonic reptiles.

    Science.gov (United States)

    Thompson, Michael B; Speake, Brian K

    2002-11-01

    Most reptiles are oviparous, with the developing embryos relying on the contents of the yolk to sustain development until hatching (lecithotrophy). The yolk is composed primarily of lipid and protein, which act as an energy source and the essential components to build embryonic tissue. Nevertheless, yolk and the resulting embryos contain many other nutrients, including inorganic ions, vitamins, carotenoids, water and hormones. Apart from water and oxygen, which may be taken up by eggs, and some inorganic ions that can come from the eggshell or even from outside the egg, everything required by the embryo must be in the egg when it is laid. Approximately 20% of squamate reptiles are viviparous, exhibiting a variety of placental complexities. Species with complex placentae have reduced yolk volumes, with the mother augmenting embryonic nutrition by provision across the placenta (placentotrophy). Despite assumed advantages of placentotrophy, only 5 out of approximately 100 lineages of viviparous squamates exhibit substantial placentotrophy. This paper reviews available and recent information on the yolk contents of a variety of squamate reptiles to ask the question, how are nutrients transported from the yolk to the embryo or across the placenta? Although, current available data suggest that, in broad terms, yolk is taken up by embryos without discrimination of the nutrients, there are some apparent exceptions, including the very long chain polyunsaturated fatty acids. In addition, fundamental differences in the patterns of energy utilisation in lizards and snakes suggest fundamental differences in lipid profiles in these taxa, which appear to reflect the differences between placentotrophic and lecithotrophic viviparous lizards.

  12. Evidence for stage-specific modulation of specific microRNAs (miRNAs) and miRNA processing components in zygotic embryo and female gametophyte of loblolly pine (Pinus taeda).

    Science.gov (United States)

    Oh, Thomas J; Wartell, Roger M; Cairney, John; Pullman, Gerald S

    2008-01-01

    MicroRNAs (miRNAs) are known to regulate plant development, but have not been studied in gymnosperm seed tissues. The presence and characteristics of several miRNAs were examined in zygotic embryos (ZEs) and female gametophytes (FGs) of Pinus taeda (loblolly pine). Evidence for miRNAs was obtained using northern analyses and quantitative reverse transcription polymerase chain reaction (qRT-PCR) mediated with poly(A) polymerase. Partial sequences of two miRNAs were verified. Three regions of putative mRNA targets were analyzed by qRT-PCR to monitor the occurrence of stage-dependent miRNA-mediated cleavage. Five miRNAs were identified in ZEs and FGs along with partial sequences of Pta-miR166 and Pta-miR167. Both miRNAs showed differing degrees of tissue-specific and stage-specific modulation. Analysis of HB15L mRNA (a potential Pta-miR166 target) suggested miRNA-guided cleavage in ZEs and FGs. Analysis of ARF8L mRNA (a potential Pta-miR167 target) implied cleavage in ZEs but not in FGs. Argonaute9-like mRNA (ptAGO9L) showed stage-specific modulation of expression in ZEs that appeared to be inverted in the corresponding FGs. MicroRNAs and argonaute genes varied spatiotemporally during seed development. The peak levels of Pta-miR166 in FGs and ptAGO9L in embryos occurred at stage 9.1, a critical transition point during embryo development and a point where somatic embryo maturation often stops. MicroRNAs identified in FG tissue may play a role in embryogenesis.

  13. 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.

  14. Switching of the core structures of glycosphingolipids from globo- and lacto- to ganglio-series upon human embryonic stem cell differentiation.

    Science.gov (United States)

    Liang, Yuh-Jin; Kuo, Huan-Hsien; Lin, Chi-Hung; Chen, Yen-Ying; Yang, Bei-Chia; Cheng, Yuan-Yuan; Yu, Alice L; Khoo, Kay-Hooi; Yu, John

    2010-12-28

    A systematic survey of expression profiles of glycosphingolipids (GSLs) in two hESC lines and their differentiated embryoid body (EB) outgrowth with three germ layers was carried out using immunofluorescence, flow cytometry, and MALDI-MS and MS/MS analyses. In addition to the well-known hESC-specific markers stage-specific embryonic antigen 3 (SSEA-3) and SSEA-4, we identified several globosides and lacto-series GSLs, previously unrevealed in hESCs, including Gb(4)Cer, Lc(4)Cer, fucosyl Lc(4)Cer, Globo H, and disialyl Gb(5)Cer. During hESC differentiation into EBs, MS analysis revealed a clear-cut switch in the core structures of GSLs from globo- and lacto- to ganglio-series, which was not as evident by immunostaining with antibodies against SSEA-3 and SSEA-4, owing to their cross-reactivities with various glycosphingolipids. Such a switch was attributable to altered expression of key glycosyltransferases (GTs) in the biosynthetic pathways by the up-regulation of ganglio-series-related GTs with simultaneous down-regulation of globo- and lacto-series-related GTs. Thus, these results provide insights into the unique stage-specific transition and mechanism for alterations of GSL core structures during hESC differentiation. In addition, unique glycan structures uncovered by MS analyses may serve as surface markers for further delineation of hESCs and help identify of their functional roles not only in hESCs but also in cancers.

  15. Function of JARID2 in bovines during early embryonic development

    Directory of Open Access Journals (Sweden)

    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

  16. Evolution of embryonic development in nematodes

    Directory of Open Access Journals (Sweden)

    Schulze Jens

    2011-09-01

    Full Text Available Abstract Background Nematodes can be subdivided into basal Enoplea (clades 1 and 2 and more derived Chromadorea (clades 3 to 12. Embryogenesis of Caenorhabditis elegans (clade 9 has been analyzed in most detail. Their establishment of polarity and asymmetric cleavage requires the differential localization of PAR proteins. Earlier studies on selected other nematodes revealed that embryonic development of nematodes is more diverse than the essentially invariant development of C. elegans and the classic study object Ascaris had suggested. To obtain a more detailed picture of variations and evolutionary trends we compared embryonic cell lineages and pattern formation in embryos of all 12 nematode clades. Methods The study was conducted using 4-D microscopy and 3-D modeling of developing embryos. Results We found dramatic differences compared to C. elegans in Enoplea but also considerable variations among Chromadorea. We discovered 'Polarity Organizing Centers' (POCs that orient cleavage spindles along the anterior-posterior axis in distinct cells over consecutive cell generations. The resulting lineally arranged blastomeres represent a starting point for the establishment of bilateral symmetry within individual lineages. We can discern six different early cleavage types and suggest that these variations are due to modifications in the activity of the POCs in conjunction with changes in the distribution of PAR proteins. In addition, our studies indicate that lineage complexity advanced considerably during evolution, that is we observe trends towards an increase of somatic founder cells, from monoclonal to polyclonal lineages and from a variable (position-dependent to an invariable (lineage-dependent way of cell fate specification. In contrast to the early phase of embryogenesis, the second half ('morphogenesis' appears similar in all studied nematodes. Comparison of early cleavage between the basal nematode Tobrilus stefanskii and the tardigrade

  17. Growth inhibition of mouse embryonic stem (ES) cells on the feeders ...

    African Journals Online (AJOL)

    ajl4

    Mouse embryonic stem cells (mESCs) can be propagated in vitro on the feeders of mouse embryonic fibroblasts. In this study, we found growth inhibition of mESCs cultured on embryonic fibroblast feeders derived from different livestock animals. Under the same condition, mESCs derived from mouse embryonic fibroblast ...

  18. A toolbox to explore the mechanics of living embryonic tissues.

    Science.gov (United States)

    Campàs, Otger

    2016-07-01

    The sculpting of embryonic tissues and organs into their functional morphologies involves the spatial and temporal regulation of mechanics at cell and tissue scales. Decades of in vitro work, complemented by some in vivo studies, have shown the relevance of mechanical cues in the control of cell behaviors that are central to developmental processes, but the lack of methodologies enabling precise, quantitative measurements of mechanical cues in vivo have hindered our understanding of the role of mechanics in embryonic development. Several methodologies are starting to enable quantitative studies of mechanics in vivo and in situ, opening new avenues to explore how mechanics contributes to shaping embryonic tissues and how it affects cell behavior within developing embryos. Here we review the present methodologies to study the role of mechanics in living embryonic tissues, considering their strengths and drawbacks as well as the conditions in which they are most suitable. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    treated mouse embryonic fibroblast (MEF) feeder cells in knockout ..... 501. Liu H, Ye Z, Kim Y, Sharkis S, Jang YY (2010). Generation of endoderm-derived human induced pluripotent stem cells from primary hepatocytes.

  20. Graphene for enhanced embryonic stem cell photo-transfection efficiency

    CSIR Research Space (South Africa)

    Mthunzi, P

    2013-04-01

    Full Text Available Due to their pluripotency properties, embryonic stem (ES) cells possess great potential in regenerative therapy. Since reported a promising tissue engineering scaffold material, here, graphene is demonstrated to significantly improve the ES cell...

  1. 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.

  2. A toolbox to explore the mechanics of living embryonic tissues

    Science.gov (United States)

    Campàs, Otger

    2016-01-01

    The sculpting of embryonic tissues and organs into their functional morphologies involves the spatial and temporal regulation of mechanics at cell and tissue scales. Decades of in vitro work, complemented by some in vivo studies, have shown the relevance of mechanical cues in the control of cell behaviors that are central to developmental processes, but the lack of methodologies enabling precise, quantitative measurements of mechanical cues in vivo have hindered our understanding of the role of mechanics in embryonic development. Several methodologies are starting to enable quantitative studies of mechanics in vivo and in situ, opening new avenues to explore how mechanics contributes to shaping embryonic tissues and how it affects cell behavior within developing embryos. Here we review the present methodologies to study the role of mechanics in living embryonic tissues, considering their strengths and drawbacks as well as the conditions in which they are most suitable. PMID:27061360

  3. The epigenomics of embryonic stem cell differentiation.

    Science.gov (United States)

    Kraushaar, Daniel C; Zhao, Keji

    2013-01-01

    Embryonic stem cells (ESCs) possess an open and highly dynamic chromatin landscape, which underlies their plasticity and ultimately maintains ESC pluripotency. The ESC epigenome must not only maintain the transcription of pluripotency-associated genes but must also, through gene priming, facilitate rapid and cell type-specific activation of developmental genes upon lineage commitment. Trans-generational inheritance ensures that the ESC chromatin state is stably transmitted from one generation to the next; yet at the same time, epigenetic marks are highly dynamic, reversible and responsive to extracellular cues. Once committed to differentiation, the ESC epigenome is remodeled and resolves into a more compact chromatin state. A thorough understanding of the role of chromatin modifiers in ESC fate and differentiation will be important if they are to be used for therapeutic purposes. Recent technical advances, particularly in next-generation sequencing technologies, have provided a genome-scale view of epigenetic marks and chromatin modifiers. More affordable and faster sequencing platforms have led to a comprehensive characterization of the ESC epigenome and epigenomes of differentiated cell types. In this review, we summarize and discuss the recent progress that has highlighted the central role of histone modifications, histone variants, DNA methylation and chromatin modifiers in ESC pluripotency and ESC fate. We provide a detailed and comprehensive discussion of genome-wide studies that are pertinent to our understanding of mammalian development.

  4. Targeting embryonic signaling pathways in cancer therapy.

    Science.gov (United States)

    Harris, Pamela Jo; Speranza, Giovanna; Dansky Ullmann, Claudio

    2012-01-01

    The embryonic signaling pathways (ESP), Hedgehog, Notch and Wnt, are critical for the regulation of normal stem cells and cellular development processes. They are also activated in the majority of cancers. ESP are operational in putative cancer stem cells (CSC), which drive initial tumorigenesis and sustain cancer progression and recurrence in non-CSC bulk subpopulations. ESP represent novel therapeutic targets. A variety of inhibitors and targeting strategies are being developed. This review discusses the rationale for targeting ESP for cancer treatment, as well as specific inhibitors under development; mainly focusing on those approaching clinical use and the challenges that lie ahead. The data sources utilized are several database search engines (PubMed, Google, Clinicaltrials.gov), and the authors' involvement in the field. CSC research is rapidly evolving. Expectations regarding their therapeutic targeting are rising quickly. Further definition of what constitutes a true CSC, proper validation of CSC markers, a better understanding of cross-talk among ESP and other pathways, and interactions with tumor non-CSC and the tumor microenvironment are needed. The appropriate patient population, the right clinical setting and combination strategies to test these therapies, as well as the proper pharmacodynamic markers to measure, need to be further established.

  5. Avian embryonic development in hyperdynamic environments

    Science.gov (United States)

    Abbott, U. K.; Smith, A. H.

    1983-01-01

    Embryos which developed for 24 hours in the oviduct of hens maintained at 2 G and which were subsequently incubated at Earth gravity had a 14% reduction in hatchability. Increased mortality during the first 4 days, and an increase in embryonic abnormalities were of the types usually found during the first mortality peak (2-3 days). Embryos in eggs that were produced at Earth gravity and continued their development on the centrifuge at fields of 2 G or less did not appear to be greatly affected by the treatment. At 4 G, 91% of the embryos died, mostly on the first and second days of incubation. Abnormalities prominent in the centrifuged eggs include: (a) a failure of the primitive streak to develop; (b) interference with the development of the axial skeleton; (c) multiple hemorrhages, mostly petechial which is consistent with capillary fragility; and (d) retardation of embryo growth, possibly caused by an interference with gaseous diffusion, the result of an acceleration-induced increase in gas density in the centrifuging incubator.

  6. Programmed cell senescence during mammalian embryonic development.

    Science.gov (United States)

    Muñoz-Espín, Daniel; Cañamero, Marta; Maraver, Antonio; Gómez-López, Gonzalo; Contreras, Julio; Murillo-Cuesta, Silvia; Rodríguez-Baeza, Alfonso; Varela-Nieto, Isabel; Ruberte, Jesús; Collado, Manuel; Serrano, Manuel

    2013-11-21

    Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-β/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Monoclonal antibodies to carcino-embryonic antigen

    International Nuclear Information System (INIS)

    Teh, Jinghee; McKenzie, I.F.C.

    1990-01-01

    With the aim of producing new MoAb to colorectal carcinoma, immunization with cell suspensions of a fresh colonic tumour was performed and MoAb 17C4 was obtained. To produce other MoAb to colon cancer, an immunization protocol using fresh tumour, colonic cell lines and sera from patients with colonic tumours was employed and resulted in MoAb JGT-13, LK-4 and XPX-13. MoAb I-1 and O-1 were raised against sera from patients with colon cancer to produce MoAb directed against circulating tumour associated antigens. The six antibodies gave a range of reactions with normal and malignant tissues, indicating that they most likely reacted with different epitopes. Thus, apart from the reactions of 17C4, LK-4 and XPX-13 with fresh and formalin-fixed granulocytes, none of the antibodies reacted with formalin-fixed normal tissues. Despite the apparent specificity of these MoAb for colon cancer, serum testing using MoAb gave similar results to carcino-embryonic antigen polyclonal antibodies, that is the MoAb gave no obvious advantage. 9 refs., 1 tab., 3 figs

  8. Ovarian Embryonal Carcinoma in a Dog.

    Science.gov (United States)

    Banco, B; Ferrari, R; Stefanello, D; Groppetti, D; Pecile, A; Faverzani, S; Longo, M; Zani, D D; Ravasio, G; Caniatti, M; Grieco, V

    2017-11-01

    A 17-month-old female doberman pinscher was referred for an abdominal mass and ascites. Exploratory laparotomy revealed the presence of a large neoplastic mass replacing the right ovary and associated with multiple mesovarian, mesometrial and peritoneal nodules. An ovariohysterectomy was performed. Grossly, the tumour was soft and multilocular with large areas of haemorrhage and necrosis. Microscopically, it was infiltrative and composed of round and polygonal cells arranged respectively in solid sheets or forming distorted tubular structures separated by thick fibrovascular septae. Tubules contained necrotic debris, proteinaceous fluid or small endoluminal papillary structures. Marked cellular atypia, multiple neoplastic emboli and high mitotic count were observed. Immunohistochemically, the round cells uniformly expressed placental alkaline phosphatase, while the polygonal cells arranged in tubules and papillae expressed cytokeratin (CK) AE1/AE3 and CK7. A final diagnosis of metastasizing ovarian embryonal carcinoma (EC), a primitive germ cell tumour characterized by rudimentary epithelial differentiation was made. Canine ovarian EC should be considered as a differential diagnosis for undifferentiated aggressive ovarian tumours in young dogs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Microglia Modulate Wiring of the Embryonic Forebrain

    Directory of Open Access Journals (Sweden)

    Paola Squarzoni

    2014-09-01

    Full Text Available Dysfunction of microglia, the tissue macrophages of the brain, has been associated with the etiology of several neuropsychiatric disorders. Consistently, microglia have been shown to regulate neurogenesis and synaptic maturation at perinatal and postnatal stages. However, microglia invade the brain during mid-embryogenesis and thus could play an earlier prenatal role. Here, we show that embryonic microglia, which display a transiently uneven distribution, regulate the wiring of forebrain circuits. Using multiple mouse models, including cell-depletion approaches and cx3cr1−/−, CR3−/−, and DAP12−/− mutants, we find that perturbing microglial activity affects the outgrowth of dopaminergic axons in the forebrain and the laminar positioning of subsets of neocortical interneurons. Since defects in both dopamine innervation and cortical networks have been linked to neuropsychiatric diseases, our study provides insights into how microglial dysfunction can impact forebrain connectivity and reveals roles for immune cells during normal assembly of brain circuits.

  10. Embryonic diapause is conserved across mammals.

    Directory of Open Access Journals (Sweden)

    Grazyna E Ptak

    Full Text Available Embryonic diapause (ED is a temporary arrest of embryo development and is characterized by delayed implantation in the uterus. ED occurs in blastocysts of less than 2% of mammalian species, including the mouse (Mus musculus. If ED were an evolutionarily conserved phenomenon, then it should be inducible in blastocysts of normally non-diapausing mammals, such as domestic species. To prove this hypothesis, we examined whether blastocysts from domestic sheep (Ovis aries could enter into diapause following their transfer into mouse uteri in which diapause conditions were induced. Sheep blastocysts entered into diapause, as demonstrated by growth arrest, viability maintenance and their ED-specific pattern of gene expression. Seven days after transfer, diapausing ovine blastocysts were able to resume growth in vitro and, after transfer to surrogate ewe recipients, to develop into normal lambs. The finding that non-diapausing ovine embryos can enter into diapause implies that this phenomenon is phylogenetically conserved and not secondarily acquired by embryos of diapausing species. Our study questions the current model of independent evolution of ED in different mammalian orders.

  11. Embryonic Diapause Is Conserved across Mammals

    Science.gov (United States)

    Ptak, Grazyna E.; Tacconi, Emanuela; Czernik, Marta; Toschi, Paola; Modlinski, Jacek A.; Loi, Pasqualino

    2012-01-01

    Embryonic diapause (ED) is a temporary arrest of embryo development and is characterized by delayed implantation in the uterus. ED occurs in blastocysts of less than 2% of mammalian species, including the mouse (Mus musculus). If ED were an evolutionarily conserved phenomenon, then it should be inducible in blastocysts of normally non-diapausing mammals, such as domestic species. To prove this hypothesis, we examined whether blastocysts from domestic sheep (Ovis aries) could enter into diapause following their transfer into mouse uteri in which diapause conditions were induced. Sheep blastocysts entered into diapause, as demonstrated by growth arrest, viability maintenance and their ED-specific pattern of gene expression. Seven days after transfer, diapausing ovine blastocysts were able to resume growth in vitro and, after transfer to surrogate ewe recipients, to develop into normal lambs. The finding that non-diapausing ovine embryos can enter into diapause implies that this phenomenon is phylogenetically conserved and not secondarily acquired by embryos of diapausing species. Our study questions the current model of independent evolution of ED in different mammalian orders. PMID:22427933

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

    African Journals Online (AJOL)

    At 33 days one of 31 (3.2%) pregnancies showed cardiac activity and by 36days 18 out of 21 (85.8) pregnancies had cardiac activity. By day 37 all pregnancies showed embryonic cardiac activity using the LMP. From days 33 to 56, mean embryonic cardiac activity rose from 84 at day 33 to 173.9 bear per minutes. This study ...

  13. Embryonic development of Anodontites trapesialis (Lamarck, 1819) (Bivalvia: Mycetopodidae)

    OpenAIRE

    Silva-Souza,AT.; Guardia-Felipi,P.; Arrebola,NR.

    2011-01-01

    The phases of embryonic development of Anodontites trapesialis lasidia are described for the first time. Adult specimens were obtained from two fish farms located in Londrina, Paraná, Brazil. The internal demibranchs of 120 individuals were studied using a routine histological technique; 70 of these carried eggs and/or larvae in the marsupium and were utilized for the description of the phases of embryonic development. The demibranchs of five specimens were evaluated by scanning electron micr...

  14. Embryonic miRNA Profiles of Normal and Ectopic Pregnancies

    OpenAIRE

    Dominguez, Francisco; Moreno-Moya, Juan Manuel; Lozoya, Teresa; Romero, Ainhoa; Martínez, Sebastian; Monterde, Mercedes; Gurrea, Marta; Ferri, Blanca; Núñez, Maria Jose; Simón, Carlos; Pellicer, Antonio

    2014-01-01

    Our objective was to investigate the miRNA profile of embryonic tissues in ectopic pregnancies (EPs) and controlled abortions (voluntary termination of pregnancy; VTOP). Twenty-three patients suffering from tubal EP and twenty-nine patients with a normal ongoing pregnancy scheduled for a VTOP were recruited. Embryonic tissue samples were analyzed by miRNA microarray and further validated by real time PCR. Microarray studies showed that four miRNAs were differentially downregulated (hsa-mir-19...

  15. Classic and current opinion in embryonic organ transplantation.

    Science.gov (United States)

    Hammerman, Marc R

    2014-04-01

    Here, we review the rationale for the use of organs from embryonic donors, antecedent investigations and recent work from our own laboratory, exploring the utility for transplantation of embryonic kidney and pancreas as an organ replacement therapy. Ultrastructurally precise kidneys differentiate in situ in rats following xenotransplantation in mesentery of embryonic pig renal primordia. The developing organ attracts its blood supply from the host. Engraftment of pig renal primordia requires host immune suppression. However, beta cells originating from embryonic pig pancreas obtained very early following initiation of organogenesis [embryonic day 28 (E28)] engraft long term in nonimmune-suppressed diabetic rats or rhesus macaques. Engraftment of morphologically similar cells originating from adult porcine islets of Langerhans occurs in animals previously transplanted with E28 pig pancreatic primordia. Organ primordia engraft, attract a host vasculature and differentiate following transplantation to ectopic sites. Attempts have been made to exploit these characteristics to achieve clinically relevant endpoints for end-stage renal disease and diabetes mellitus using animal models. We and others have focused on use of the embryonic pig as a donor.

  16. 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.

  17. Brief embryonic strychnine exposure in zebrafish causes long-term adult behavioral impairment with indications of embryonic synaptic changes.

    Science.gov (United States)

    Roy, Nicole M; Arpie, Brianna; Lugo, Joseph; Linney, Elwood; Levin, Edward D; Cerutti, Daniel

    2012-01-01

    Zebrafish provide a powerful model of the impacts of embryonic toxicant exposure on neural development that may result in long-term behavioral dysfunction. In this study, zebrafish embryos were treated with 1.5mM strychnine for short embryonic time windows to induce transient changes in inhibitory neural signaling, and were subsequently raised in untreated water until adulthood. PCR analysis showed indications that strychnine exposure altered expression of some genes related to glycinergic, GABAergic and glutamatergic neuronal synapses during embryonic development. In adulthood, treated fish showed significant changes in swimming speed and tank diving behavior compared to controls. Taken together, these data show that a short embryonic exposure to a neurotoxicant can alter development of neural synapses and lead to changes in adult behavior. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Cell- and stage-specific chromatin structure across the Complement receptor 2 (CR2/CD21) promoter coincide with CBF1 and C/EBP-beta binding in B cells.

    Science.gov (United States)

    Cruickshank, Mark N; Fenwick, Emily; Karimi, Mahdad; Abraham, Lawrence J; Ulgiati, Daniela

    2009-08-01

    Stringent developmental transcription requires multiple transcription factor (TF) binding sites, cell-specific expression of signaling molecules, TFs and co-regulators and appropriate chromatin structure. During B-lymphopoiesis, human Complement receptor 2 (CR2/CD21) is detected on immature and mature B cells but not on B cell precursors and plasma cells. We examined cell- and stage-specific human CR2 gene regulation using cell lines modeling B-lymphopoiesis. Chromatin accessibility assays revealed a region between -409 and -262 with enhanced accessibility in mature B cells and pre-B cells, compared to either non-lymphoid or plasma cell-types, however, accessibility near the transcription start site (TSS) was elevated only in CR2-expressing B cells. A correlation between histone acetylation and CR2 expression was observed, while histone H3K4 dimethylation was enriched near the TSS in both CR2-expressing B cells and non-expressing pre-B cells. Candidate sites within the CR2 promoter were identified which could regulate chromatin, including a matrix attachment region associated with CDP, SATB1/BRIGHT and CEBP-beta sites as well as two CBF1 sites. ChIP assays verified that both CBF1 and C/EBP-beta bind the CR2 promoter in B cells raising the possibility that these factors facilitate or respond to alterations in chromatin structure to control the timing and/or level of CR2 transcription.

  19. Comparison of Teratoma Formation between Embryonic Stem Cells and Parthenogenetic Embryonic Stem Cells by Molecular Imaging

    Directory of Open Access Journals (Sweden)

    Hongyan Tao

    2018-01-01

    Full Text Available With their properties of self-renewal and differentiation, embryonic stem (ES cells hold great promises for regenerative therapy. However, teratoma formation and ethical concerns of ES cells may restrict their potential clinical applications. Currently, parthenogenetic embryonic stem (pES cells have attracted the interest of researchers for its self-renewing and pluripotent differentiation while eliciting less ethic concerns. In this study, we established a model with ES and pES cells both stably transfected with a double-fusion reporter gene containing renilla luciferase (Rluc and red fluorescent protein (RFP to analyze the mechanisms of teratoma formation. Transgenic Vegfr2-luc mouse, which expresses firefly luciferase (Fluc under the promoter of vascular endothelial growth factor receptor 2 (Vegfr2-luc, was used to trace the growth of new blood vessel recruited by transplanted cells. Bioluminescence imaging (BLI of Rluc/Fluc provides an effective tool in estimating the growth and angiogenesis of teratoma in vivo. We found that the tumorigenesis and angiogenesis capacity of ES cells were higher than those of pES cells, in which VEGF/VEGFR2 signal pathway plays an important role. In conclusion, pES cells have the decreased potential of teratoma formation but meanwhile have similar differentiating capacity compared with ES cells. These data demonstrate that pES cells provide an alternative source for ES cells with the risk reduction of teratoma formation and without ethical controversy.

  20. Nucleosome Organization in Human Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Puya G Yazdi

    Full Text Available The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a "ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this "ground state" by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational

  1. Extracellular matrix, cell skeletons, and embryonic development.

    Science.gov (United States)

    Hay, E D

    1989-09-01

    During embryonic development, the extracellular matrix (ECM) promotes the production of differentiated products by epithelial cells and the migration of mesenchymal cells, and probably also plays a role in epithelial-mesenchymal transformation. Here we examine the role of the cell skeleton (actin, microtubules, intermediate filaments) in mediating matrix effects on mesenchymal cell morphology, migration, and formation. The interaction of both epithelial cells and mesenchymal cells with ECM seems to involve the actin cortex, which is best developed in the base of the epithelial cell, where it attaches to underlying matrix via membrane-intercalated receptors. To interact with the matrix, the fibroblast has appropriate ECM receptors and an actin cortex around the whole cell. The actin cortex is absolutely required for assumption of bipolar shape, elongation, and movement through the matrix. Since the cortex seems to be anchored to the matrix, it is unlikely that it moves during cell migration. A new hypothesis states that the microtubule- and intermediate filament-rich endoplasm, containing the nucleus, moves past the actin cortex-receptor-matrix complex into the newly synthesized front end of the mesenchymal cell to effect forward movement. When epithelial cells transform into mesenchyme in the embryo, or when they are induced to do this in vitro, they switch from the keratin intermediate filament profile to one rich in vimentin, and the effect of cell matrix interaction on cell shape is profoundly altered. Vimentin-actin interactions with ECM may be a major factor in the ability of a cell to become mesenchymal.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. 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

  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. Transfer of microRNAs by embryonic stem cell microvesicles.

    Directory of Open Access Journals (Sweden)

    Alex Yuan

    Full Text Available Microvesicles are plasma membrane-derived vesicles released into the extracellular environment by a variety of cell types. Originally characterized from platelets, microvesicles are a normal constituent of human plasma, where they play an important role in maintaining hematostasis. Microvesicles have been shown to transfer proteins and RNA from cell to cell and they are also believed to play a role in intercellular communication. We characterized the RNA and protein content of embryonic stem cell microvesicles and show that they can be engineered to carry exogenously expressed mRNA and protein such as green fluorescent protein (GFP. We demonstrate that these engineered microvesicles dock and fuse with other embryonic stem cells, transferring their GFP. Additionally, we show that embryonic stem cells microvesicles contain abundant microRNA and that they can transfer a subset of microRNAs to mouse embryonic fibroblasts in vitro. Since microRNAs are short (21-24 nt, naturally occurring RNAs that regulate protein translation, our findings open up the intriguing possibility that stem cells can alter the expression of genes in neighboring cells by transferring microRNAs contained in microvesicles. Embryonic stem cell microvesicles may be useful therapeutic tools for transferring mRNA, microRNAs, protein, and siRNA to cells and may be important mediators of signaling within stem cell niches.

  5. 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.

  6. 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.)

  7. Congenital diaphragmatic hernia candidate genes derived from embryonic transcriptomes

    DEFF Research Database (Denmark)

    Russell, Meaghan K; Longoni, Mauro; Wells, Julie

    2012-01-01

    expression profiling of developing embryonic diaphragms would help identify genes likely to be associated with diaphragm defects. We generated a time series of whole-transcriptome expression profiles from laser captured embryonic mouse diaphragms at embryonic day (E)11.5 and E12.5 when experimental...... perturbations lead to CDH phenotypes, and E16.5 when the diaphragm is fully formed. Gene sets defining biologically relevant pathways and temporal expression trends were identified by using a series of bioinformatic algorithms. These developmental sets were then compared with a manually curated list of genes...... previously shown to cause diaphragm defects in humans and in mouse models. Our integrative filtering strategy identified 27 candidates for CDH. We examined the diaphragms of knockout mice for one of the candidate genes, pre-B-cell leukemia transcription factor 1 (Pbx1), and identified a range of previously...

  8. 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.

  9. In vitro pancreas organogenesis from dispersed mouse embryonic progenitors

    DEFF Research Database (Denmark)

    Greggio, Chiara; De Franceschi, Filippo; Figueiredo-Larsen, Evan Manuel

    2014-01-01

    The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells (1). The whole embryonic organ can be cultured at multiple stages...... expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how...... the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature...

  10. Contributions of Noncanonical Smoothened Signaling During Embryonic Development.

    Science.gov (United States)

    Pandit, Tanushree; Ogden, Stacey K

    2017-01-01

    The Sonic Hedgehog (Shh) signaling pathway is active during embryonic development in metazoans, and provides instructional cues necessary for proper tissue patterning. The pathway signal transducing component, Smoothened (Smo), is a G protein-coupled receptor (GPCR) that has been demonstrated to signal through at least two effector routes. The first is a G protein-independent canonical route that signals to Gli transcriptional effectors to establish transcriptional programs specifying cell fate during early embryonic development. The second, commonly referred to as the noncanonical Smo signal, induces rapid, transcription-independent responses that are essential for establishing and maintaining distinct cell behaviors during development. Herein, we discuss contributions of this noncanonical route during embryonic development. We also highlight important open questions regarding noncanonical Smo signal route selection during development, and consider implications of noncanonical signal corruption in disease.

  11. Contributions of Noncanonical Smoothened Signaling During Embryonic Development

    Directory of Open Access Journals (Sweden)

    Tanushree Pandit

    2017-10-01

    Full Text Available The Sonic Hedgehog (Shh signaling pathway is active during embryonic development in metazoans, and provides instructional cues necessary for proper tissue patterning. The pathway signal transducing component, Smoothened (Smo, is a G protein-coupled receptor (GPCR that has been demonstrated to signal through at least two effector routes. The first is a G protein–independent canonical route that signals to Gli transcriptional effectors to establish transcriptional programs specifying cell fate during early embryonic development. The second, commonly referred to as the noncanonical Smo signal, induces rapid, transcription-independent responses that are essential for establishing and maintaining distinct cell behaviors during development. Herein, we discuss contributions of this noncanonical route during embryonic development. We also highlight important open questions regarding noncanonical Smo signal route selection during development, and consider implications of noncanonical signal corruption in disease.

  12. Impact of nutritional stress on early embryonic survival

    Directory of Open Access Journals (Sweden)

    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.

  13. 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.

  14. Expansion of human embryonic stem cells in vitro.

    Science.gov (United States)

    Costa, Magdaline; Sourris, Koula; Hatzistavrou, Tanya; Elefanty, Andrew G; Stanley, Edouard G

    2008-05-01

    This unit describes a protocol for the large-scale expansion of karyotypically normal human embryonic stem cells (hESCs). hESCs can be maintained indefinitely as dense colonies that are mechanically cut into pieces, which are subsequently transferred to fresh organ culture dishes seeded with primary mouse embryonic fibroblasts (MEFs). hESCs can also be enzymatically passaged (bulk culture); however, over time, this style of culturing may lead to the acquisition of chromosomal abnormalities. Nevertheless, enzymatic passaging can be used for short periods (up to 25 passages) without the appearance of cells with abnormal karyotypes. Copyright 2008 by John Wiley & Sons, Inc.

  15. Embryonic stem cell research in Iran: status and ethics.

    Science.gov (United States)

    Saniei, Mansooreh; De Vries, Raymond

    2008-01-01

    Various successes in regenerative medicine by therapeutic cloning have given rise to expectations that treatments will soon be developed for incurable diseases. But using embryonic stem cells for this purpose raises many ethical dilemmas including those about the beginning of human life. Arguments concerning stem cell research and therapeutic cloning in different countries are influenced by both the religious and bioethical traditions which dominate in these cultures. This article examines how these traditions have influenced stem cell research in Iran through an account of scientific advances and the development of regulations on embryonic stem cell research in Iran.

  16. Differentiation of Mouse Embryonic Stem Cells into Ventral Foregut Precursors

    DEFF Research Database (Denmark)

    Rothová, Michaela; Hölzenspies, Jurriaan J; Livigni, Alessandra

    2016-01-01

    Anterior definitive endoderm (ADE), the ventral foregut precursor, is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other organs. Here, a method is described for the differentiation of mouse embryonic stem cells (mESCs) to definitive...... endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (embryoid body) culture or in a serum-free adherent monolayer culture. ESC-derived ADE cells are committed to endodermal fates and can undergo further differentiation in vitro towards ventral foregut...

  17. 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.

  18. Photo-transfection of mouse embryonic stem cells with plasmid DNA using femtosecond laser pulses

    CSIR Research Space (South Africa)

    Thobakgale, Lebogang

    2017-01-01

    Full Text Available This presentation is about the photo-transfection of mouse embryonic stem cells with plasmid DNA using femtosecond laser pulses. It outlines the background on embryonic stem cells (ES) and phototransfection....

  19. 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....

  20. Aspects on properties, use and ethical considerations of embryonic stem cells – A short review

    OpenAIRE

    Borge, Ole Johan; Evers, Kathinka

    2003-01-01

    Mammalian embryonic stem cells have the potential to differentiate into all cell types of an adult individual. The culturing of human embryonic stem cells renders possible studies that were previously only available in animal models. Embryonic stem cells constitute a particularly attractive tool for studies of self-renewal, commitment, differentiation, maturation and cell-cell interaction. There is currently considerable hope that studies of embryonic stem cells will lead to new therapies; ei...

  1. Gene targeting in embryonic stem cells, II: conditional technologies

    Science.gov (United States)

    Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombi...

  2. Expression Pattern of Antioxidant Enzyme Genes in Embryonic Axes

    African Journals Online (AJOL)

    lin

    2012-07-17

    Jul 17, 2012 ... that the embryonic axes of sacred lotus maintain a protective and recovery mechanism from heat damage during and after ..... germinated in distilled water for H2O2 accumulation detection. Browning of cotyledons and .... the leak of single electrons from the electron transport chain to O2 (Cvetkovska and ...

  3. Dynamics of nuclear matrix proteome during embryonic development

    Indian Academy of Sciences (India)

    2011-07-08

    Jul 8, 2011 ... Embryonic development is a complex and dynamic process that involves spatiotemporal expression of genes in a highly coordinated manner. Multiple levels of nuclear architecture maintain the fidelity of gene expression programme. One of the components of nuclear architecture, which is believed to play ...

  4. A developmental program drives aggressive embryonal brain tumors.

    Science.gov (United States)

    Archer, Tenley C; Pomeroy, Scott L

    2014-01-01

    Embryonal tumors with multilayered rosettes (ETMRs) are primitive neuroectodermal tumors arising in infants. A new study shows that these tumors are universally driven by fusion of the promoter of a gene with brain-specific expression, TTYH1, to C19MC, the largest human microRNA cluster, activating a fetal neural development program.

  5. Characterization of embryonic stem cell transplantation immunobiology using molecular imaging

    NARCIS (Netherlands)

    Swijnenburg, Rutger-Jan

    2009-01-01

    Given their self-renewing and pluripotent capabilities, embryonic stem cells (ESCs) are well-poised as a cellular source for tissue regeneration therapy. Successful in vitro differentiation of both mouse (m) and human (h) ESCs into multiple somatic cell types has been reported, including

  6. Ca2+ signalling and early embryonic patterning during zebrafish development.

    Science.gov (United States)

    Webb, Sarah E; Miller, Andrew L

    2007-09-01

    1. It has been proposed that Ca2+ signalling, in the form of pulses, waves and steady gradients, may play a crucial role in key pattern-forming events during early vertebrate development. 2. With reference to the embryo of the zebrafish (Danio rerio), herein we review the Ca2+ transients reported from the cleavage to segmentation periods. This time-window includes most of the major pattern-forming events of early development, which transform a single-cell zygote into a complex multicellular embryo with established primary germ layers and body axes. 3. Data are presented to support our proposal that intracellular Ca2+ waves are an essential feature of embryonic cytokinesis and that propagating intercellular Ca2+ waves (both long and short range) may play a crucial role in: (i) the establishment of the embryonic periderm and the coordination of cell movements during epiboly, convergence and extension; (ii) the establishment of the basic embryonic axes and germ layers; and (iii) definition of the morphological boundaries of specific tissue domains and embryonic structures, including future organ anlagen. 4. The potential downstream targets of these Ca2+ transients are also discussed, as well as how they may integrate with other pattern-forming signalling pathways known to modulate early developmental events.

  7. Can embryonic skipper frogs (Euphlyctis cyanophlyctis) learn to ...

    Indian Academy of Sciences (India)

    Surprisingly,larval E. cyanophlyctis could learn to recognise kairomones through association during embryonic stages evenbefore the development of a nervous system. Although larval E. cyanophlyctis lack the innate ability to recognise kairomones,they were able to recognise conspecific alarm cues on the first encounter, ...

  8. 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

  9. Improved genetic manipulation of human embryonic stem cells.

    NARCIS (Netherlands)

    Braam, S.R.; Denning, C.; van den Brink, S.; Kats, P.; Hochstenbach, R.; Passier, R.; Mummery, C.L.

    2008-01-01

    Low efficiency of transfection limits the ability to genetically manipulate human embryonic stem cells (hESCs), and differences in cell derivation and culture methods require optimization of transfection protocols. We transiently transferred multiple independent hESC lines with different growth

  10. 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 ...

  11. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  12. optimization of protocol for m apical meristem of embryonic axes

    African Journals Online (AJOL)

    userpc

    source of dietary protein with both its dry gra and vegetative parts containing 23 and other important food .... percentage of total embryonic axes cultured) were taken at regular intervals. Number of elongated shoots per ... or de novoorganogenesis has earlier bee reported from various explants (for a review se. Saniet al.

  13. Innovative virtual reality measurements for embryonic growth and development

    NARCIS (Netherlands)

    C.M. Verwoerd-Dikkeboom (Christine); A.H.J. Koning (Anton); W.C.J. Hop (Wim); P.J. van der Spek (Peter); N. Exalto (Niek); R.P.M. Steegers-Theunissen (Régine)

    2010-01-01

    textabstractBackground Innovative imaging techniques, using up-to-date ultrasonic equipment, necessitate specific biometry. The aim of our study was to test the possibility of detailed human embryonic biometry using a virtual reality (VR) technique. Methods In a longitudinal study, three-dimensional

  14. Embryonic development and hatchlings of Illex argentinus derived ...

    African Journals Online (AJOL)

    The embryonic and early post-hatching development of artificially fertilized eggs of Illex argentinus was observed at several temperatures from 8.5 to 23.2°C. During the fertilization procedure, oviducal gland jelly was added to eggs (about 1.0 mm long). Chorion expansion began 20 minutes after fertilization and continued ...

  15. Alterations to embryonic serotonin change aggression and fearfulness

    Science.gov (United States)

    Prenatal environment, including maternal hormones, affects the development of the serotonin (5-HT) system, with long-lasting effects on mood and behavioral exhibition in children and adults. The chicken provides a unique animal model to study the effects of embryonic development on childhood and ado...

  16. Cell surface carbohydrate changes during embryonic and fetal skin development

    DEFF Research Database (Denmark)

    Dabelsteen, Erik; Holbrook, K; Clausen, H

    1986-01-01

    Monoclonal antibodies to four type 2 chain carbohydrate antigens were used for immunohistochemical studies of embryonic and fetal skin. The antibodies detected N-acetyllactosamine and 3 fucosyl substitutes of this, blood group antigen H, Lex, and Ley. Periderm consistently stained for N-acetyllac...

  17. Calcium metabolism in olive ridley turtle eggs during embryonic development.

    Science.gov (United States)

    Sahoo, G; Sahoo, R K; Mohanty-Hejmadi, P

    1998-09-01

    Analyses of calcium, magnesium, sulphur, potassium and phosphorus content of the eggshell, yolk-albumen and embryos of olive ridley turtle, Lepidochelys olivacea, have been carried out at various stages of embryonic development. Calcium is the major inorganic constituent in the egg (shell and yolk-albumen) and embryos. Other elements are present either in trace or in minute trace amounts. The egg contents (yolk and albumen) provide only 40% of the embryonic calcium requirement of the hatchling. The remaining 60% is provided by the eggshell. The eggshell also undergoes a similar reduction in its calcium content from laying to hatching. Elements other than calcium present in the yolk-albumen are sufficient for normal embryonic development. The movement of calcium from the eggshell to the embryo starts at about the 40th day of development at 29.5 degrees C. Birds, turtles and crocodiles use their eggshell as the secondary source of embryonic calcium requirement. This dependence on the eggshell varies in different groups which is highest in birds and lowest in crocodiles.

  18. Deciphering caudal embryonic defects: embryological analysis and reviewing literature data.

    Science.gov (United States)

    Arvind Athavale, Sunita

    2012-01-01

    A number of syndromes÷associations involving the caudal region have been described in the literature. Each of them is characterized by a set of morphological features. Reports on difficulties in delineation and an ever-increasing constellation of defects in recent past call for a comprehensive study into the morphologic presentations and pathogenesis of caudal embryonic defects. The present article describes a case of the OEIS complex--a combination of omphalocele, exstrophy of bladder, imperforate anus and spinal defects. Literature search was performed and morphologic presentations, as described in literature, of all syndromes and associations affecting the caudal region of the embryo have been compared. Morphologic presentations were analyzed embryologically. A remarkable overlap of symptom complex was observed. Embryological analysis of the phenotypic presentations of all these syndromes points towards a common pathogenesis, early in the embryonic life. The embryologic analysis suggests that these defects are a result of defects in proliferation, migration or subsequent differentiation of any of the three subdivisions of intra-embryonic mesoderm. Based on the analysis a new hypothesis for the causation of caudal defects is proposed. This hypothesis suggests that a local internal environmental imbalance, at the site of implantation, can cause nutritional insult to the embryo during gastrulation, during the third and the early fourth week of embryonic life.

  19. Management of undifferentiated embryonal sarcoma of the liver in ...

    African Journals Online (AJOL)

    Background. Undifferentiated embryonal sarcoma of the liver (UESL) is a rare neoplasm, and the third-most common paediatric hepatic malignancy. However, no treatment guidelines exist. No randomised, controlled trials support specific combinations of therapy. Objective. To compare presentation and management of ...

  20. Tracking the embryonic stem cell transition from ground state pluripotency

    NARCIS (Netherlands)

    Kalkan, T.; Olova, N.; Roode, M.; Mulas, C.; Lee, H.J.; Nett, I.; Marks, H.; Walker, R.; Stunnenberg, H.; Lilley, K.S.; Nichols, J.; Reik, W.; Bertone, P.; Smith, A.

    2017-01-01

    Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition process. The ES cell

  1. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Studies have shown that embryonic stem (ES) cells can be successfully differentiated into liver cells, which offer the potential unlimited cell source for a variety of end-stage liver disease. In our study, in order to induce mouse ES cells to differentiate into hepatocyte-like cells under chemically defined conditions, ES cells ...

  2. Cryopreservation of embryonic axes of maize (Zea mays L.) by ...

    African Journals Online (AJOL)

    GREGORY

    2010-12-21

    Dec 21, 2010 ... Cryopreservation of embryonic axes of maize (Zea mays L.) by vitrification protocol. I. S. Usman* and M. M. Abdulmalik. Department of Plant Science, Ahmadu Bello University,PMB 1044, Zaria, Kaduna State, Nigeria. Accepted 27 April, 2010. A storage protocol at cryogenic temperature was established for ...

  3. Can embryonic skipper frogs (Euphlyctis cyanophlyctis) learn to ...

    Indian Academy of Sciences (India)

    Swapnil C Supekar

    2017-06-12

    Jun 12, 2017 ... prisingly, larval E. cyanophlyctis could learn to recognise kairomones through association during embryonic stages even before the development of a nervous system. Although larval E. cyanophlyctis lack the innate ability to recognise kair- omones, they were able to recognise conspecific alarm cues on the ...

  4. Directional differentiation of chicken embryonic stem cells into ...

    African Journals Online (AJOL)

    Chicken embryonic stem (ES) cells are useful for producing transgenic chickens and preserving genetic material in avian species. In this study, the differentiation potential of chicken ES cells was investigated in vitro. Chicken ES cells were differentiated into osteoblasts cultured for 15 to 21 days in the induction media ...

  5. File list: ALL.Emb.05.AllAg.Embryonic_eye [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.05.AllAg.Embryonic_eye mm9 All antigens Embryo Embryonic eye SRX804055,SRX1...038028,SRX804056,SRX804054,SRX745813,SRX804057,SRX1038027 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.05.AllAg.Embryonic_eye.bed ...

  6. File list: His.Emb.20.AllAg.Embryonic_eye [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.20.AllAg.Embryonic_eye mm9 Histone Embryo Embryonic eye SRX804054,SRX804056...,SRX1038028,SRX1038027 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.20.AllAg.Embryonic_eye.bed ...

  7. File list: ALL.Emb.10.AllAg.Embryonic_eye [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.10.AllAg.Embryonic_eye mm9 All antigens Embryo Embryonic eye SRX804056,SRX8...04054,SRX1038028,SRX804057,SRX804055,SRX745813,SRX1038027 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.10.AllAg.Embryonic_eye.bed ...

  8. File list: ALL.Emb.50.AllAg.Embryonic_eye [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.50.AllAg.Embryonic_eye mm9 All antigens Embryo Embryonic eye SRX804056,SRX8...04054,SRX745813,SRX804057,SRX804055,SRX1038028,SRX1038027 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.50.AllAg.Embryonic_eye.bed ...

  9. File list: InP.Emb.05.AllAg.Embryonic_eye [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.05.AllAg.Embryonic_eye mm9 Input control Embryo Embryonic eye SRX804055,SRX...804057 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.05.AllAg.Embryonic_eye.bed ...

  10. File list: His.Emb.05.AllAg.Embryonic_eye [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.05.AllAg.Embryonic_eye mm9 Histone Embryo Embryonic eye SRX1038028,SRX80405...6,SRX804054,SRX1038027 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.05.AllAg.Embryonic_eye.bed ...

  11. File list: His.Emb.10.AllAg.Embryonic_eye [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.10.AllAg.Embryonic_eye mm9 Histone Embryo Embryonic eye SRX804056,SRX804054...,SRX1038028,SRX1038027 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.10.AllAg.Embryonic_eye.bed ...

  12. File list: InP.Emb.20.AllAg.Embryonic_eye [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.20.AllAg.Embryonic_eye mm9 Input control Embryo Embryonic eye SRX804057,SRX...804055 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.20.AllAg.Embryonic_eye.bed ...

  13. File list: InP.Emb.10.AllAg.Embryonic_eye [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.10.AllAg.Embryonic_eye mm9 Input control Embryo Embryonic eye SRX804057,SRX...804055 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.10.AllAg.Embryonic_eye.bed ...

  14. File list: His.Emb.50.AllAg.Embryonic_eye [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.50.AllAg.Embryonic_eye mm9 Histone Embryo Embryonic eye SRX804056,SRX804054...,SRX1038028,SRX1038027 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.50.AllAg.Embryonic_eye.bed ...

  15. File list: ALL.Emb.20.AllAg.Embryonic_eye [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.20.AllAg.Embryonic_eye mm9 All antigens Embryo Embryonic eye SRX804054,SRX8...04056,SRX745813,SRX1038028,SRX804057,SRX804055,SRX1038027 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.20.AllAg.Embryonic_eye.bed ...

  16. File list: InP.Emb.50.AllAg.embryonic_skin [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.50.AllAg.embryonic_skin mm9 Input control Embryo embryonic skin SRX1062967,...SRX1062972 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.50.AllAg.embryonic_skin.bed ...

  17. 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 ...

  18. File list: InP.Emb.20.AllAg.embryonic_skin [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.20.AllAg.embryonic_skin mm9 Input control Embryo embryonic skin SRX1062967,...SRX1062972 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.20.AllAg.embryonic_skin.bed ...

  19. 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 ...

  20. 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 ...

  1. File list: InP.Emb.10.AllAg.embryonic_skin [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.10.AllAg.embryonic_skin mm9 Input control Embryo embryonic skin SRX1062972,...SRX1062967 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.10.AllAg.embryonic_skin.bed ...

  2. 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 ...

  3. 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 ...

  4. Towards embryonic scaffolds for regenerative medicine. Glycosaminoglycans, effector molecules and collagen

    NARCIS (Netherlands)

    Uijtdewilligen, P.J.E.

    2018-01-01

    Based on the observation that skin heals without a scar during embryonic development, we investigated the possibilities of using principles found in embryonic development to design novel constructs. Such constructs may induce embryonic-like processes, potentially, and result in scarless healing when

  5. 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 ...

  6. 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 ...

  7. File list: InP.Emb.50.AllAg.Embryonic_eye [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.50.AllAg.Embryonic_eye mm9 Input control Embryo Embryonic eye SRX804057,SRX...804055 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.50.AllAg.Embryonic_eye.bed ...

  8. File list: InP.Emb.50.AllAg.Embryonic_limb [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.50.AllAg.Embryonic_limb mm9 Input control Embryo Embryonic limb SRX804047,S...69,SRX083262,SRX083272 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.50.AllAg.Embryonic_limb.bed ...

  9. File list: InP.Emb.50.AllAg.Embryonic_flank [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.50.AllAg.Embryonic_flank mm9 Input control Embryo Embryonic flank SRX804059... http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.50.AllAg.Embryonic_flank.bed ...

  10. File list: NoD.Emb.20.AllAg.Embryonic_trunk [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.20.AllAg.Embryonic_trunk mm9 No description Embryo Embryonic trunk ERX40226...7,ERX402264 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.20.AllAg.Embryonic_trunk.bed ...

  11. 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 ...

  12. 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 ...

  13. 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 ...

  14. 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.

  15. File list: InP.Emb.50.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.50.AllAg.Embryonic_heart mm9 Input control Embryo Embryonic heart SRX143735...RX377685,SRX377687,SRX967654,SRX077933,SRX377683,SRX967652,SRX244285 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.50.AllAg.Embryonic_heart.bed ...

  16. File list: ALL.Emb.50.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.50.AllAg.Embryonic_heart mm9 All antigens Embryo Embryonic heart SRX112938,...52,SRX967653,SRX112936,SRX1100405,SRX112937,SRX185857,SRX244285,SRX022494,SRX337963 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.50.AllAg.Embryonic_heart.bed ...

  17. File list: NoD.Emb.20.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.20.AllAg.Embryonic_heart mm9 No description Embryo Embryonic heart SRX11004...02,SRX1100404,SRX1100405 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.20.AllAg.Embryonic_heart.bed ...

  18. File list: InP.Emb.10.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.10.AllAg.Embryonic_heart mm9 Input control Embryo Embryonic heart SRX967652...RX967654,SRX377683,SRX185886,SRX698167,SRX244285,SRX377687,SRX377685 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.10.AllAg.Embryonic_heart.bed ...

  19. 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 ...

  20. 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 ...

  1. 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 ...

  2. 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 ...

  3. 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 ...

  4. File list: NoD.Emb.50.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.50.AllAg.Embryonic_heart mm9 No description Embryo Embryonic heart SRX11004...02,SRX1100404,SRX1100405 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.50.AllAg.Embryonic_heart.bed ...

  5. File list: ALL.Emb.10.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.10.AllAg.Embryonic_heart mm9 All antigens Embryo Embryonic heart SRX1437350...RX1437340,SRX1437357,SRX1437344,SRX1437336,SRX1437356,SRX377685,SRX022494,SRX337963 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.10.AllAg.Embryonic_heart.bed ...

  6. File list: NoD.Emb.10.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.10.AllAg.Embryonic_heart mm9 No description Embryo Embryonic heart SRX11004...02,SRX1100404,SRX1100405 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.10.AllAg.Embryonic_heart.bed ...

  7. File list: InP.Emb.05.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.05.AllAg.Embryonic_heart mm9 Input control Embryo Embryonic heart SRX967652...RX698167,SRX377681,SRX967654,SRX377683,SRX377685,SRX377687,SRX244285 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.05.AllAg.Embryonic_heart.bed ...

  8. File list: NoD.Emb.05.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.05.AllAg.Embryonic_heart mm9 No description Embryo Embryonic heart SRX11004...04,SRX1100402,SRX1100405 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.05.AllAg.Embryonic_heart.bed ...

  9. 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 ...

  10. File list: InP.Emb.20.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.20.AllAg.Embryonic_heart mm9 Input control Embryo Embryonic heart SRX967652...RX077933,SRX377683,SRX377685,SRX377681,SRX377687,SRX967654,SRX244285 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.20.AllAg.Embryonic_heart.bed ...

  11. 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 ...

  12. File list: ALL.Emb.05.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.05.AllAg.Embryonic_heart mm9 All antigens Embryo Embryonic heart SRX967652,...4,SRX1437348,SRX377683,SRX377685,SRX377687,SRX190172,SRX244285,SRX1100405,SRX337963 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.05.AllAg.Embryonic_heart.bed ...

  13. 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 ...

  14. File list: InP.Emb.10.AllAg.Embryonic_testis [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.10.AllAg.Embryonic_testis mm9 Input control Embryo Embryonic testis SRX1491...74,SRX149168,SRX957136,SRX149172,SRX149166,SRX1175150,SRX1175149 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.10.AllAg.Embryonic_testis.bed ...

  15. 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 ...

  16. Dynamic changes in energy metabolism upon embryonic stem cell differentiation support developmental toxicant identification

    NARCIS (Netherlands)

    Dartel, van D.A.M.; Schulpen, S.H.; Theunissen, P.T.; Bunschoten, A.; Piersma, A.H.; Keijer, J.

    2014-01-01

    Embryonic stem cells (ESC) are widely used to study embryonic development and to identify developmental toxicants. Particularly, the embryonic stem cell test (EST) is well known as in vitro model to identify developmental toxicants. Although it is clear that energy metabolism plays a crucial role in

  17. Caffeine exposure alters cardiac gene expression in embryonic cardiomyocytes

    Science.gov (United States)

    Fang, Xiefan; Mei, Wenbin; Barbazuk, William B.; Rivkees, Scott A.

    2014-01-01

    Previous studies demonstrated that in utero caffeine treatment at embryonic day (E) 8.5 alters DNA methylation patterns, gene expression, and cardiac function in adult mice. To provide insight into the mechanisms, we examined cardiac gene and microRNA (miRNA) expression in cardiomyocytes shortly after exposure to physiologically relevant doses of caffeine. In HL-1 and primary embryonic cardiomyocytes, caffeine treatment for 48 h significantly altered the expression of cardiac structural genes (Myh6, Myh7, Myh7b, Tnni3), hormonal genes (Anp and BnP), cardiac transcription factors (Gata4, Mef2c, Mef2d, Nfatc1), and microRNAs (miRNAs; miR208a, miR208b, miR499). In addition, expressions of these genes were significantly altered in embryonic hearts exposed to in utero caffeine. For in utero experiments, pregnant CD-1 dams were treated with 20–60 mg/kg of caffeine, which resulted in maternal circulation levels of 37.3–65.3 μM 2 h after treatment. RNA sequencing was performed on embryonic ventricles treated with vehicle or 20 mg/kg of caffeine daily from E6.5-9.5. Differential expression (DE) analysis revealed that 124 genes and 849 transcripts were significantly altered, and differential exon usage (DEU) analysis identified 597 exons that were changed in response to prenatal caffeine exposure. Among the DE genes identified by RNA sequencing were several cardiac structural genes and genes that control DNA methylation and histone modification. Pathway analysis revealed that pathways related to cardiovascular development and diseases were significantly affected by caffeine. In addition, global cardiac DNA methylation was reduced in caffeine-treated cardiomyocytes. Collectively, these data demonstrate that caffeine exposure alters gene expression and DNA methylation in embryonic cardiomyocytes. PMID:25354728

  18. Caffeine exposure alters cardiac gene expression in embryonic cardiomyocytes.

    Science.gov (United States)

    Fang, Xiefan; Mei, Wenbin; Barbazuk, William B; Rivkees, Scott A; Wendler, Christopher C

    2014-12-15

    Previous studies demonstrated that in utero caffeine treatment at embryonic day (E) 8.5 alters DNA methylation patterns, gene expression, and cardiac function in adult mice. To provide insight into the mechanisms, we examined cardiac gene and microRNA (miRNA) expression in cardiomyocytes shortly after exposure to physiologically relevant doses of caffeine. In HL-1 and primary embryonic cardiomyocytes, caffeine treatment for 48 h significantly altered the expression of cardiac structural genes (Myh6, Myh7, Myh7b, Tnni3), hormonal genes (Anp and BnP), cardiac transcription factors (Gata4, Mef2c, Mef2d, Nfatc1), and microRNAs (miRNAs; miR208a, miR208b, miR499). In addition, expressions of these genes were significantly altered in embryonic hearts exposed to in utero caffeine. For in utero experiments, pregnant CD-1 dams were treated with 20-60 mg/kg of caffeine, which resulted in maternal circulation levels of 37.3-65.3 μM 2 h after treatment. RNA sequencing was performed on embryonic ventricles treated with vehicle or 20 mg/kg of caffeine daily from E6.5-9.5. Differential expression (DE) analysis revealed that 124 genes and 849 transcripts were significantly altered, and differential exon usage (DEU) analysis identified 597 exons that were changed in response to prenatal caffeine exposure. Among the DE genes identified by RNA sequencing were several cardiac structural genes and genes that control DNA methylation and histone modification. Pathway analysis revealed that pathways related to cardiovascular development and diseases were significantly affected by caffeine. In addition, global cardiac DNA methylation was reduced in caffeine-treated cardiomyocytes. Collectively, these data demonstrate that caffeine exposure alters gene expression and DNA methylation in embryonic cardiomyocytes. Copyright © 2014 the American Physiological Society.

  19. Proteomic Analysis of Chicken Skeletal Muscle during Embryonic Development

    Directory of Open Access Journals (Sweden)

    Hongjia Ouyang

    2017-05-01

    Full Text Available Embryonic growth and development of skeletal muscle is a major determinant of muscle mass, and has a significant effect on meat production in chicken. To assess the protein expression profiles during embryonic skeletal muscle development, we performed a proteomics analysis using isobaric tags for relative and absolute quantification (iTRAQ in leg muscle tissues of female Xinghua chicken at embryonic age (E 11, E16, and 1-day post hatch (D1. We identified 3,240 proteins in chicken embryonic muscle and 491 of them were differentially expressed (fold change ≥ 1.5 or ≤ 0.666 and p < 0.05. There were 19 up- and 32 down-regulated proteins in E11 vs. E16 group, 238 up- and 227 down-regulated proteins in E11 vs. D1 group, and 13 up- and 5 down-regulated proteins in E16 vs. D1 group. Protein interaction network analyses indicated that these differentially expressed proteins were mainly involved in the pathway of protein synthesis, muscle contraction, and oxidative phosphorylation. Integrative analysis of proteome and our previous transcriptome data found 189 differentially expressed proteins that correlated with their mRNA level. The interactions between these proteins were also involved in muscle contraction and oxidative phosphorylation pathways. The lncRNA-protein interaction network found four proteins DMD, MYL3, TNNI2, and TNNT3 that are all involved in muscle contraction and may be lncRNA regulated. These results provide several candidate genes for further investigation into the molecular mechanisms of chicken embryonic muscle development, and enable us to better understanding their regulation networks and biochemical pathways.

  20. Differential effects of high and low strength magnetic fields on mouse embryonic development and vasculogenesis of embryonic stem cells.

    Science.gov (United States)

    Bekhite, Mohamed M; Finkensieper, Andreas; Abou-Zaid, Fouad A; El-Shourbagy, Ibrahim K; El-Fiky, Nabil K; Omar, Khaled M; Sauer, Heinrich; Wartenberg, Maria

    2016-10-01

    Man-made magnetic fields (MFs) may exert adverse effects on mammalian embryonic development. Herein, we analysed the effect of 10mT 50Hz sinusoidal (AC) or static (DC) MFs versus 1mT MFs on embryonic development of mice. Exposure for 20days during gestation to 10mT MFs increased resorptions and dead fetuses, decreased crown-rump length and fresh weight, reduced blood vessel differentiation and caused histological changes, accompanied with diminished vascular endothelial growth factor (VEGF) protein expression in several organs. In embryonic stem (ES) cell-derived embryoid bodies exposure towards 10mT MFs increased reactive oxygen species (ROS), decreased vascular marker as well as VEGF expression and enhanced apoptosis. In conclusion, our combined data from in vivo and in vitro experiments identified VEGF as an important mediator during embryonic development that can be influenced by high strength MFs, which in consequence leads to severe abnormalities in fetus organs and blood vessel formation. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. 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.

  2. Autotaxin overexpression causes embryonic lethality and vascular defects.

    Directory of Open Access Journals (Sweden)

    Hiroshi Yukiura

    Full Text Available Autotaxin (ATX is a secretory protein, which converts lysophospholipids to lysophosphatidic acid (LPA, and is essential for embryonic vascular formation. ATX is abundantly detected in various biological fluids and its level is elevated in some pathophysiological conditions. However, the roles of elevated ATX levels remain to be elucidated. In this study, we generated conditional transgenic (Tg mice overexpressing ATX and examined the effects of excess LPA signalling. We found that ATX overexpression in the embryonic period caused severe vascular defects and was lethal around E9.5. ATX was conditionally overexpressed in the neonatal period using the Cre/loxP system, which resulted in a marked increase in the plasma LPA level. This resulted in retinal vascular defects including abnormal vascular plexus and increased vascular regression. Our findings indicate that the ATX level must be carefully regulated to ensure coordinated vascular formation.

  3. Autotaxin Overexpression Causes Embryonic Lethality and Vascular Defects

    Science.gov (United States)

    Yukiura, Hiroshi; Kano, Kuniyuki; Kise, Ryoji; Inoue, Asuka; Aoki, Junken

    2015-01-01

    Autotaxin (ATX) is a secretory protein, which converts lysophospholipids to lysophosphatidic acid (LPA), and is essential for embryonic vascular formation. ATX is abundantly detected in various biological fluids and its level is elevated in some pathophysiological conditions. However, the roles of elevated ATX levels remain to be elucidated. In this study, we generated conditional transgenic (Tg) mice overexpressing ATX and examined the effects of excess LPA signalling. We found that ATX overexpression in the embryonic period caused severe vascular defects and was lethal around E9.5. ATX was conditionally overexpressed in the neonatal period using the Cre/loxP system, which resulted in a marked increase in the plasma LPA level. This resulted in retinal vascular defects including abnormal vascular plexus and increased vascular regression. Our findings indicate that the ATX level must be carefully regulated to ensure coordinated vascular formation PMID:25992708

  4. Analysis of Coronary Vessels in Cleared Embryonic Hearts.

    Science.gov (United States)

    Ivins, Sarah; Roberts, Catherine; Vernay, Bertrand; Scambler, Peter J

    2016-12-07

    Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

  5. Analysis of Coronary Vessels in Cleared Embryonic Hearts

    Science.gov (United States)

    Ivins, Sarah; Roberts, Catherine; Vernay, Bertrand; Scambler, Peter J.

    2016-01-01

    Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos. PMID:28060348

  6. Embryonic development of Anodontites trapesialis (Lamarck, 1819) (Bivalvia: Mycetopodidae).

    Science.gov (United States)

    Silva-Souza, A T; Guardia-Felipi, P; Arrebola, N R

    2011-02-01

    The phases of embryonic development of Anodontites trapesialis lasidia are described for the first time. Adult specimens were obtained from two fish farms located in Londrina, Paraná, Brazil. The internal demibranchs of 120 individuals were studied using a routine histological technique; 70 of these carried eggs and/or larvae in the marsupium and were utilized for the description of the phases of embryonic development. The demibranchs of five specimens were evaluated by scanning electron microscopy to detail the morphology of the larvae. Five phases of development were established: phase I, corresponding to the initial stage of cleavage with the formation of apical cells; phase II, including the stages of the morula and blastula; phase III, where the gastrula forms; phase IV, where the larva formed is still inside the egg envelope; and phase V, where the lasidium can still be identified immediately after eclosion.

  7. Embryonic development of Anodontites trapesialis (Lamarck, 1819 (Bivalvia: Mycetopodidae

    Directory of Open Access Journals (Sweden)

    AT. Silva-Souza

    Full Text Available The phases of embryonic development of Anodontites trapesialis lasidia are described for the first time. Adult specimens were obtained from two fish farms located in Londrina, Paraná, Brazil. The internal demibranchs of 120 individuals were studied using a routine histological technique; 70 of these carried eggs and/or larvae in the marsupium and were utilized for the description of the phases of embryonic development. The demibranchs of five specimens were evaluated by scanning electron microscopy to detail the morphology of the larvae. Five phases of development were established: phase I, corresponding to the initial stage of cleavage with the formation of apical cells; phase II, including the stages of the morula and blastula; phase III, where the gastrula forms; phase IV, where the larva formed is still inside the egg envelope; and phase V, where the lasidium can still be identified immediately after eclosion.

  8. Primary hepatic embryonal sarcoma masquerading as metastatic ovarian cancer

    Directory of Open Access Journals (Sweden)

    Praseedom Raaj

    2009-06-01

    Full Text Available Abstract Background Hepatic embryonal sarcoma (HES is a rare but aggressive primary tumor of the liver occurring most frequently in childhood. Case presentation We report a case of a 52 year old woman having previously undergone treatment for ovarian serous papillary carcinoma who subsequently presented with a large solitary mass in the liver. Initially this was presumed to be metastasis from the ovarian primary however, on further examination it was shown to be a primary hepatic embryonal sarcoma. Conclusion Primary liver tumors should be considered in differential diagnoses in patients with ovarian cancer who subsequently present with liver tumors. This is particularly important when there is no direct evidence of recurrence of ovarian cancer.

  9. 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.

  10. 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.

  11. Epigenetic regulation of osteogenesis: human embryonic palatal mesenchymal cells

    OpenAIRE

    Barkhordarian, Andre; Sison, Jay; Cayabyab, Riana; Mahanian, Nicole; Chiappelli, Francesco

    2011-01-01

    Mesenchymal stem cells (MSCs) provide an appropriate model to study epigenetic changes during osteogenesis and bone regeneration due to their differentiation potential. Since there are no unique markers for MSCs, methods of identification are limited. The complex morphology of human embryonic palatal mesenchyme stem cell (HEPM) requires analysis of fractal dimensions to provide an objective quantification of self-similarity, a statistical transformation of cellular shape and border complexity...

  12. A toolbox to explore the mechanics of living embryonic tissues

    OpenAIRE

    Camp?s, Otger

    2016-01-01

    The sculpting of embryonic tissues and organs into their functional morphologies involves the spatial and temporal regulation of mechanics at cell and tissue scales. Decades of in vitro work, complemented by some in vivo studies, have shown the relevance of mechanical cues in the control of cell behaviors that are central to developmental processes, but the lack of methodologies enabling precise, quantitative measurements of mechanical cues in vivo have hindered our understanding of the role ...

  13. Derivation of human embryonic stem cells in defined conditions.

    Science.gov (United States)

    Ludwig, Tenneille E; Levenstein, Mark E; Jones, Jeffrey M; Berggren, W Travis; Mitchen, Erika R; Frane, Jennifer L; Crandall, Leann J; Daigh, Christine A; Conard, Kevin R; Piekarczyk, Marian S; Llanas, Rachel A; Thomson, James A

    2006-02-01

    We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.

  14. Integration of embryonic stem cells in metanephric kidney organ culture.

    Science.gov (United States)

    Steenhard, Brooke M; Isom, Kathryn S; Cazcarro, Patricia; Dunmore, Judy H; Godwin, Alan R; St John, Patricia L; Abrahamson, Dale R

    2005-06-01

    Many stages of nephrogenesis can be studied using cultured embryonic kidneys, but there is no efficient technique available to readily knockdown or overexpress transgenes for rapid evaluation of resulting phenotypes. Embryonic stem (ES) cells have unlimited developmental potential and can be manipulated at the molecular genetic level by a variety of methods. The aim of this study was to determine if ES cells could respond to developmental signals within the mouse embryonic day 12 to embryonic day 13 (E12 to E13) kidney microenvironment and incorporate into kidney structures. ROSA26 ES cells were shown to express beta-galactosidase ubiquitously when cultured in the presence of leukemia inhibitory factor to suppress differentiation. When these cells were microinjected into E12 to E13 metanephroi and then placed in transwell organ culture, ES cell-derived, beta-galactosidase-positive cells were identified in epithelial structures resembling tubules. On rare occasions, individual ES cells were observed in structures resembling glomerular tufts. Electron microscopy showed that the ES cell-derived tubules were surrounded by basement membrane and had apical microvilli and junctional complexes. Marker analysis revealed that a subset of these epithelial tubules bound Lotus tetragonolobus and expressed alpha(1) Na(+)/K(+) ATPase. ES cells were infected before injection with a cytomegalovirus promoter-green fluorescence protein (GFP) adenovirus and GFP expression was found as early as 18 h, persisting for up to 48 h in cultured kidneys. This ES cell technology may achieve the objective of obtaining a versatile cell culture system in which molecular interventions can be used in vitro and consequences of these perturbations on the normal kidney development program in vivo can be studied.

  15. Surface landmark quantification of embryonic mouse craniofacial morphogenesis

    OpenAIRE

    Percival, Christopher J; Green, Rebecca; Marcucio, Ralph; Hallgrímsson, Benedikt

    2014-01-01

    Background Morphometric quantification of subtle craniofacial variation in studies of experimentally modified embryonic mice has proved valuable in determining the effects of developmental perturbations on craniofacial morphogenesis. The direct comparison of landmark coordinate data from embryos of many different mouse strains and mouse models can advance our understanding of the bases for craniofacial variation. We propose a standard set of craniofacial surface landmarks, for use with embryo...

  16. Fibroblast growth factor signaling in embryonic and cancer stem cells

    Czech Academy of Sciences Publication Activity Database

    Dvořák, Petr; Dvořáková, D.; Hampl, Aleš

    2006-01-01

    Roč. 580, - (2006), s. 2869-2874 ISSN 0014-5793 R&D Projects: GA MŠk 1M0538; GA ČR GA301/03/1122 Institutional research plan: CEZ:AV0Z50390512 Keywords : Fibroblast growth factor 2 * Embryonic stem cell * Hematopoietic progenitor cell Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.372, year: 2006

  17. Hemihypertrophy and a poorly differentiated embryonal rhabdomyosarcoma of the pelvis.

    Science.gov (United States)

    Samuel, D P; Tsokos, M; DeBaun, M R

    1999-01-01

    Asymmetry of the limbs (conventionally known as hemihypertrophy) is one of the overgrowth syndromes occurring sporadically in the general population at a frequency of approximately 1:86,000. Hemihypertrophy is also reported as part of the Beckwith-Wiedemann syndrome which has as its cardinal features omphalocele, macroglossia and gigantism with hypoglycemia, organomegaly, renal anomalies, hemihypertrophy, and embryonal tumors occurring less frequently. Various neoplasms are also associated with isolated hemihypertrophy. Wilms tumor, adrenocortical carcinoma, and hepatoblastoma are the most frequent. Rhabdomyosarcoma, neuroblastoma, phaeochromocytoma, and undifferentiated sarcoma of the lung are encountered only rarely. Loss of heterozygosity (LOH) of chromosome 11p15.5 is strongly associated with childhood embryonal tumors, particularly Wilms tumor, hepatoblastoma, and rhabdomyosarcoma. In this article, we describe an adolescent male with congenital asymmetry of the lower limbs who presented with a large poorly differentiated pelvic sarcoma. Conventional histologic, immunohistochemical, and ultrastructural studies of this tumor were insufficient for accurate subclassfication. However, positive staining for MyoD1 (a recently identified embryonically expressed marker of muscle differentiation) and LOH at the tyrosine hydroxylase locus of chromosome 11p15.5 by molecular analysis favored the diagnosis of embryonal rhabdomyosarcoma over an undifferentiated sarcoma. This case stresses the importance of pursuing clinical findings when they occur in conditions with an increased risk of developing cancer, which in this case was asymmetry of a limb. Also illustrated by this patient is the need for early consideration of molecular diagnostic tests where available, to refine an uncertain pathologic diagnosis that may ultimately have an impact on treatment and prognosis.

  18. CULTURE OF EMBRYONIC CELLS OF DROSOPHILA MELANOGASTER IN VITRO.

    Science.gov (United States)

    HORIKAWA, M; FOX, A S

    1964-09-25

    Embryonic cells isolated from eggs ofDrosophila melanogasterhave been cultured continuously in a new medium. Generation time for cell division is 30 hours. Chromosome number remains constant for at least 10 days. Cells from embryos of the mutant maroon-like grow at the same rate as those from wild-type embryos, but cells from rosy-2 grow slower and at a lower optimum temperature.

  19. Contributions of Noncanonical Smoothened Signaling During Embryonic Development

    OpenAIRE

    Pandit, Tanushree; Ogden, Stacey K.

    2017-01-01

    The Sonic Hedgehog (Shh) signaling pathway is active during embryonic development in metazoans, and provides instructional cues necessary for proper tissue patterning. The pathway signal transducing component, Smoothened (Smo), is a G protein-coupled receptor (GPCR) that has been demonstrated to signal through at least two effector routes. The first is a G protein–independent canonical route that signals to Gli transcriptional effectors to establish transcriptional programs specifying cell fa...

  20. Embryonic miRNA profiles of normal and ectopic pregnancies.

    Directory of Open Access Journals (Sweden)

    Francisco Dominguez

    Full Text Available Our objective was to investigate the miRNA profile of embryonic tissues in ectopic pregnancies (EPs and controlled abortions (voluntary termination of pregnancy; VTOP. Twenty-three patients suffering from tubal EP and twenty-nine patients with a normal ongoing pregnancy scheduled for a VTOP were recruited. Embryonic tissue samples were analyzed by miRNA microarray and further validated by real time PCR. Microarray studies showed that four miRNAs were differentially downregulated (hsa-mir-196b, hsa-mir-30a, hsa-mir-873, and hsa-mir-337-3p and three upregulated (hsa-mir-1288, hsa-mir-451, and hsa-mir-223 in EP compared to control tissue samples. Hsa-miR-196, hsa-miR-223, and hsa-miR-451 were further validated by real time PCR in a wider population of EP and control samples. We also performed a computational analysis to identify the gene targets and pathways which might be modulated by these three differentially expressed miRNAs. The most significant pathways found were the mucin type O-glycan biosynthesis and the ECM-receptor-interaction pathways. We also checked that the dysregulation of these three miRNAs was able to alter the expression of the gene targets in the embryonic tissues included in these pathways such as GALNT13 and ITGA2 genes. In conclusion, analysis of miRNAs in ectopic and eutopic embryonic tissues shows different expression patterns that could modify pathways which are critical for correct implantation, providing new insights into the understanding of ectopic implantation in humans.

  1. Polycomb enables primitive endoderm lineage priming in embryonic stem cells

    DEFF Research Database (Denmark)

    Illingworth, Robert S; Hölzenspies, Jurriaan J; Roske, Fabian V

    2016-01-01

    Mouse embryonic stem cells (ESCs), like the blastocyst from which they are derived, contain precursors of the epiblast (Epi) and primitive endoderm (PrEn) lineages. While transient in vivo, these precursor populations readily interconvert in vitro. We show that altered transcription is the driver...... polycomb with dynamic changes in transcription and stalled lineage commitment, allowing cells to explore alternative choices prior to a definitive decision....

  2. Comparative metal oxide nanoparticle toxicity using embryonic zebrafish

    OpenAIRE

    Wehmas, Leah C.; Anders, Catherine; Chess, Jordan; Punnoose, Alex; Pereira, Cliff B.; Greenwood, Juliet A.; Tanguay, Robert L.

    2015-01-01

    Engineered metal oxide nanoparticles (MO NPs) are finding increasing utility in the medical field as anticancer agents. Before validation of in vivo anticancer efficacy can occur, a better understanding of whole-animal toxicity is required. We compared the toxicity of seven widely used semiconductor MO NPs made from zinc oxide (ZnO), titanium dioxide, cerium dioxide and tin dioxide prepared in pure water and in synthetic seawater using a five-day embryonic zebrafish assay. We hypothesized tha...

  3. Epigenetic stability, adaptability, and reversibility in human embryonic stem cells

    OpenAIRE

    Tompkins, Joshua D.; Hall, Christine; Chen, Vincent Chang-yi; Li, Arthur Xuejun; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

    2012-01-01

    The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for a...

  4. Embryonic miRNA Profiles of Normal and Ectopic Pregnancies

    Science.gov (United States)

    Dominguez, Francisco; Moreno-Moya, Juan Manuel; Lozoya, Teresa; Romero, Ainhoa; Martínez, Sebastian; Monterde, Mercedes; Gurrea, Marta; Ferri, Blanca; Núñez, Maria Jose; Simón, Carlos; Pellicer, Antonio

    2014-01-01

    Our objective was to investigate the miRNA profile of embryonic tissues in ectopic pregnancies (EPs) and controlled abortions (voluntary termination of pregnancy; VTOP). Twenty-three patients suffering from tubal EP and twenty-nine patients with a normal ongoing pregnancy scheduled for a VTOP were recruited. Embryonic tissue samples were analyzed by miRNA microarray and further validated by real time PCR. Microarray studies showed that four miRNAs were differentially downregulated (hsa-mir-196b, hsa-mir-30a, hsa-mir-873, and hsa-mir-337-3p) and three upregulated (hsa-mir-1288, hsa-mir-451, and hsa-mir-223) in EP compared to control tissue samples. Hsa-miR-196, hsa-miR-223, and hsa-miR-451 were further validated by real time PCR in a wider population of EP and control samples. We also performed a computational analysis to identify the gene targets and pathways which might be modulated by these three differentially expressed miRNAs. The most significant pathways found were the mucin type O-glycan biosynthesis and the ECM-receptor-interaction pathways. We also checked that the dysregulation of these three miRNAs was able to alter the expression of the gene targets in the embryonic tissues included in these pathways such as GALNT13 and ITGA2 genes. In conclusion, analysis of miRNAs in ectopic and eutopic embryonic tissues shows different expression patterns that could modify pathways which are critical for correct implantation, providing new insights into the understanding of ectopic implantation in humans. PMID:25013942

  5. Rat embryonic palatal shelves respond to TCDD in organ culture

    International Nuclear Information System (INIS)

    Abbott, B.D.; Birnbaum, L.S.

    1990-01-01

    TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a highly toxic environmental contaminant, is teratogenic in mice, inducing cleft palate (CP) and hydronephrosis at doses which are not overtly maternally or embryo toxic. Palatal shelves of embryonic mice respond to TCDD, both in vivo and in organ culture, with altered differentiation of medial epithelial cells. By contrast, in the rat TCDD produces substantial maternal, embryonic, and fetal toxicity, including fetal lethality, with few malformations. In this study the possible effects of maternal toxicity on induction of cleft palate were eliminated by exposure of embryonic rat palatal shelves in organ culture. The shelves were examined for specific TCDD-induced alterations in differentiation of the medial cells. On Gestation Day (GD) 14 or 15 palatal shelves from embryonic F344 rats were placed in organ culture for 2 to 3 days (IMEM:F12 medium, 5% FBS, 0.1% DMSO) containing 0, 1 x 10(-8), 1 x 10(-9), 1 x 10(-10), or 5 x 10(-11) M TCDD. The medial epithelial peridermal cells degenerated on shelves exposed to control media or 5 x 10(-11) M TCDD. Exposure to 10(-10), 10(-9), and 10(-8) M TCDD inhibited this degeneration in 20, 36, and 60% of the shelves, respectively, and was statistically significant at the two highest doses. A normally occurring decrease in [3H]TdR incorporation was inhibited in some GD 15 shelves cultured with 10(-10) and 10(-9) M TCDD. The medial cells of TCDD-exposed shelves continued to express high levels of immunohistochemically detected EGF receptors. The altered differentiation of rat medial epithelium is similar to that reported for TCDD-exposed mouse medial cells in vivo and in vitro. However, in order to obtain these responses, the cultured rat shelves require much higher concentrations of TCDD than the mouse shelves

  6. Case Study: Organotypic human in vitro models of embryonic ...

    Science.gov (United States)

    Morphogenetic fusion of tissues is a common event in embryonic development and disruption of fusion is associated with birth defects of the eye, heart, neural tube, phallus, palate, and other organ systems. Embryonic tissue fusion requires precise regulation of cell-cell and cell-matrix interactions that drive proliferation, differentiation, and morphogenesis. Chemical low-dose exposures can disrupt morphogenesis across space and time by interfering with key embryonic fusion events. The Morphogenetic Fusion Task uses computer and in vitro models to elucidate consequences of developmental exposures. The Morphogenetic Fusion Task integrates multiple approaches to model responses to chemicals that leaad to birth defects, including integrative mining on ToxCast DB, ToxRefDB, and chemical structures, advanced computer agent-based models, and human cell-based cultures that model disruption of cellular and molecular behaviors including mechanisms predicted from integrative data mining and agent-based models. The purpose of the poster is to indicate progress on the CSS 17.02 Virtual Tissue Models Morphogenesis Task 1 products for the Board of Scientific Counselors meeting on Nov 16-17.

  7. In vitro pancreas organogenesis from dispersed mouse embryonic progenitors.

    Science.gov (United States)

    Greggio, Chiara; De Franceschi, Filippo; Figueiredo-Larsen, Manuel; Grapin-Botton, Anne

    2014-07-19

    The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells (1). The whole embryonic organ can be cultured at multiple stages of development (2-4). These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell´s tensegrity.

  8. Morphometric human embryonic brain features according to developmental stage.

    Science.gov (United States)

    Kobayashi, Ami; Ishizu, Koichi; Yamada, Shigehito; Uwabe, Chigako; Kose, Katsumi; Takakuwa, Tetsuya

    2016-04-01

    The present study investigated linear, area, and volume measurements of human brain samples according to Carnegie stages (CS) in an attempt to select suitable morphometric features that reflect embryonic development. Using magnetic resonance imaging, we measured seven linear segments, three separate areas, and three regional volumes in 101 samples between CS13 and 23. Brain volume was determined via manual segmentation of the magnetic resonance image, whereby a formula was generated to estimate the volume of each linear measurement. All parameters correlated with crown-rump length. Bitemporal length and mesencephalic height increased linearly according to the CS, and a high correlation between bitemporal length and both whole-brain (r = 0.98) and prosencephalon (r = 0.99) volumes was found when brain cavity volume was excluded. Morphometric data related to human embryonic stages are valuable for correcting and comparing sonographic data. The present approach may contribute to improvements in prenatal diagnostics by enabling the selection of more suitable measurements during early embryonic stages. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

  9. Effects of embryonic cyclosporine exposures on brain development and behavior.

    Science.gov (United States)

    Clift, Danielle E; Thorn, Robert J; Passarelli, Emily A; Kapoor, Mrinal; LoPiccolo, Mary K; Richendrfer, Holly A; Colwill, Ruth M; Creton, Robbert

    2015-04-01

    Cyclosporine, a calcineurin inhibitor, is successfully used as an immunosuppressant in transplant medicine. However, the use of this pharmaceutical during pregnancy is concerning since calcineurin is thought to play a role in neural development. The risk for human brain development is difficult to evaluate because of a lack of basic information on the sensitive developmental times and the potentially pleiotropic effects on brain development and behavior. In the present study, we use zebrafish as a model system to examine the effects of embryonic cyclosporine exposures. Early embryonic exposures reduced the size of the eyes and brain. Late embryonic exposures did not affect the size of the eyes or brain, but did lead to substantial behavioral defects at the larval stages. The cyclosporine-exposed larvae displayed a reduced avoidance response to visual stimuli, low swim speeds, increased resting, an increase in thigmotaxis, and changes in the average distance between larvae. Similar results were obtained with the calcineurin inhibitor FK506, suggesting that most, but not all, effects on brain development and behavior are mediated by calcineurin inhibition. Overall, the results show that cyclosporine can induce either structural or functional brain defects, depending on the exposure window. The observed functional brain defects highlight the importance of quantitative behavioral assays when evaluating the risk of developmental exposures. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Demethylating agent, 5-azacytidine, reverses differentiation of embryonic stem cells

    International Nuclear Information System (INIS)

    Tsuji-Takayama, Kazue; Inoue, Toshiya; Ijiri, Yoshihiro; Otani, Takeshi; Motoda, Ryuichi; Nakamura, Shuji; Orita, Kunzo

    2004-01-01

    The de novo methylation activity is essential for embryonic development as well as embryonic stem (ES) cell differentiation, where the intensive and extensive DNA methylation was detected. In this study, we investigated the effects of a demethylating agent, 5-azacytidine (5-AzaC), on differentiated ES cells in order to study the possibility of reversing the differentiation process. We first induced differentiation of ES cells by forming embryoid bodies, and then the cells were treated with 5-AzaC. The cells showed some undifferentiated features such as stem cell-like morphology with unclear cell-to-cell boundary and proliferative responsiveness to LIF. Moreover, 5-AzaC increased the expressions of ES specific markers, SSEA-1, and alkaline phosphatase activity as well as ES specific genes, Oct4, Nanog, and Sox2. We also found that 5-AzaC demethylated the promoter region of H19 gene, a typical methylated gene during embryonic differentiation. These results indicate that 5-AzaC reverses differentiation state of ES cells through its DNA demethylating activity to differentiation related genes

  11. MRI diagnosis of embryonal tumors in the spinal canal

    International Nuclear Information System (INIS)

    Sun Jilin; Zhang Xinchuan; Zhang Huaning; Liu Lianxiang; Wu Yujin

    1997-01-01

    To evaluate MRI diagnostic value of the embryonal tumors in the spinal canal. Materials and methods: The MRI appearances of 15 cases of histologically confirmed embryonal tumors in the spinal canal were analyzed. (1) Lipoma (3 cases) had characteristic MRI appearance, demonstrating high signal intensity on T 1 WI, and moderately high signal on T 2 WI. High signal intensity of the lipoma was turned into low signal intensity by fat suppression technique. (2) Dermoids (2 cases) and epidermoid (7 cases) exhibiting low or iso-low signal on T 1 WI and high or iso-high signal on T 2 WI. All had an iso-intense capsule on T 1 WI. However, the two tumors could not be distinguished from each other. (3) Teratoma (3 cases) appeared as a mass of inhomo-generous signals in the spinal canal including soft tissue, fatty tissue and calcification within the same tumor. The diagnosis of embryonal tumors in the spinal canal mainly depend on their MRI appearances, specific tumor location and patient's age

  12. Embryonic cerebrospinal fluid in brain development: neural progenitor control.

    Science.gov (United States)

    Gato, Angel; Alonso, M Isabel; Martín, Cristina; Carnicero, Estela; Moro, José Antonio; De la Mano, Aníbal; Fernández, José M F; Lamus, Francisco; Desmond, Mary E

    2014-08-28

    Due to the effort of several research teams across the world, today we have a solid base of knowledge on the liquid contained in the brain cavities, its composition, and biological roles. Although the cerebrospinal fluid (CSF) is among the most relevant parts of the central nervous system from the physiological point of view, it seems that it is not a permanent and stable entity because its composition and biological properties evolve across life. So, we can talk about different CSFs during the vertebrate life span. In this review, we focus on the CSF in an interesting period, early in vertebrate development before the formation of the choroid plexus. This specific entity is called "embryonic CSF." Based on the structure of the compartment, CSF composition, origin and circulation, and its interaction with neuroepithelial precursor cells (the target cells) we can conclude that embryonic CSF is different from the CSF in later developmental stages and from the adult CSF. This article presents arguments that support the singularity of the embryonic CSF, mainly focusing on its influence on neural precursor behavior during development and in adult life.

  13. 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

  14. Maternal Embryonic Leucine Zipper Kinase (MELK: A Novel Regulator in Cell Cycle Control, Embryonic Development, and Cancer

    Directory of Open Access Journals (Sweden)

    Pengfei Jiang

    2013-10-01

    Full Text Available Maternal embryonic leucine zipper kinase (MELK functions as a modulator of intracellular signaling and affects various cellular and biological processes, including cell cycle, cell proliferation, apoptosis, spliceosome assembly, gene expression, embryonic development, hematopoiesis, and oncogenesis. In these cellular processes, MELK functions by binding to numerous proteins. In general, the effects of multiple protein interactions with MELK are oncogenic in nature, and the overexpression of MELK in kinds of cancer provides some evidence that it may be involved in tumorigenic process. In this review, our current knowledge of MELK function and recent discoveries in MELK signaling pathway were discussed. The regulation of MELK in cancers and its potential as a therapeutic target were also described.

  15. Contested embryonic culture in Japan--public discussion, and human embryonic stem cell research in an aging welfare society.

    Science.gov (United States)

    Sleeboom-Faulkner, Margaret

    2010-01-01

    This article explores the reasons for the lack of a broad discussion on bioethical regulation of human embryonic stem cell research (hESR) in Japan and asks why scientists experience difficulties accessing resources for hESR despite the acclaimed indifference of dominant Japanese culture to embryo research. The article shows how various social actors express their views on the embryo and oocyte donation in terms of dominant Japanese culture, foiled against what is regarded as Western culture. Second, it shows how the lack of concern with hESR should be understood in the context of public health policies and communications and bioethics decision making in Japan. Finally, it interprets the meaning of the embryo in the context of Japan as an aging modern welfare society, explaining how policymakers have come to emphasize the urgency of infertility problems over issues around abortion and embryonic life.

  16. Diverging functions of Scr between embryonic and post-embryonic development in a hemimetabolous insect, Oncopeltus fasciatus.

    Science.gov (United States)

    Chesebro, John; Hrycaj, Steven; Mahfooz, Najmus; Popadić, Aleksandar

    2009-05-01

    Hemimetabolous insects undergo an ancestral mode of development in which embryos hatch into first nymphs that resemble miniature adults. While recent studies have shown that homeotic (hox) genes establish segmental identity of first nymphs during embryogenesis, no information exists on the function of these genes during post-embryogenesis. To determine whether and to what degree hox genes influence the formation of adult morphologies, we performed a functional analysis of Sex combs reduced (Scr) during post-embryonic development in Oncopeltus fasciatus. The main effect was observed in prothorax of Scr-RNAi adults, and ranged from significant alterations in its size and shape to a near complete transformation of its posterior half toward a T2-like identity. Furthermore, while the consecutive application of Scr-RNAi at both of the final two post-embryonic stages (fourth and fifth) did result in formation of ectopic wings on T1, the individual applications at each of these stages did not. These experiments provide two new insights into evolution of wings. First, the role of Scr in wing repression appears to be conserved in both holo- and hemimetabolous insects. Second, the prolonged Scr-depletion (spanning at least two nymphal stages) is both necessary and sufficient to restart wing program. At the same time, other structures that were previously established during embryogenesis are either unaffected (T1 legs) or display only minor changes (labium) in adults. These observations reveal a temporal and spatial divergence of Scr roles during embryonic (main effect in labium) and post-embryonic (main effect in prothorax) development.

  17. The Fos-Related Antigen 1–JUNB/Activator Protein 1 Transcription Complex, a Downstream Target of Signal Transducer and Activator of Transcription 3, Induces T Helper 17 Differentiation and Promotes Experimental Autoimmune Arthritis

    Directory of Open Access Journals (Sweden)

    Young-Mee Moon

    2017-12-01

    Full Text Available Dysfunction of T helper 17 (Th17 cells leads to chronic inflammatory disorders. Signal transducer and activator of transcription 3 (STAT3 orchestrates the expression of proinflammatory cytokines and pathogenic cell differentiation from interleukin (IL-17-producing Th17 cells. However, the pathways mediated by STAT3 signaling are not fully understood. Here, we observed that Fos-related antigen 1 (FRA1 and JUNB are directly involved in STAT3 binding to sites in the promoters of Fosl1 and Junb. Promoter binding increased expression of IL-17 and the development of Th17 cells. Overexpression of Fra1 and Junb in mice resulted in susceptibility to collagen-induced arthritis and an increase in Th17 cell numbers and inflammatory cytokine production. In patients with rheumatoid arthritis, FRA1 and JUNB were colocalized with STAT3 in the inflamed synovium. These observations suggest that FRA1 and JUNB are associated closely with STAT3 activation, and that this activation leads to Th17 cell differentiation in autoimmune diseases and inflammation.

  18. Differentiation and Transplantation of Embryonic Stem Cell-Derived Cone Photoreceptors into a Mouse Model of End-Stage Retinal Degeneration

    Directory of Open Access Journals (Sweden)

    Kamil Kruczek

    2017-06-01

    Full Text Available The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs. Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor β2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1−/− mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.

  19. Selection of embryonic stem cell-derived enhanced green fluorescent protein-positive dopamine neurons using the tyrosine hydroxylase promoter is confounded by reporter gene expression in immature cell populations.

    Science.gov (United States)

    Hedlund, Eva; Pruszak, Jan; Ferree, Andrew; Viñuela, Angel; Hong, Sunghoi; Isacson, Ole; Kim, Kwang-Soo

    2007-05-01

    Transplantation of mouse embryonic stem (mES) cells can restore function in Parkinson disease models, but can generate teratomas. Purification of dopamine neurons derived from embryonic stem cells by fluorescence-activated cell sorting (FACS) could provide a functional cell population for transplantation while eliminating the risk of teratoma formation. Here we used the tyrosine hydroxylase (TH) promoter to drive enhanced green fluorescent protein (eGFP) expression in mES cells. First, we evaluated 2.5-kilobase (kb) and 9-kb TH promoter fragments and showed that clones generated using the 9-kb fragment produced significantly more eGFP+/TH+ neurons. We selected the 9-kb TH clone with the highest eGFP/TH overlap for further differentiation, FACS, and transplantation experiments. Grafts contained large numbers of eGFP+ dopamine neurons of an appropriate phenotype. However, there were also numerous eGFP+ cells that did not express TH and did not have a neuronal morphology. In addition, we found cells in the grafts representing all three germ layers. Based on these findings, we examined the expression of stem cell markers in our eGFP+ population. We found that a majority of eGFP+ cells were stage-specific embryonic antigen-positive (SSEA-1+) and that the genetically engineered clones contained more SSEA-1+ cells after differentiation than the original D3 mES cells. By negative selection of SSEA-1, we could isolate a neuronal eGFP+ population of high purity. These results illustrate the complexity of using genetic selection to purify mES cell-derived dopamine neurons and provide a comprehensive analysis of cell selection strategies based on tyrosine hydroxylase expression. Disclosure of potential conflicts of interest is found at the end of this article.

  20. 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.

  1. Surface landmark quantification of embryonic mouse craniofacial morphogenesis.

    Science.gov (United States)

    Percival, Christopher J; Green, Rebecca; Marcucio, Ralph; Hallgrímsson, Benedikt

    2014-07-24

    Morphometric quantification of subtle craniofacial variation in studies of experimentally modified embryonic mice has proved valuable in determining the effects of developmental perturbations on craniofacial morphogenesis. The direct comparison of landmark coordinate data from embryos of many different mouse strains and mouse models can advance our understanding of the bases for craniofacial variation. We propose a standard set of craniofacial surface landmarks, for use with embryonic day (E) 10.5-12.5 mice, to serve as the foundation for this type of data compilation and analysis. We quantify the intra- and inter-observer landmark placement variation associated with each landmark and determine how the results of a simple ontogenetic analysis might be influenced by selection of landmark set. Intraobserver landmark placement error for experienced landmarkers generally remains below 0.1 mm, with some landmarks exhibiting higher values at E11.5 and E12.5. Interobserver error tends to increase with embryonic age and those landmarks defined on wide inflections of curves or facial processes exhibit the highest error. Landmarks with highest intra- or inter-observer are identified and we determine that their removal from the dataset does not significantly change the vectors of craniofacial shape change associated with an ontogenetic regression. Our quantification of landmark placement error demonstrates that it is preferable for a single observer to identify all landmark coordinates within a single study and that significant training and experience are necessary before a landmarker can produce data for use in larger meta-analyses. However, we are confident that this standard landmark set, once landmarks with higher error are removed, can serve as a foundation for a comparative dataset of facial morphogenesis across various mouse populations to help identify the developmental bases for phenotypic variation in the craniofacial complex.

  2. Can physics help to explain embryonic development? An overview.

    Science.gov (United States)

    Fleury, V

    2013-10-01

    Recent technical advances including digital imaging and particle image velocimetry can be used to extract the full range of embryonic movements that constitute the instantaneous 'morphogenetic fields' of a developing animal. The final shape of the animal results from the sum over time (integral) of the movements that make up the velocity fields of all the tissue constituents. In vivo microscopy can be used to capture the details of vertebrate development at the earliest embryonic stages. The movements thus observed can be quantitatively compared to physical models that provide velocity fields based on simple hypotheses about the nature of living matter (a visco-elastic gel). This approach has cast new light on the interpretation of embryonic movement, folding, and organisation. It has established that several major discontinuities in development are simple physical changes in boundary conditions. In other words, with no change in biology, the physical consequences of collisions between folds largely explain the morphogenesis of the major structures (such as the head). Other discontinuities result from changes in physical conditions, such as bifurcations (changes in physical behaviour beyond specific yield points). For instance, beyond a certain level of stress, a tissue folds, without any new gene being involved. An understanding of the physical features of movement provides insights into the levers that drive evolution; the origin of animals is seen more clearly when viewed under the light of the fundamental physical laws (Newton's principle, action-reaction law, changes in symmetry breaking scale). This article describes the genesis of a vertebrate embryo from the shapeless stage (round mass of tissue) to the development of a small, elongated, bilaterally symmetric structure containing vertebral precursors, hip and shoulder enlarges, and a head. Copyright © 2013. Published by Elsevier Masson SAS.

  3. Glycosylation analysis and protein structure determination of murine fetal antigen 1 (mFA1)--the circulating gene product of the delta-like protein (dlk), preadipocyte factor 1 (Pref-1) and stromal-cell-derived protein 1 (SCP-1) cDNAs

    DEFF Research Database (Denmark)

    Krogh, T N; Bachmann, E; Teisner, B

    1997-01-01

    By means of sequence analysis, murine fetal antigen 1 (mFA1) isolated from Mus musculus amniotic fluid was shown to be the circulating protein of the delta-like protein, stromal-cell-derived protein 1 (SCP-1) and preadipocyte factor 1 (Pref-1) gene products. The protein contains 36 cysteine resid...

  4. Mathematical Modeling of Flow Characteristics in the Embryonic Chick Heart

    DEFF Research Database (Denmark)

    Heebøll-Christensen, Jesper

    modified inertia, and resistance due to friction and curvature of the multilayered tubular heart. Through the modeling, flow conditions in the embryonic heart are characterized. The models suggest that eccentric rather than concentric deformation of the beating heart is optimal for mean flows induced...... the models are not conclusive on this point. In addition the Liebau effect is investigated in a simpler system containing two elastic tubes joined to form a liquid filled ring, with a compression pump at an asymmetric location. Through comparison to other reports the system validates model construction...

  5. Raman microscopy of individual living human embryonic stem cells

    DEFF Research Database (Denmark)

    Novikov, Sergey M.; Beermann, Jonas; Bozhevolnyi, Sergey I.

    2010-01-01

    We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing...... cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal...

  6. Human Embryonic Stem Cells Suffer from Centrosomal Amplification

    Czech Academy of Sciences Publication Activity Database

    Holubcová, Z.; Matula, P.; Sedláčková, M.; Vinarský, Vladimír; Doležalová, Dáša; Bárta, Tomáš; Dvořák, Petr; Hampl, Aleš

    2011-01-01

    Roč. 29, č. 1 (2011), s. 46-56 ISSN 1066-5099 R&D Projects: GA ČR GA204/09/2044 Grant - others:GA MŠk(CZ) 1M0538; GA MŠk(CZ) 2B06052; EU FP6 project ESTOOLS(XE) LSHG-CT-2006-018739 Program:1M Institutional research plan: CEZ:AV0Z50390703 Keywords : human embryonic stem cells * centrosome * chromosome Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.781, year: 2011

  7. Egg size, embryonic development time and ovoviviparity in Drosophila species.

    Science.gov (United States)

    Markow, T A; Beall, S; Matzkin, L M

    2009-02-01

    Lengths, widths and volumes of eggs from 11 species of Drosophila whose genomes have been fully sequenced exhibit significant variation that is not explained by their phylogenetic relationships. Furthermore, egg size differences are unrelated to embryonic development time in these species. In addition, two of the species, Drosophila sechellia and, to a lesser degree, D. yakuba, both ecological specialists, exhibit ovoviviparity, suggesting that female control over oviposition in these species differs from what is observed in D. melanogaster. The interspecific differences in these reproductive characters, coupled with the availability of whole genome sequences for each, provide an unprecedented opportunity to examine their evolution.

  8. Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

    Science.gov (United States)

    Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

    2015-02-25

    Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Reprogramming Malignant Cancer Cells toward a Benign Phenotype following Exposure to Human Embryonic Stem Cell Microenvironment

    Science.gov (United States)

    Arena, Vincenzo; Arena, Manuel; Arena, Goffredo Orazio

    2017-01-01

    The embryonic microenvironment is well known to be non-permissive for tumor development because early developmental signals naturally suppress the expression of proto-oncogenes. In an analogous manner, mimicking an early embryonic environment during embryonic stem cell culture has been shown to suppress oncogenic phenotypes of cancer cells. Exosomes derived from human embryonic stem cells harbor substances that mirror the content of the cells of origin and have been reported to reprogram hematopoietic stem/progenitor cells via horizontal transfer of mRNA and proteins. However, the possibility that these embryonic stem cells-derived exosomes might be the main effectors of the anti-tumor effect mediated by the embryonic stem cells has not been explored yet. The present study aims to investigate whether exosomes derived from human embryonic stem cells can reprogram malignant cancer cells to a benign stage and reduce their tumorigenicity. We show that the embryonic stem cell-conditioned medium contains factors that inhibit cancer cell growth and tumorigenicity in vitro and in vivo. Moreover, we demonstrate that exosomes derived from human embryonic stem cells display anti-proliferation and pro-apoptotic effects, and decrease tumor size in a xenograft model. These exosomes are also able to transfer their cargo into target cancer cells, inducing a dose-dependent increase in SOX2, OCT4 and Nanog proteins, leading to a dose-dependent decrease of cancer cell growth and tumorigenicity. This study shows for the first time that human embryonic stem cell-derived exosomes play an important role in the tumor suppressive activity displayed by human embryonic stem cells. PMID:28068409

  10. 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.

  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. The ethical dilemma of embryonic stem cell research.

    Science.gov (United States)

    Manzar, Nabeel; Manzar, Bushra; Hussain, Nuzhat; Hussain, M Fawwad Ahmed; Raza, Sajjad

    2013-03-01

    To determine the knowledge, attitude, and ethical concerns of medical students and graduates with regard to Embryonic Stem Cell (ESC) research. This questionnaire based descriptive study was conducted at the Civil Hospital Karachi (CHK), Pakistan from February to July 2008. A well structured questionnaire was administered to medical students and graduate doctors, which included their demographic profile as well as questions in line with the study objective. Informed consent was taken and full confidentiality was assured to the participants. Data were entered in a Statistical Package for Social Sciences (SPSS version.12) and analyzed. A total of 204 male and 216 female medical students and doctors were administered questionnaires out of which 105 males (51.4%) and 108 females (50%) were aware of the embryonic stem cell research and its ethical implications. Forty percent males and 47% of females were of the opinion that life begins at conception. Forty-six percent males and 39% females were in favor of stem cell research while only 31% males and 28% females supported the ESC research. Less than 1/3 of students supported using frozen embryos for research purposes while more than 2/3 indicated that they were unlikely to support abortion for stem cell research purposes. The majority of the students were in favor of stem cell research with some reservations regarding ESC research. A sizeable number of students withheld their views, reflecting their poor understanding of medical ethics. The result of the study indicates a need for incorporating bioethics into the medical curriculum.

  13. Wnt inhibition promotes vascular specification of embryonic cardiac progenitors.

    Science.gov (United States)

    Reichman, David E; Park, Laura; Man, Limor; Redmond, David; Chao, Kenny; Harvey, Richard P; Taketo, Makoto M; Rosenwaks, Zev; James, Daylon

    2018-01-08

    Several studies have demonstrated a multiphasic role for Wnt signaling during embryonic cardiogenesis and developed protocols that enrich for cardiac derivatives during in vitro differentiation of human pluripotent stem cells (hPSCs). However, few studies have investigated the role of Wnt signaling in the specification of cardiac progenitor cells (CPCs) toward downstream fates. Using transgenic mice and hPSCs, we tracked endothelial cells (ECs) that originated from CPCs expressing NKX2.5. Analysis of EC-fated CPCs at discrete phenotypic milestones during hPSC differentiation identified reduced Wnt activity as a hallmark of EC specification, and the enforced activation or inhibition of Wnt reduced or increased, respectively, the degree of vascular commitment within the CPC population during both hPSC differentiation and mouse embryogenesis. Wnt5a, which has been shown to exert an inhibitory influence on Wnt signaling during cardiac development, was dynamically expressed during vascular commitment of hPSC-derived CPCs, and ectopic Wnt5a promoted vascular specification of hPSC-derived and mouse embryonic CPCs. © 2018. Published by The Company of Biologists Ltd.

  14. Ethical and regulatory aspects of embryonic stem cell research.

    Science.gov (United States)

    Jain, Kewal K

    2002-12-01

    Ethical and regulatory issues concerning embryonic stem (ES) cell research are reviewed here a year after the controversy became a public and political issue in the US. The background of various issues are examined and the current regulations in various countries are reviewed. In the US, the debate is linked with abortion, as well as the status of a fetus as a human being, and is politically driven. Obtaining stem cells from embryonic tissues involves destruction of the embryo, to which objections are raised. Religious beliefs are examined and no serious impediments to ES cell research could be identified. Regulations vary from one country to another and it is unlikely that there will ever be any universally uniform ethical and regulatory standards for ES cell research. Currently, the most liberal and favourable environments for ES cell research are in the UK, Singapore, Sweden, India, Israel and China. Unless the US liberalises ES cell research, it may lose its lead in ES cell research and investments in this area may drift to countries with better environments for research. Suggestions are offered in this review to improve the ethical environment for ES cell research.

  15. Localization in Oogenesis of Maternal Regulators of Embryonic Development.

    Science.gov (United States)

    Escobar-Aguirre, Matias; Elkouby, Yaniv M; Mullins, Mary C

    2017-01-01

    Cell polarity generates intracellular asymmetries and functional regionalization in tissues and morphogenetic processes. Cell polarity in development often relies on mechanisms of RNA localization to specific subcellular domains to define the identity of future developing tissues. The totipotent egg of most animals illustrates in a grand way the importance of cell polarity and RNA localization in regulating multiple crucial developmental events. The polarization of the egg arises during its development in oogenesis. RNAs localize asymmetrically in the early oocyte defining its animal-vegetal (AV) axis, which upon further elaboration in mid- and late-oogenesis stages produces a mature egg with specific localized factors along its AV axis. These localized factors will define the future anterior-posterior (AP) and dorsal-ventral (DV) axes of the embryo. Furthermore, AV polarity confines germ cell determinants to the vegetal pole, from where they redistribute to the cleavage furrows of the 2- and 4-cell stage embryo, ultimately specifying the primordial germ cells (PGCs). The sperm entry region during fertilization is also defined by the AV axis. In frogs and fish, sperm enters through the animal pole, similar to the mouse where it enters predominantly in the animal half. Thus, AV polarity establishment and RNA localization are involved in all the major events of early embryonic development. In this chapter, we will review the RNA localization mechanisms in vertebrate oocytes that are key to embryonic patterning, referring to some of the groundbreaking studies in frog oocytes and incorporating the current genetic evidence from the zebrafish.

  16. Urokinase production by electrophoretically separated cultured human embryonic kidney cells

    Science.gov (United States)

    Kunze, M. E.; Plank, L. D.; Giranda, V.; Sedor, K.; Todd, P. W.

    1985-01-01

    Urokinase is a plasminogen activator found in urine. Relatively pure preparations have been tested in Europe, Japan and the United States for the treatment of deep vein thrombosis and other dangerous blood clots. Human embryonic kidney cell cultures have been found to produce urokinase at much higher concentrations, but less than 5% of the cells in typical cultures are producers. Since human diploid cells become senescent in culture the selection of clones derived from single cells will not provide enough material to be useful, so a bulk purification method is needed for the isolation of urokinase producing cell populations. Preparative cell electrophoresis was chosen as the method, since evidence exists that human embryonic cell cultures are richly heterogeneous with respect to electrophoretic mobility, and preliminary electrophoretic separations on the Apollo-Soyuz space flight produced cell populations that were rich in urokinase production. Similarly, erythropoietin is useful in the treatment of certain anemias and is a kidney cell duct, and electrophoretically enriched cell populations producing this product have been reported. Thus, there is a clear need for diploid human cells that produce these products, and there is evidence that such cells should be separable by free-flow cell electrophoresis.

  17. Generation of stomach tissue from mouse embryonic stem cells.

    Science.gov (United States)

    Noguchi, Taka-aki K; Ninomiya, Naoto; Sekine, Mari; Komazaki, Shinji; Wang, Pi-Chao; Asashima, Makoto; Kurisaki, Akira

    2015-08-01

    Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, β-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.

  18. Cell surface carbohydrate changes during embryonic and fetal skin development

    DEFF Research Database (Denmark)

    Dabelsteen, Erik; Holbrook, K; Clausen, H

    1986-01-01

    Monoclonal antibodies to four type 2 chain carbohydrate antigens were used for immunohistochemical studies of embryonic and fetal skin. The antibodies detected N-acetyllactosamine and 3 fucosyl substitutes of this, blood group antigen H, Lex, and Ley. Periderm consistently stained for N-acetyllac......Monoclonal antibodies to four type 2 chain carbohydrate antigens were used for immunohistochemical studies of embryonic and fetal skin. The antibodies detected N-acetyllactosamine and 3 fucosyl substitutes of this, blood group antigen H, Lex, and Ley. Periderm consistently stained for N......-acetyllactosamine, Lex and Ley. The H antigen showed a variable and weak expression on peridermal cells from day 57 to day 84 estimated gestation age (EGA). After this period the H antigen was no longer expressed at peridermal cells. In the epidermis, N-acetyllactosamine was present on all cells until the age of 15...... weeks EGA. After this period N-acetyllactosamine could only be demonstrated on basal cells after treatment with neuraminidase, indicating a masking of N-acetyllactosamine by sialic acid. The H antigen could not be demonstrated in the epithelium before 14 weeks EGA. At this time it appeared on spinous...

  19. Vascular Endothelial Growth Factor from Embryonic Status to Cardiovascular Pathology

    Directory of Open Access Journals (Sweden)

    Mohsen Azimi-Nezhad

    2014-05-01

    Full Text Available Vascular endothelial growth factor (VEGF is a multifunctional cytokine with distinct functions in angiogenesis, lymphangiogenesis, vascular permeability, and hematopoiesis. VEGF is a highly conserved, disulfide-bonded dimeric glycoprotein of 34 to 45 kDa produced by several cell types including fibroblasts, neutrophils, endothelial cells, and peripheral blood mononuclear cells, particularly T lymphocytes and macrophages. Six VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene, consisting of 121, 145, 165, 183, 189, or 206 amino acids. VEGF121, VEGF145, and VEGF165 are secreted whereas VEGF183, VEGF189, and VEGF206 are cell membrane-bound. VEGF145 has a key role during the vascularization of the human ovarian follicle and corpus luteum, in the placentation and embryonic periods, and in bone and wound healing, while VEGF165 is the most abundant and biologically active isoform. VEGF has been linked with a number of vascular pathologies including cardiovascular diseases such ischemic heart disease, heart failure, stroke, and diabetes and its related complications. In this review we aimed to present some important roles of VEGF in a number of clinical issues and indicate its involvement in several phenomena from the initial steps of the embryonic period to cardiovascular diseases.

  20. Stepwise Embryonic Toxicity of Silver Nanoparticles on Oryzias latipes

    Science.gov (United States)

    Cho, Jae-Gu; Kim, Kyung-Tae; Lee, Jae-woo; Kim, Ji-Eun; Kim, Jungkon; Lee, Byoung-Cheun; Jo, Eun-Hye; Yoon, Junheon; Eom, Ig-chun; Choi, Kyunghee; Kim, Pilje

    2013-01-01

    The developmental toxicity of silver nanoparticles (AgNPs) was investigated following exposure of Oryzias latipes (medaka) embryos to 0.1−1 mg/L of homogeneously dispersed AgNPs for 14 days. During this period, developmental endpoints, including lethality, heart rate, and hatching rate, were evaluated by microscopy for different stages of medaka embryonic development. To compare toxic sensitivity, acute adult toxicity was assessed. There was no difference in acute lethal toxicity between embryo and adult medaka. Interestingly, we found that the increase in stepwise toxicity was dependent on the developmental stage of the embryo. Lethal embryonic toxicity increased from exposure days 1 to 3 and exposure days 5 to 8, whereas there was no change from exposure days 3 to 5. In addition, 7 d exposure to 0.8 mg/L AgNPs resulted in significant heart beat retardation in medaka embryos. AgNPs also caused a dose-dependent decrease in the hatching rate and body length of larvae. These results indicate that AgNP exposure causes severe developmental toxicity to medaka embryos and that toxicity levels are enhanced at certain developmental stages, which should be taken into consideration in assessments of metallic NPs toxicity to embryos. PMID:23984374

  1. Stepwise Embryonic Toxicity of Silver Nanoparticles on Oryzias latipes

    Directory of Open Access Journals (Sweden)

    Jae-Gu Cho

    2013-01-01

    Full Text Available The developmental toxicity of silver nanoparticles (AgNPs was investigated following exposure of Oryzias latipes (medaka embryos to 0.1−1 mg/L of homogeneously dispersed AgNPs for 14 days. During this period, developmental endpoints, including lethality, heart rate, and hatching rate, were evaluated by microscopy for different stages of medaka embryonic development. To compare toxic sensitivity, acute adult toxicity was assessed. There was no difference in acute lethal toxicity between embryo and adult medaka. Interestingly, we found that the increase in stepwise toxicity was dependent on the developmental stage of the embryo. Lethal embryonic toxicity increased from exposure days 1 to 3 and exposure days 5 to 8, whereas there was no change from exposure days 3 to 5. In addition, 7 d exposure to 0.8 mg/L AgNPs resulted in significant heart beat retardation in medaka embryos. AgNPs also caused a dose-dependent decrease in the hatching rate and body length of larvae. These results indicate that AgNP exposure causes severe developmental toxicity to medaka embryos and that toxicity levels are enhanced at certain developmental stages, which should be taken into consideration in assessments of metallic NPs toxicity to embryos.

  2. Expression of smoothened in mouse embryonic maxillofacial development.

    Science.gov (United States)

    Du, J; Fan, Z; Ma, X; Wu, Y; Liu, S; Gao, Y; Shen, Y; Fan, M; Wang, S

    2012-04-01

    Hedgehog (Hh) signaling plays many key roles in the development of Drosophila and vertebrate embryos including regulation of craniofacial development. The seven-transmembrane protein, smoothened (Smo) transduces the Hh signal across the plasma membrane as an essential receptor of PTCHED1/2. There are few studies that evaluate the detailed expression of Smo in mouse embryonic craniofacial development. We investigated the expression patterns of Smo during murine embryonic craniofacial development using in situ hybridization (ISH), studies of whole-mounts and sections, immunohistochemistry, quantitative real time PCR, and Western blot analysis. We found that Smo mRNA was expressed in the face of mouse embryos at 11 and 12.5 days post coitum (dpc). After 13.5 dpc, the expression decreased to a low level and was faintly detected after birth. Smo protein could be detected also in embryos at 11, 12.5, and 14.5 dpc. After 15.5 dpc, the expression was very faint and paralleled the gene expression studies. No expression was detected in whisker follicle during facial development and faint signal was detected in Meckel's cartilage. These findings concerning Smo expression should guide further investigation of sonic Hh signaling pathway gene function during maxillofacial development.

  3. Strategies to reduce embryonic mortality in buffalo cows

    Directory of Open Access Journals (Sweden)

    M.J. D’Occhio

    2010-02-01

    Full Text Available The aim of the present study was to examine whether treatment with a GnRH agonist, hCG or P4 on Day 25 after AI increased P4 concentrations and reduced the incidence of embryonic mortality (EM in pregnant buffaloes mated in mid-winter in a Mediterranean environment. The trial was carried out in two farms characterized, in previous years, by low (LEM Group, 153 buffaloes (DIM=150±7 days, and high (HEM Group, 284 buffaloes (DIM=163±5 days, incidence of embryo mortality. Animals were synchronized by Ovsynch-TAI Program and artificially inseminated. On day 25, pregnant buffaloes were randomly assigned to four groups: Control (no treatment, GnRH agonist (buserelin acetate, 12.6 μg, hCG (1500 IU and P4 (341 mg of P4 i.m. every 4 days for three times. Progesterone (pg/ml was determined in milk whey on Days 10, 20 and 25 after AI in all buffaloes and in Days 30 and 45 only in buffaloes pregnant on day 25 and assigned to four groups of treatment. Pregnancy diagnosis was undertaken on Day 45 by ultrasound. All treatments increased P4 milk whey and reduced embryonic mortality in buffalo cows bred in the farm characterized by high EM.

  4. Production of Buffalo Embryonic Stem Cell from HMC Embryos

    Directory of Open Access Journals (Sweden)

    M. Zandi

    2016-06-01

    Full Text Available Embryonic stem cells (ESCs are derived from the inner cell mass (ICM of blastocyst and differentiate into all three embryonic germ layers: ectoderm, endoderm, and mesoderm. In this study, ESCs are derived from Hand Made Cloning (HMG blastocysts and their efficiencies compared to ESCs derived from In Vitro Fertilization (IVF embryos. Feeder layer was used for ESCs culture, and culture medium consisting of Knockout- Dulbecco’s Modified Eagle’s Medium (Ko-DMEM supplemented with Knockout Serum Replacement (KSR, Leukemia Inhibitory Factor (LIF, Basic Fibroblast Growth Factor-2 (FGF-2, L-glutamine, nonessential amino acids and gentamicin. The cell surface antigens used for characterization were the SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81 and the pluripotency markers were NANOG, OCT3/4 and SOX2. Results showed that, the growth rate of ESCs colonies in ESCs from IVF embryos was significantly higher than ESCs from HMG embryos (120% compared with 65%, respectively. Not only real-time PCR results revealed the same expression level of SOX2, OCT3/4 and cMYC between them, but also ESCs from HMG embryos resulted to higher expression of NANOG. Both of ESCs groups maintain in pluripotency state for more than two years and differentiated to the different types of cells like neuron, epithelial, lipid and muscle cells.

  5. Early gene regulation of osteogenesis in embryonic stem cells

    KAUST Repository

    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.

  6. Embryonic modulation of maternal steroids in European starlings (Sturnus vulgaris).

    Science.gov (United States)

    Paitz, Ryan T; Bowden, Rachel M; Casto, Joseph M

    2011-01-07

    In birds, maternally derived yolk steroids are a proposed mechanism by which females can adjust individual offspring phenotype to prevailing conditions. However, when interests of mother and offspring differ, parent-offspring conflict will arise and embryonic interests, not those of the mother, should drive offspring response to maternal steroids in eggs. Because of this potential conflict, we investigated the ability of developing bird embryos to process maternally derived yolk steroids. We examined how progesterone, testosterone and oestradiol levels changed in both the yolk/albumen (YA) and the embryo of European starling eggs during the first 10 days of development. Next, we injected tritiated testosterone into eggs at oviposition to characterize potential metabolic pathways during development. Ether extractions separated organic and aqueous metabolites in both the embryo and YA homogenate, after which major steroid metabolites were identified. Results indicate that the concentrations of all three steroids declined during development in the YA homogenate. Exogenous testosterone was primarily metabolized to an aqueous form of etiocholanolone that remained in the YA. These results clearly demonstrate that embryos can modulate their local steroid environment, setting up the potential for parent-offspring conflict. Embryonic regulation must be considered when addressing the evolutionary consequences of maternal steroids in eggs.

  7. Effect of radiation on the embryonic midgut of the silkworm

    International Nuclear Information System (INIS)

    Miki, Mutsuo; Ohtuki, Yoshiki; Murakami, Akio.

    1979-01-01

    In order to determine the effect of X-rays on embryonic midguts of the silkworm (Bombyx mori L.), embryos of C108 strain were X-irradiated (180 kVp, 25 mA, 1.0 mm Al filter and a dose-rate at 300 R/min) at different stages ranging from 44 to 128 hours after oviposition to the stage organogenesis. Histological observation was made on the midguts from treated or nontreated embryos which had grown up to the stage of abdominal pigmentation. The midgut of embryos younger than 80 hours was remarkably damaged following X-irradiations with Ld 50 dose-range (1.5 KR). The midgut of embryos from 44 to 68 hours after oviposition or in the very early phase of organogenesis was most radiosensitive the midgut of embryos older than 92 hours was almost completed and X-irradiation caused slight damage. No significant histological damage was observed in the midgut of embryos older than 104 hours even after the treatment with a high dose of X-rays (4.0 or 6.0 KR). The sensitivity of the midgut to X-rays correlated with the embryonic lethality. Whether the midgut is a principal target of radiation was discussed. (author)

  8. Tracking the mechanical dynamics of human embryonic stem cell chromatin

    Directory of Open Access Journals (Sweden)

    Hinde Elizabeth

    2012-12-01

    Full Text Available Abstract Background A plastic chromatin structure has emerged as fundamental to the self-renewal and pluripotent capacity of embryonic stem (ES cells. Direct measurement of chromatin dynamics in vivo is, however, challenging as high spatiotemporal resolution is required. Here, we present a new tracking-based method which can detect high frequency chromatin movement and quantify the mechanical dynamics of chromatin in live cells. Results We use this method to study how the mechanical properties of chromatin movement in human embryonic stem cells (hESCs are modulated spatiotemporally during differentiation into cardiomyocytes (CM. Notably, we find that pluripotency is associated with a highly discrete, energy-dependent frequency of chromatin movement that we refer to as a ‘breathing’ state. We find that this ‘breathing’ state is strictly dependent on the metabolic state of the cell and is progressively silenced during differentiation. Conclusions We thus propose that the measured chromatin high frequency movements in hESCs may represent a hallmark of pluripotency and serve as a mechanism to maintain the genome in a transcriptionally accessible state. This is a result that could not have been observed without the high spatial and temporal resolution provided by this novel tracking method.

  9. Effects of simulated microgravity on embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Yulan Wang

    Full Text Available There have been many studies on the biological effects of simulated microgravity (SMG on differentiated cells or adult stem cells. However, there has been no systematic study on the effects of SMG on embryonic stem (ES cells. In this study, we investigated various effects (including cell proliferation, cell cycle distribution, cell differentiation, cell adhesion, apoptosis, genomic integrity and DNA damage repair of SMG on mouse embryonic stem (mES cells. Mouse ES cells cultured under SMG condition had a significantly reduced total cell number compared with cells cultured under 1 g gravity (1G condition. However, there was no significant difference in cell cycle distribution between SMG and 1G culture conditions, indicating that cell proliferation was not impaired significantly by SMG and was not a major factor contributing to the total cell number reduction. In contrast, a lower adhesion rate cultured under SMG condition contributed to the lower cell number in SMG. Our results also revealed that SMG alone could not induce DNA damage in mES cells while it could affect the repair of radiation-induced DNA lesions of mES cells. Taken together, mES cells were sensitive to SMG and the major alterations in cellular events were cell number expansion, adhesion rate decrease, increased apoptosis and delayed DNA repair progression, which are distinct from the responses of other types of cells to SMG.

  10. Fusion of Epstein-Barr virus nuclear antigen-1-derived glycine-alanine repeat to trans-dominant HIV-1 Gag increases inhibitory activities and survival of transduced cells in vivo.

    Science.gov (United States)

    Hammer, Diana; Wild, Jens; Ludwig, Christine; Asbach, Benedikt; Notka, Frank; Wagner, Ralf

    2008-06-01

    Trans-dominant human immunodeficiency virus type 1 (HIV-1) Gag derivatives have been shown to efficiently inhibit late steps of HIV-1 replication in vitro by interfering with Gag precursor assembly, thus ranking among the interesting candidates for gene therapy approaches. However, efficient antiviral activities of corresponding transgenes are likely to be counteracted in particular by cell-mediated host immune responses toward the transgene-expressing cells. To decrease this potential immunogenicity, a 24-amino acid Gly-Ala (GA) stretch derived from Epstein-Barr virus nuclear antigen-1 (EBNA1) and known to overcome proteasomal degradation was fused to a trans-dominant Gag variant (sgD1). To determine the capacity of this fusion polypeptide to repress viral replication, PM-1 cells were transduced with sgD1 and GAsgD1 transgenes, using retroviral gene transfer. Challenge of stably transfected permissive cell lines with various viral strains indicated that N-terminal GA fusion even enhanced the inhibitory properties of sgD1. Further studies revealed that the GA stretch increased protein stability by blocking proteasomal degradation of Gag proteins. Immunization of BALB/c mice with a DNA vaccine vector expressing sgD1 induced substantial Gag-specific immune responses that were, however, clearly diminished in the presence of GA. Furthermore, recognition of cells expressing the GA-fused transgene by CD8(+) T cells was drastically reduced, both in vitro and in vivo, resulting in prolonged survival of the transduced cells in recipient mice.

  11. MG132 plus apoptosis antigen-1 (APO-1) antibody cooperate to restore p53 activity inducing autophagy and p53-dependent apoptosis in HPV16 E6-expressing keratinocytes.

    Science.gov (United States)

    Lagunas-Martínez, Alfredo; García-Villa, Enrique; Arellano-Gaytán, Magaly; Contreras-Ochoa, Carla O; Dimas-González, Jisela; López-Arellano, María E; Madrid-Marina, Vicente; Gariglio, Patricio

    2017-01-01

    The E6 oncoprotein can interfere with the ability of infected cells to undergo programmed cell death through the proteolytic degradation of proapoptotic proteins such as p53, employing the proteasome pathway. Therefore, inactivation of the proteasome through MG132 should restore the activity of several proapoptotic proteins. We investigated whether in HPV16 E6-expressing keratinocytes (KE6 cells), the restoration of p53 levels mediated by MG132 and/or activation of the CD95 pathway through apoptosis antigen-1 (APO-1) antibody are responsible for the induction of apoptosis. We found that KE6 cells underwent apoptosis mainly after incubation for 24 h with MG132 alone or APO-1 plus MG132. Both treatments activated the extrinsic and intrinsic apoptosis pathways. Autophagy was also activated, principally by APO-1 plus MG132. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in the elevation of p53 protein levels and its phosphorylation in Ser46 and Ser20; the p53 protein was localized mainly at nucleus after treatment with MG132 or APO-1 plus MG132. In addition, induction of its transcriptional target genes such as p21, Bax and TP53INP was observed 3 and 6 h after treatment. Also, LC3 mRNA was induced after 3 and 6 h, which correlates with lipidation of LC3B protein and induction of autophagy. Finally, using pifithrin alpha we observed a decrease in apoptosis induced by MG132, and by APO-1 plus MG132, suggesting that restoration of APO-1 sensitivity occurs in part through an increase in both the levels and the activity of p53. The use of small molecules to inhibit the proteasome pathway might permit the activation of cell death, providing new opportunities for CC treatment.

  12. Molecular fingerprinting of TGFbeta-treated embryonic maxillary mesenchymal cells.

    Science.gov (United States)

    Pisano, M M; Mukhopadhyay, P; Greene, R M

    2003-11-01

    The transforming growth factor-beta (TGF(beta)) family represents a class of signaling molecules that plays a central role in normal embryonic development, specifically in development of the craniofacial region. Members of this family are vital to development of the secondary palate where they regulate maxillary and palate mesenchymal cell proliferation and extracellular matrix synthesis. The function of this growth factor family is particularly critical in that perturbation of either process results in a cleft of the palate. While the cellular and phenotypic effects of TGF(beta) on embryonic craniofacial tissue have been extensively cataloged, the specific genes that function as downstream mediators of TGF(beta) in maxillary/palatal development are poorly defined. Gene expression arrays offer the ability to conduct a rapid, simultaneous assessment of hundreds to thousands of differentially expressed genes in a single study. Inasmuch as the downstream sequelae of TGF(beta) action are only partially defined, a complementary DNA (cDNA) expression array technology (Clontech's Atlas Mouse cDNA Expression Arrays), was utilized to delineate a profile of differentially expressed genes from TGF(beta)-treated primary cultures of murine embryonic maxillary mesenchymal cells. Hybridization of a membrane-based cDNA array (1178 genes) was performed with 32P-labeled cDNA probes synthesized from RNA isolated from either TGF(beta)-treated or vehicle-treated embryonic maxillary mesenchymal cells. Resultant phosphorimages were subject to AtlasImage analysis in order to determine differences in gene expression between control and TGF(beta)-treated maxillary mesenchymal cells. Of the 1178 arrayed genes, 552 (47%) demonstrated detectable levels of expression. Steady state levels of 22 genes were up-regulated, while those of 8 other genes were down-regulated, by a factor of twofold or greater in response to TGF(beta). Affected genes could be grouped into three general functional

  13. LIN28A immunoreactivity is a potent diagnostic marker of embryonal tumor with multilayered rosettes (ETMR)

    NARCIS (Netherlands)

    Korshunov, Andrey; Ryzhova, Marina; Jones, David T. W.; Northcott, Paul A.; van Sluis, Peter; Volckmann, Richard; Koster, Jan; Versteeg, Rogier; Cowdrey, Cynthia; Perry, Arie; Picard, Daniel; Rosenblum, Marc; Giangaspero, Felice; Aronica, Eleonora; Schüller, Ulrich; Hasselblatt, Martin; Collins, V. Peter; von Deimling, Andreas; Lichter, Peter; Huang, Annie; Pfister, Stefan M.; Kool, Marcel

    2012-01-01

    Embryonal tumor with multilayered rosettes (ETMR, previously known as ETANTR) is a highly aggressive embryonal CNS tumor, which almost exclusively affects infants and is associated with a dismal prognosis. Accurate diagnosis is of critical clinical importance because of its poor response to current

  14. Case Study: Organotypic human in vitro models of embryonic morphogenetic fusion

    Science.gov (United States)

    Morphogenetic fusion of tissues is a common event in embryonic development and disruption of fusion is associated with birth defects of the eye, heart, neural tube, phallus, palate, and other organ systems. Embryonic tissue fusion requires precise regulation of cell-cell and cell...

  15. Influence of muscular activity on local mineralization patterns in metatarsals of the embryonic mouse

    NARCIS (Netherlands)

    Tanck, E.; Blankevoort, L.; Haaijman, A.; Burger, E. H.; Huiskes, R.

    2000-01-01

    This study addressed the theory that local mechanical loading may influence the development of embryonic long bones. Embryonic mouse metatarsal rudiments were cultured as whole organs, and the geometry of the primary ossification center was compared with that of rudiments that had developed in

  16. Enhanced Differentiation of Human Embryonic Stem Cells Toward Definitive Endoderm on Ultrahigh Aspect Ratio Nanopillars

    DEFF Research Database (Denmark)

    Rasmussen, Camilla Holzmann; Reynolds, Paul M.; Petersen, Dorthe Roenn

    2016-01-01

    Differentiation of human embryonic stem cells is widely studied as a potential unlimited source for cell replacement therapy to treat degenerative diseases such as diabetes. The directed differentiation of human embryonic stem cells relies mainly on soluble factors. Although, some studies have hi...

  17. Undifferentiated Embryonic Cell Transcription Factor 1 Regulates ESC Chromatin Organization and Gene Expression

    NARCIS (Netherlands)

    Kooistra, Susanne M.; van den Boom, Vincent; Thummer, Rajkumar P.; Johannes, Frank; Wardenaar, Rene; Tesson, Bruno M.; Veenhoff, Liesbeth M.; Fusetti, Fabrizia; O'Neill, Laura P.; Turner, Bryan M.; de Haan, Gerald; Eggen, Bart J. L.; O’Neill, Laura P.

    2010-01-01

    Previous reports showed that embryonic stem (ES) cells contain hyperdynamic and globally transcribed chromatin-properties that are important for ES cell pluripotency and differentiation. Here, we demonstrate a role for undifferentiated embryonic cell transcription factor 1 (UTF1) in regulating ES

  18. Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1

    DEFF Research Database (Denmark)

    Robert-Moreno, Àlex; Robert-Moreno, Àlex; Guiu, Jordi

    2008-01-01

    Specific deletion of Notch1 and RBPjκ in the mouse results in abrogation of definitive haematopoiesis concomitant with the loss of arterial identity at embryonic stage. As prior arterial determination is likely to be required for the generation of embryonic haematopoiesis, it is difficult...

  19. Gro/TLE enables embryonic stem cell differentiation by repressing pluripotent gene expression

    DEFF Research Database (Denmark)

    Laing, Adam F; Lowell, Sally; Brickman, Joshua M

    2015-01-01

    Gro/TLE proteins (TLE1-4) are a family of transcriptional corepressors acting downstream of multiple signalling pathways. Several TLEs are expressed in a dynamic manner throughout embryonic development and at high levels in embryonic stem cells (ESCs). Here we find that Gro/TLE is not required...

  20. Childhood Central Nervous System Embryonal Tumors Treatment (PDQ®)—Patient Version

    Science.gov (United States)

    Childhood embryonal tumors (medulloblastoma and nonmedulloblastoma) of the brain and spinal cord are treated with surgery, radiation therapy, and chemotherapy. Get information about symptoms, tests to diagnose, prognosis, and treatment of newly diagnosed and recurrent childhood embryonal tumors, in this expert-reviewed summary.

  1. 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...

  2. [Effects of different culture system of isolating and passage of sheep embryonic stem-like cells].

    Science.gov (United States)

    Bai, Changming; Liu, Chousheng; Wang, Zhigang; Wang, Xinzhuang

    2008-07-01

    In this research, we use mouse embryonic fibroblasts as feeder layers. To eliminate the influence of serum and mouse embryonic stem cells (ESCs) conditioned medium (ESCCM) on self-renewal of sheep embryonic stem-like cells, knockout serum replacement (KSR) was used to replace serum, then supplanted with ESCCM for the isolation and cloning of sheep embryonic stem-like cells. We found when inner cell masses (ICMs) cultured in the control group with medium supplanted with fetal bovine serum (FBS), sheep ES-like cells could not survive for more than 3 passages. However, sheep embryonic stem-like cells could remain undifferentiated for 5 passages when cultured in the medium that FBS was substituted by KSR. The result indicates that KSR culture system was more suitable for the isolation and cloning of sheep embryonic stem-like cells compared to FBS culture system. Finally we applied medium with 15% KSR as basic medium supplanted with 40% ESCCM as a new culture system to isolate sheep embryonic stem-like cells, we found one embryonic stem-like cell line still maintained undifferentiating for 8 passages, which characterized with a normal and stable karyotype and high expression of alkaline phosphatase. These results suggest that it is suitable to culture sheep ICM in the new culture system with 15% KSR as basic medium and supplanted with 40% ESCCM, which indicated that mouse ES cells might secrete factors playing important roles in promoting sheep ES-like cells' self-renewal.

  3. Embryonic development rates of northern grasshoppers (Orthoptera: Acrididae): implications for climate change and habitat management

    Science.gov (United States)

    Temperature-dependent rates of embryonic development are a primary determinant of the life cycle of many species of grasshoppers which, in cold climates, spend two winters in the egg stage. Knowledge of embryonic developmental rates is important for an assessment of the effects of climate change and...

  4. Diabetes-induced effects on cardiomyocytes in chick embryonic heart micromass and mouse embryonic D3 differentiated stem cells.

    Science.gov (United States)

    Mohammed, Omar J; Latif, Muhammad Liaque; Pratten, Margaret K

    2017-04-01

    Diabetes mellitus during pregnancy is a considerable medical challenge, since it is related to ‎augmented morbidity and mortality concerns for both the fetus ‎and the pregnant woman. Records show that the etiology of diabetic ‎embryopathy is complicated, as many teratological factors might be involved ‎in the mechanisms of diabetes mellitus-induced congenital malformation. ‎In this study, the potential cardiotoxic effect of hyperglycemia with hyperketonemia was investigated by using two in vitro models; primary chick embryonic cardiomyocytes and stem cell derived cardiomyocytes, where adverse effects were recorded in both systems. The cells were evaluated by changes in beating activity, cell activity, protein content, ROS production, DNA damage and differentiating stem cell migration. The diabetic formulae used produced an increase in DNA damage and a decline in cell migration in mouse embryonic stem cells. These results provide an additional insight into adverse effects during gestational diabetes mellitus and a recommendation for expectant mothers and maternity staff to monitor glycaemic levels months ahead of conception. This study also supports the recommendation of using antioxidants during pregnancy to prevent DNA damage by the production of ROS, which might result in heart defects as well as other developmental anomalies. Copyright © 2017. Published by Elsevier Inc.

  5. 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

  6. Embryonic pig liver, pancreas, and lung as a source for transplantation: Optimal organogenesis without teratoma depends on distinct time windows

    OpenAIRE

    Eventov-Friedman, Smadar; Katchman, Helena; Shezen, Elias; Aronovich, Anna; Tchorsh, Dalit; Dekel, Benjamin; Freud, Enrique; Reisner, Yair

    2005-01-01

    Pig embryonic tissues represent an attractive option for organ transplantation. However, the achievement of optimal organogenesis after transplantation, namely, maximal organ growth and function without teratoma development, represents a major challenge. In this study, we determined distinct gestational time windows for the growth of pig embryonic liver, pancreas, and lung precursors. Transplantation of embryonic-tissue precursors at various gestational ages [from E (embryonic day) 21 to E100...

  7. Gene-manipulated embryonic stem cells for rat transgenesis.

    Science.gov (United States)

    Kawamata, Masaki; Ochiya, Takahiro

    2011-06-01

    Embryonic stem cells (ESCs) are derived from blastocysts and are capable of differentiating into whole tissues and organs. Transplantation of ESCs into recipient blastocysts leads to the generation of germline-competent chimeras in mice. Transgenic, knockin, and knockout gene manipulations are available in mouse ESCs, enabling the production of genetically modified animals. Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. However, in contrast to mouse ESCs, rat ESCs were not established until 2008 because of the difficulty of maintaining pluripotency. Although the use of signaling inhibitors has allowed the generation of rat ESCs, the production of genetically modified rats has been difficult due to problems in rat ESCs after gene introduction. In this review, we will focus on some well-documented examples of gene manipulation in rat ESCs.

  8. Auxetic nuclei in embryonic stem cells exiting pluripotency

    Science.gov (United States)

    Pagliara, Stefano; Franze, Kristian; McClain, Crystal R.; Wylde, George W.; Fisher, Cynthia L.; Franklin, Robin J. M.; Kabla, Alexandre J.; Keyser, Ulrich F.; Chalut, Kevin J.

    2014-06-01

    Embryonic stem cells (ESCs) self-renew in a state of naïve pluripotency in which they are competent to generate all somatic cells. It has been hypothesized that, before irreversibly committing, ESCs pass through at least one metastable transition state. This transition would represent a gateway for differentiation and reprogramming of somatic cells. Here, we show that during the transition, the nuclei of ESCs are auxetic: they exhibit a cross-sectional expansion when stretched and a cross-sectional contraction when compressed, and their stiffness increases under compression. We also show that the auxetic phenotype of transition ESC nuclei is driven at least in part by global chromatin decondensation. Through the regulation of molecular turnover in the differentiating nucleus by external forces, auxeticity could be a key element in mechanotransduction. Our findings highlight the importance of nuclear structure in the regulation of differentiation and reprogramming.

  9. Distinct Regulatory Mechanisms Govern Embryonic versus Adult Adipocyte Maturation

    Science.gov (United States)

    Wang, Qiong A.; Tao, Caroline; Jiang, Lei; Shao, Mengle; Ye, Risheng; Zhu, Yi; Gordillo, Ruth; Ali, Aktar; Lian, Yun; Holland, William L.; Gupta, Rana K.; Scherer, Philipp E.

    2015-01-01

    Pathological expansion of adipose tissue contributes to the metabolic syndrome. Distinct depots develop at various times under different physiological conditions. The transcriptional cascade mediating adipogenesis is established in vitro, and centers around a core program involving PPARγ and C/EBPα. We developed an inducible, adipocyte-specific knockout system to probe the requirement of key adipogenic transcription factors at various stages of adipogenesis in vivo. C/EBPα is essential for all white adipogenic conditions in the adult stage, such as adipose tissue regeneration, adipogenesis in muscle and unhealthy expansion of white adipose tissue during high fat feeding or due to leptin deficiency. Surprisingly, terminal embryonic adipogenesis is fully C/EBPα independent, does depend however on PPARγ; cold-induced beige adipogenesis is also C/EBPα independent. Moreover, C/EBPα is not vital for adipocyte survival in the adult stage. We reveal a surprising diversity of transcriptional signals required at different stages of adipogenesis in vivo. PMID:26280538

  10. Molecular mechanisms controlling the cell cycle in embryonic stem cells.

    Science.gov (United States)

    Abdelalim, Essam M

    2013-12-01

    Embryonic stem (ES) cells are originated from the inner cell mass of a blastocyst stage embryo. They can proliferate indefinitely, maintain an undifferentiated state (self-renewal), and differentiate into any cell type (pluripotency). ES cells have an unusual cell cycle structure, consists mainly of S phase cells, a short G1 phase and absence of G1/S checkpoint. Cell division and cell cycle progression are controlled by mechanisms ensuring the accurate transmission of genetic information from generation to generation. Therefore, control of cell cycle is a complicated process, involving several signaling pathways. Although great progress has been made on the molecular mechanisms involved in the regulation of ES cell cycle, many regulatory mechanisms remain unknown. This review summarizes the current knowledge about the molecular mechanisms regulating the cell cycle of ES cells and describes the relationship existing between cell cycle progression and the self-renewal.

  11. The role of flow in the morphodynamics of embryonic heart

    Science.gov (United States)

    Gharib, Morteza

    2017-11-01

    Nature has shown us that some hearts do not require valves to achieve unidirectional flow. In its earliest stages, the vertebrate heart consists of a primitive tube that drives blood through a simple vascular network nourishing tissues and other developing organ systems. We have shown that in the case of the embryonic zebrafish heart, an elastic wave resonance mechanism based on impedance mismatches at the boundaries of the heart tube is the likely mechanism responsible for the valveless pumping behavior. When functioning normally, mature heart valves prevent intracardiac retrograde blood flow; before valves develop there is considerable regurgitation, resulting in oscillatory flow between the atrium and ventricle. We show that reversing flows are particularly strong stimuli to endothelial cells and that heart valves form as a developmental response to oscillatory blood flow through the maturing heart.

  12. Embryonic transcriptome of the brackishwater amphipod Gammarus chevreuxi.

    Science.gov (United States)

    Truebano, Manuela; Tills, Oliver; Spicer, John I

    2016-08-01

    Environmental change can dramatically alter the development of aquatic organisms. While the effect of such change on physiological and morphological ontogenies is becoming clearer, the molecular mechanisms underpinning them are largely unexplored. Characterizing these mechanisms is often limited by the lack of molecular resources. We have applied Illumina HiSeq sequencing to RNA isolated from different developmental stages of the brackishwater amphipod Gammarus chevreuxi. Over 52.6M paired-end reads were assembled de novo into 172,081 contigs, representing 118,812 potential genes. The assembly generated constitutes a reference embryonic transcriptome for an ecologically-important aquatic shredder species. This resource will contribute to our understanding of the mechanisms underpinning the development of physiological function through functional, comparative and quantitative expression studies. It will also allow the identification of candidate biomarkers for assessing the impact of environmental stressors in estuarine systems. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Transcription pausing regulates mouse embryonic stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Melodi Tastemel

    2017-12-01

    Full Text Available The pluripotency of embryonic stem cells (ESCs relies on appropriate responsiveness to developmental cues. Promoter-proximal pausing of RNA polymerase II (Pol II has been suggested to play a role in keeping genes poised for future activation. To identify the role of Pol II pausing in regulating ESC pluripotency, we have generated mouse ESCs carrying a mutation in the pause-inducing factor SPT5. Genomic studies reveal genome-wide reduction of paused Pol II caused by mutant SPT5 and further identify a tight correlation between pausing-mediated transcription effect and local chromatin environment. Functionally, this pausing-deficient SPT5 disrupts ESC differentiation upon removal of self-renewal signals. Thus, our study uncovers an important role of Pol II pausing in regulating ESC differentiation and suggests a model that Pol II pausing coordinates with epigenetic modification to influence transcription during mESC differentiation.

  14. Embryonic stem cells and the challenge of transplantation tolerance.

    Science.gov (United States)

    Fairchild, Paul J; Cartland, Siân; Nolan, Kathleen F; Waldmann, Herman

    2004-09-01

    Isolated from early blastocysts, embryonic stem (ES) cells capture the brief moment of pluripotency in the developing embryo, as evidenced by their differentiation into many somatic cell types in vitro. Although these properties might help meet the growing demand for 'spare parts' to replace diseased or worn-out tissues, their use in so-called cell replacement therapy (CRT) poses several challenges, not least of which is the prevention of their subsequent rejection. Here we explore the notion that ES cells might spawn cell types necessary for the treatment of disease while acting as a plentiful source of hematopoietic stem cells (HSCs) or terminally differentiated dendritic cells (DCs) that might facilitate the induction of transplantation tolerance to the replacement tissues.

  15. Quantitative analysis of chromatin accessibility in mouse embryonic fibroblasts.

    Science.gov (United States)

    Zhuo, Baowen; Yu, Juan; Chang, Luyuan; Lei, Jiafan; Wen, Zengqi; Liu, Cuifang; Mao, Guankun; Wang, Kehui; Shen, Jie; Xu, Xueqing

    2017-11-04

    Genomic DNA of eukaryotic cells is hierarchically packaged into chromatin by histones. The dynamic organization of chromatin fibers plays a critical role in the regulation of gene transcription and other DNA-associated biological processes. Recently, numerous approaches have been developed to map the chromatin organization by characterizing chromatin accessibilities in genome-wide. However, reliable methods to quantitatively map chromatin accessibility are not well-established, especially not on a genome-wide scale. Here, we developed a modified MNase-seq for mouse embryonic fibroblasts, wherein chromatin was partially digested at multiple digestion times using micrococcal nuclease (MNase), allowing quantitative analysis of local yet genome-wide chromatin compaction. Our results provide strong evidence that the chromatin accessibility at promoter regions are positively correlated with gene activity. In conclusion, our assay is an ideal tool for the quantitative study of gene regulation in the perspective of chromatin accessibility. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. GATA-1 directly regulates Nanog in mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Wen-Zhong; Ai, Zhi-Ying [College of Life Sciences, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Wang, Zhi-Wei [School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui 230027 (China); Chen, Lin-Lin [College of Life Sciences, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Guo, Ze-Kun, E-mail: gzknwaf@126.com [College of Veterinary Medicine, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Zhang, Yong, E-mail: zylabnwaf@126.com [College of Veterinary Medicine, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China)

    2015-09-25

    Nanog safeguards pluripotency in mouse embryonic stem cells (mESCs). Insight into the regulation of Nanog is important for a better understanding of the molecular mechanisms that control pluripotency of mESCs. In a silico analysis, we identify four GATA-1 putative binding sites in Nanog proximal promoter. The Nanog promoter activity can be significantly repressed by ectopic expression of GATA-1 evidenced by a promoter reporter assay. Mutation studies reveal that one of the four putative binding sites counts for GATA-1 repressing Nanog promoter activity. Direct binding of GATA-1 on Nanog proximal promoter is confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Our data provide new insights into the expanded regulatory circuitry that coordinates Nanog expression. - Highlights: • The Nanog proximal promoter conceives functional element for GATA-1. • GATA-1 occupies the Nanog proximal promoter in vitro and in vivo. • GATA-1 transcriptionally suppresses Nanog.

  17. Raman microscopy of individual living human embryonic stem cells

    DEFF Research Database (Denmark)

    Novikov, Sergey M.; Beermann, Jonas; Bozhevolnyi, Sergey I.

    2010-01-01

    We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing...... cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal...... scanning Raman microscope (Alpha300R) from Witec and sub-μm spatially resolved Raman images were obtained using a 532 nm excitation wavelength....

  18. Evolution of the mechanisms that establish the embryonic axes.

    Science.gov (United States)

    Stern, Claudio D

    2006-08-01

    A long-standing debate in developmental biology concerns the extent to which embryos are largely 'mosaic' (cell fates are allocated by localization of maternal determinants that are inherited differentially) or 'regulative' (cell interactions determine cell fates). Generally, it has been thought that amniotes, especially birds and mammals, are at the extreme regulative end of the spectrum, whereas most invertebrates, lower chordates and anamnia are more mosaic. Various studies have identified additional differences, including egg size, the timing of zygotic transcription and the speed of development. However, new research is starting to reveal among the vertebrate classes an astonishing degree of conservation in the intercellular signalling mechanisms that regulate cell fate and embryonic polarity before gastrulation.

  19. Incubation times of dinosaur eggs via embryonic metabolism

    Science.gov (United States)

    Lee, Scott A.

    2016-08-01

    The incubation times for the eggs of 21 dinosaurs are determined from an estimate of their embyronic metabolic rate and the mass of the hatchlings via a mass growth model based on conservation of energy. Embryos in extant birds and crocodiles are studied in order to determine the best model for embryonic metabolism and growth. These results are used to develop a theoretical model that predicts the incubation times of an egg. This model is applied to dinosaur eggs and provides a unique window into dinosaur reproduction. The dinosaurs studied come from both Saurischia and Ornithischia. The incubation times vary from about 28 days for Archaeopteryx lithographica to about 76 days for Alamosaurus sanjuanensis.

  20. 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....

  1. Dynamic heterogeneity and DNA methylation in embryonic stem cells.

    KAUST Repository

    Singer, Zakary S

    2014-07-01

    Cell populations can be strikingly heterogeneous, composed of multiple cellular states, each exhibiting stochastic noise in its gene expression. A major challenge is to disentangle these two types of variability and to understand the dynamic processes and mechanisms that control them. Embryonic stem cells (ESCs) provide an ideal model system to address this issue because they exhibit heterogeneous and dynamic expression of functionally important regulatory factors. We analyzed gene expression in individual ESCs using single-molecule RNA-FISH and quantitative time-lapse movies. These data discriminated stochastic switching between two coherent (correlated) gene expression states and burst-like transcriptional noise. We further showed that the "2i" signaling pathway inhibitors modulate both types of variation. Finally, we found that DNA methylation plays a key role in maintaining these metastable states. Together, these results show how ESC gene expression states and dynamics arise from a combination of intrinsic noise, coherent cellular states, and epigenetic regulation.

  2. The Design Space of the Embryonic Cell Cycle Oscillator.

    Science.gov (United States)

    Mattingly, Henry H; Sheintuch, Moshe; Shvartsman, Stanislav Y

    2017-08-08

    One of the main tasks in the analysis of models of biomolecular networks is to characterize the domain of the parameter space that corresponds to a specific behavior. Given the large number of parameters in most models, this is no trivial task. We use a model of the embryonic cell cycle to illustrate the approaches that can be used to characterize the domain of parameter space corresponding to limit cycle oscillations, a regime that coordinates periodic entry into and exit from mitosis. Our approach relies on geometric construction of bifurcation sets, numerical continuation, and random sampling of parameters. We delineate the multidimensional oscillatory domain and use it to quantify the robustness of periodic trajectories. Although some of our techniques explore the specific features of the chosen system, the general approach can be extended to other models of the cell cycle engine and other biomolecular networks. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. Cryoconservation du sperme et des embryons de poissons

    OpenAIRE

    Maisse, Gérard; Labbé, Catherine; Ogier de Baulny, Bénédicte; Leveroni Calvi, Sylvia; Haffray, Pierrick

    1998-01-01

    Le développement des programmes de sélection génétique en pisciculture et la protection de la biodiversité de l’ichtyofaune sauvage justifient la création de cryo-banques de sperme et d’embryons de poissons. Les travaux sur la formulation des dilueurs de congélation montrent que l’on doit tenir compte à la fois de l’espèce cible, du type cellulaire concerné et des interactions entre les différents composants du dilueur. L’aptitude à la cryoconservation du sperme est très variable suivant les ...

  4. 78 FR 25091 - Submission for OMB Review; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To...

    Science.gov (United States)

    2013-04-29

    ...; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be Approved for Use in NIH... Embryonic Stem Cell Line to be Approved for Use in NIH-Funded Research, 0925-0601, Expiration Date 04/30... Information Collection: The form is used by applicants to request that human embryonic stem cell lines be...

  5. Reidentification of avian embryonic remains from the cretaceous of mongolia.

    Directory of Open Access Journals (Sweden)

    David J Varricchio

    Full Text Available Embryonic remains within a small (4.75 by 2.23 cm egg from the Late Cretaceous, Mongolia are here re-described. High-resolution X-ray computed tomography (HRCT was used to digitally prepare and describe the enclosed embryonic bones. The egg, IGM (Mongolian Institute for Geology, Ulaanbaatar 100/2010, with a three-part shell microstructure, was originally assigned to Neoceratopsia implying extensive homoplasy among eggshell characters across Dinosauria. Re-examination finds the forelimb significantly longer than the hindlimbs, proportions suggesting an avian identification. Additional, postcranial apomorphies (strut-like coracoid, cranially located humeral condyles, olecranon fossa, slender radius relative to the ulna, trochanteric crest on the femur, and ulna longer than the humerus identify the embryo as avian. Presence of a dorsal coracoid fossa and a craniocaudally compressed distal humerus with a strongly angled distal margin support a diagnosis of IGM 100/2010 as an enantiornithine. Re-identification eliminates the implied homoplasy of this tri-laminate eggshell structure, and instead associates enantiornithine birds with eggshell microstructure composed of a mammillary, squamatic, and external zones. Posture of the embryo follows that of other theropods with fore- and hindlimbs folded parallel to the vertebral column and the elbow pointing caudally just dorsal to the knees. The size of the egg and embryo of IGM 100/2010 is similar to the two other Mongolian enantiornithine eggs. Well-ossified skeletons, as in this specimen, characterize all known enantiornithine embryos suggesting precocial hatchlings, comparing closely to late stage embryos of modern precocial birds that are both flight- and run-capable upon hatching. Extensive ossification in enantiornithine embryos may contribute to their relatively abundant representation in the fossil record. Neoceratopsian eggs remain unrecognized in the fossil record.

  6. Embryonic morphogen nodal promotes breast cancer growth and progression.

    Directory of Open Access Journals (Sweden)

    Daniela F Quail

    Full Text Available Breast cancers expressing human embryonic stem cell (hESC-associated genes are more likely to progress than well-differentiated cancers and are thus associated with poor patient prognosis. Elevated proliferation and evasion of growth control are similarly associated with disease progression, and are classical hallmarks of cancer. In the current study we demonstrate that the hESC-associated factor Nodal promotes breast cancer growth. Specifically, we show that Nodal is elevated in aggressive MDA-MB-231, MDA-MB-468 and Hs578t human breast cancer cell lines, compared to poorly aggressive MCF-7 and T47D breast cancer cell lines. Nodal knockdown in aggressive breast cancer cells via shRNA reduces tumour incidence and significantly blunts tumour growth at primary sites. In vitro, using Trypan Blue exclusion assays, Western blot analysis of phosphorylated histone H3 and cleaved caspase-9, and real time RT-PCR analysis of BAX and BCL2 gene expression, we demonstrate that Nodal promotes expansion of breast cancer cells, likely via a combinatorial mechanism involving increased proliferation and decreased apopotosis. In an experimental model of metastasis using beta-glucuronidase (GUSB-deficient NOD/SCID/mucopolysaccharidosis type VII (MPSVII mice, we show that although Nodal is not required for the formation of small (<100 cells micrometastases at secondary sites, it supports an elevated proliferation:apoptosis ratio (Ki67:TUNEL in micrometastatic lesions. Indeed, at longer time points (8 weeks, we determined that Nodal is necessary for the subsequent development of macrometastatic lesions. Our findings demonstrate that Nodal supports tumour growth at primary and secondary sites by increasing the ratio of proliferation:apoptosis in breast cancer cells. As Nodal expression is relatively limited to embryonic systems and cancer, this study establishes Nodal as a potential tumour-specific target for the treatment of breast cancer.

  7. Embryonic morphogen nodal promotes breast cancer growth and progression.

    Science.gov (United States)

    Quail, Daniela F; Zhang, Guihua; Walsh, Logan A; Siegers, Gabrielle M; Dieters-Castator, Dylan Z; Findlay, Scott D; Broughton, Heather; Putman, David M; Hess, David A; Postovit, Lynne-Marie

    2012-01-01

    Breast cancers expressing human embryonic stem cell (hESC)-associated genes are more likely to progress than well-differentiated cancers and are thus associated with poor patient prognosis. Elevated proliferation and evasion of growth control are similarly associated with disease progression, and are classical hallmarks of cancer. In the current study we demonstrate that the hESC-associated factor Nodal promotes breast cancer growth. Specifically, we show that Nodal is elevated in aggressive MDA-MB-231, MDA-MB-468 and Hs578t human breast cancer cell lines, compared to poorly aggressive MCF-7 and T47D breast cancer cell lines. Nodal knockdown in aggressive breast cancer cells via shRNA reduces tumour incidence and significantly blunts tumour growth at primary sites. In vitro, using Trypan Blue exclusion assays, Western blot analysis of phosphorylated histone H3 and cleaved caspase-9, and real time RT-PCR analysis of BAX and BCL2 gene expression, we demonstrate that Nodal promotes expansion of breast cancer cells, likely via a combinatorial mechanism involving increased proliferation and decreased apopotosis. In an experimental model of metastasis using beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII (MPSVII) mice, we show that although Nodal is not required for the formation of small (apoptosis ratio (Ki67:TUNEL) in micrometastatic lesions. Indeed, at longer time points (8 weeks), we determined that Nodal is necessary for the subsequent development of macrometastatic lesions. Our findings demonstrate that Nodal supports tumour growth at primary and secondary sites by increasing the ratio of proliferation:apoptosis in breast cancer cells. As Nodal expression is relatively limited to embryonic systems and cancer, this study establishes Nodal as a potential tumour-specific target for the treatment of breast cancer.

  8. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    Directory of Open Access Journals (Sweden)

    Rajvi H Mehta

    2014-01-01

    Full Text Available The ability to successfully derive human embryonic stem cells (hESC lines from human embryos following in vitro fertilization (IVF opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. In case a couple does not desire to ′cryopreserve′ their embryos then all the embryos remaining following embryo transfer can be considered ′spare′ or if a couple is no longer in need of the ′cryopreserved′ embryos then these also can be considered as ′spare′. But, the question raised by the ethicists is, "what about ′slightly′ over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to ′discarded′ embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of ′discarding′ embryos. What would be the criteria for discarding embryos and the potential ′use′ of ESC derived from the ′abnormal appearing′ embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

  9. Dynamic expression of manganese superoxide dismutase during mouse embryonic organogenesis.

    Science.gov (United States)

    Yon, Jung-Min; Baek, In-Jeoung; Lee, Beom Jun; Yun, Young Won; Nam, Sang-Yoon

    2011-01-01

    The balance between reactive oxygen species production and antioxidant defense enzymes in embryos is necessary for normal embryogenesis. To determine the dynamic expression profile of manganese superoxide dismutase (MnSOD) in embryos, which is an essential antioxidant enzyme in embryonic organogenesis, the expression level and distribution of MnSOD mRNA and protein were investigated in mouse embryos, as well as extraembryonic tissues on embryonic days (EDs) 7.5-18.5. MnSOD mRNA levels were remarkably high in extraembryonic tissues rather than in embryos during these periods. MnSOD protein levels were also higher in extraembryonic tissues than in embryos until ED 16.5, but the opposite trend was found after ED 17.5. MnSOD mRNA was observed in the chorion, allantois, amnion, ectoderm, ectoplacental cone and neural fold at ED 7.5 and in the neural fold, gut, ectoplacental cone, outer extraembryonic membranes and primitive heart at ED 8.5. After removing the extraembryonic tissues, the prominent expression of MnSOD mRNA in embryos was seen in the sensory organs, central nervous system and limbs on EDs 9.5-12.5 and in the ganglia, spinal cord, sensory organ epithelia, lung, blood cells and vessels, intestinal and skin epithelia, hepatocytes and thymus on EDs 13.5-18.5. Strong MnSOD immunoreactivity was observed in the choroid plexus, ganglia, myocardium, blood vessels, heapatocytes, pancreatic acinus, osteogenic tissues, brown adipose tissue, thymus and skin. These findings suggest that MnSOD is mainly produced from extraembryonic tissues and then may be utilized to protect the embryos against endogenous or exogenous oxidative stress during embryogenesis.

  10. Human embryonic stem cells form functional thyroid follicles.

    Science.gov (United States)

    Ma, Risheng; Latif, Rauf; Davies, Terry F

    2015-04-01

    The molecular events that lead to human thyroid cell speciation remain incompletely characterized. It has been shown that overexpression of the regulatory transcription factors Pax8 and Nkx2-1 (ttf-1) directs murine embryonic stem (mES) cells to differentiate into thyroid follicular cells by initiating a transcriptional regulatory network. Such cells subsequently organized into three-dimensional follicular structures in the presence of extracellular matrix. In the current study, human embryonic stem (hES) cells were studied with the aim of recapitulating this scenario and producing functional human thyroid cell lines. Reporter gene tagged pEZ-lentiviral vectors were used to express human PAX8-eGFP and NKX2-1-mCherry in the H9 hES cell line followed by differentiation into thyroid cells directed by Activin A and thyrotropin (TSH). Both transcription factors were expressed efficiently in hES cells expressing either PAX8, NKX2-1, or in combination in the hES cells, which had low endogenous expression of these transcription factors. Further differentiation of the double transfected cells showed the expression of thyroid-specific genes, including thyroglobulin (TG), thyroid peroxidase (TPO), the sodium/iodide symporter (NIS), and the TSH receptor (TSHR) as assessed by reverse transcription polymerase chain reaction and immunostaining. Most notably, the Activin/TSH-induced differentiation approach resulted in thyroid follicle formation and abundant TG protein expression within the follicular lumens. On stimulation with TSH, these hES-derived follicles were also capable of dose-dependent cAMP generation and radioiodine uptake, indicating functional thyroid epithelial cells. The induced expression of PAX8 and NKX2-1 in hES cells was followed by differentiation into thyroid epithelial cells and their commitment to form functional three-dimensional neo-follicular structures. The data provide proof of principal that hES cells can be committed to thyroid cell speciation under

  11. Reidentification of avian embryonic remains from the cretaceous of mongolia.

    Science.gov (United States)

    Varricchio, David J; Balanoff, Amy M; Norell, Mark A

    2015-01-01

    Embryonic remains within a small (4.75 by 2.23 cm) egg from the Late Cretaceous, Mongolia are here re-described. High-resolution X-ray computed tomography (HRCT) was used to digitally prepare and describe the enclosed embryonic bones. The egg, IGM (Mongolian Institute for Geology, Ulaanbaatar) 100/2010, with a three-part shell microstructure, was originally assigned to Neoceratopsia implying extensive homoplasy among eggshell characters across Dinosauria. Re-examination finds the forelimb significantly longer than the hindlimbs, proportions suggesting an avian identification. Additional, postcranial apomorphies (strut-like coracoid, cranially located humeral condyles, olecranon fossa, slender radius relative to the ulna, trochanteric crest on the femur, and ulna longer than the humerus) identify the embryo as avian. Presence of a dorsal coracoid fossa and a craniocaudally compressed distal humerus with a strongly angled distal margin support a diagnosis of IGM 100/2010 as an enantiornithine. Re-identification eliminates the implied homoplasy of this tri-laminate eggshell structure, and instead associates enantiornithine birds with eggshell microstructure composed of a mammillary, squamatic, and external zones. Posture of the embryo follows that of other theropods with fore- and hindlimbs folded parallel to the vertebral column and the elbow pointing caudally just dorsal to the knees. The size of the egg and embryo of IGM 100/2010 is similar to the two other Mongolian enantiornithine eggs. Well-ossified skeletons, as in this specimen, characterize all known enantiornithine embryos suggesting precocial hatchlings, comparing closely to late stage embryos of modern precocial birds that are both flight- and run-capable upon hatching. Extensive ossification in enantiornithine embryos may contribute to their relatively abundant representation in the fossil record. Neoceratopsian eggs remain unrecognized in the fossil record.

  12. Sourcing human embryos for embryonic stem cell lines: problems & perspectives.

    Science.gov (United States)

    Mehta, Rajvi H

    2014-11-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been 'discarded' or 'spare' fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART) and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. in case a couple does not desire to 'cryopreserve' their embryos then all the embryos remaining following embryo transfer can be considered 'spare' or if a couple is no longer in need of the 'cryopreserved' embryos then these also can be considered as 'spare'. But, the question raised by the ethicists is, "what about 'slightly' over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to 'discarded' embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of 'discarding' embryos. What would be the criteria for discarding embryos and the potential 'use' of ESC derived from the 'abnormal appearing' embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

  13. Comparative metal oxide nanoparticle toxicity using embryonic zebrafish

    Directory of Open Access Journals (Sweden)

    Leah C. Wehmas

    2015-01-01

    Full Text Available Engineered metal oxide nanoparticles (MO NPs are finding increasing utility in the medical field as anticancer agents. Before validation of in vivo anticancer efficacy can occur, a better understanding of whole-animal toxicity is required. We compared the toxicity of seven widely used semiconductor MO NPs made from zinc oxide (ZnO, titanium dioxide, cerium dioxide and tin dioxide prepared in pure water and in synthetic seawater using a five-day embryonic zebrafish assay. We hypothesized that the toxicity of these engineered MO NPs would depend on physicochemical properties. Significant agglomeration of MO NPs in aqueous solutions is common making it challenging to associate NP characteristics such as size and charge with toxicity. However, data from our agglomerated MO NPs suggests that the elemental composition and dissolution potential are major drivers of toxicity. Only ZnO caused significant adverse effects of all MO particles tested, and only when prepared in pure water (point estimate median lethal concentration = 3.5–9.1 mg/L. This toxicity was life stage dependent. The 24 h toxicity increased greatly (∼22.7 fold when zebrafish exposures started at the larval life stage compared to the 24 h toxicity following embryonic exposure. Investigation into whether dissolution could account for ZnO toxicity revealed high levels of zinc ion (40–89% of total sample were generated. Exposure to zinc ion equivalents revealed dissolved Zn2+ may be a major contributor to ZnO toxicity.

  14. Histone demethylase JMJD5 is essential for embryonic development

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Sangphil [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 (United States); Janknecht, Ralf, E-mail: ralf-janknecht@ouhsc.edu [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 (United States)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Histone demethylase JMJD5 is essential for embryogenesis. Black-Right-Pointing-Pointer Transcription of tumor suppressor p53 is upregulated in JMJD5 knockout embryos. Black-Right-Pointing-Pointer JMJD5 may antagonize p53-dependent growth inhibition and apoptosis. Black-Right-Pointing-Pointer JMJD5 is overexpressed in leukemias and breast cancer. -- Abstract: Histone lysine methylation is pivotal in regulating chromatin structure and thus profoundly affects the transcriptome. JMJD5 (jumonji C domain-containing 5) is a histone demethylase that specifically removes methyl moieties from dimethylated lysine 36 on histone H3 and exerts a pro-proliferative effect on breast cancer cells. Here, we generated JMJD5 knockout mice in order to study the physiological significance of this enzyme. Whereas heterozygous knockout mice displayed no overt phenotype, homozygous JMJD5 knockouts died around day 10 of embryonal development. JMJD5{sup -/-} embryos showed delayed development already at E8.5 and were actively resorbed at E10.5. While strong JMJD5 expression was observed only in the yolk sac at E8.5, JMJD5 was robustly expressed in E10.5 embryos at several sites, including the heart and eye. Lack of JMJD5 resulted in transcriptional upregulation of the tumor suppressor p53. Concurrently, the cell cycle inhibitor p21 and the pro-apoptotic molecule Noxa, both of which are prominent p53 target genes, became strongly upregulated in JMJD5{sup -/-} embryos. Collectively, our data indicate that JMJD5 is essential during embryonal development and a repressor of p53 expression. The latter suggests that JMJD5 has oncogenic activity and accordingly JMJD5 is upregulated in leukemias and breast cancer.

  15. Human embryonic stem cells as models for aneuploid chromosomal syndromes.

    Science.gov (United States)

    Biancotti, Juan-Carlos; Narwani, Kavita; Buehler, Nicole; Mandefro, Berhan; Golan-Lev, Tamar; Yanuka, Ofra; Clark, Amander; Hill, David; Benvenisty, Nissim; Lavon, Neta

    2010-09-01

    Syndromes caused by chromosomal aneuploidies are widely recognized genetic disorders in humans and often lead to spontaneous miscarriage. Preimplantation genetic screening is used to detect chromosomal aneuploidies in early embryos. Our aim was to derive aneuploid human embryonic stem cell (hESC) lines that may serve as models for human syndromes caused by aneuploidies. We have established 25 hESC lines from blastocysts diagnosed as aneuploid on day 3 of their in vitro development. The hESC lines exhibited morphology and expressed markers typical of hESCs. They demonstrated long-term proliferation capacity and pluripotent differentiation. Karyotype analysis revealed that two-third of the cell lines carry a normal euploid karyotype, while one-third remained aneuploid throughout the derivation, resulting in eight hESC lines carrying either trisomy 13 (Patau syndrome), 16, 17, 21 (Down syndrome), X (Triple X syndrome), or monosomy X (Turner syndrome). On the basis of the level of single nucleotide polymorphism heterozygosity in the aneuploid chromosomes, we determined whether the aneuploidy originated from meiotic or mitotic chromosomal nondisjunction. Gene expression profiles of the trisomic cell lines suggested that all three chromosomes are actively transcribed. Our analysis allowed us to determine which tissues are most affected by the presence of a third copy of either chromosome 13, 16, 17 or 21 and highlighted the effects of trisomies on embryonic development. The results presented here suggest that aneuploid embryos can serve as an alternative source for either normal euploid or aneuploid hESC lines, which represent an invaluable tool to study developmental aspects of chromosomal abnormalities in humans.

  16. Congenital vascular malformations: the persistence of marginal and embryonal veins.

    Science.gov (United States)

    Weber, J; Daffinger, N

    2006-05-01

    In about 18% of cases with conginental vascular malformations we find a perspective of an atypical truncular vein, located along the outside of the leg, frequently extended from the dorsal foot up to the bottom. In presence of a normally developed system of the deep collecting veins of the lower limb and within the pelvic outflow we are talking about a persisting marginal vein (MV). Hypoplasia or even aplasia of the main deep veins in contrary defines the persisting embryonal vein (EV). Already in childhood these truncular dysplastic veins tend to develop varicose enlargement, causing severe reflux of a huge volume of blood--even more when being associated with av-fistulas (46%). In consequence a rapidly growing chronic venous insufficiency will guide to additional injuries. We have analysed 97 patients showing a persisting MV (n: 92 ) within a total of 102 legs. A persistent embryonal vein (EV) was seen 10 times within this clientel. The persisting truncular veins, associated with phlebectasias and typical clinical symptoms have been examined in a diagnostic "step-by-step" procedure, mainly phlebographically (ascending leg phlebography and varicography), including direct venous blood pressure measurements (phlebodynamometry) and--if needed--by arteriography, showing av-shunting fistulae in 46% of cases. CT and MRI were consulted for the exact therapy planing (frequently initially offered as a non-invasive, however, inadequate key of diagnostic). Actually now these techniques cannot replace pre-operatively the angiographic imaging techniques. The analysis of clinical, morphologic and functional signs, guiding to a specific therapy-relevant classification of MV's and EV's will be presented. And a specific strategy of surgical repair, interventional treatment of av-fistulas and conservative compressive follow-up treatment attempting palliative recompensation of the diseased venous outflow will be discussed also.

  17. Non-apical membrane antigen 1 (AMA1 IgGs from Malian children interfere with functional activity of AMA1 IgGs as judged by growth inhibition assay.

    Directory of Open Access Journals (Sweden)

    Kazutoyo Miura

    Full Text Available BACKGROUND: Apical membrane antigen 1 (AMA1 is one of the best-studied blood-stage malaria vaccine candidates. When an AMA1 vaccine was tested in a malaria naïve population, it induced functionally active antibodies judged by Growth Inhibition Assay (GIA. However, the same vaccine failed to induce higher growth-inhibitory activity in adults living in a malaria endemic area. Vaccination did induce functionally active antibodies in malaria-exposed children with less than 20% inhibition in GIA at baseline, but not in children with more than that level of baseline inhibition. METHODS: Total IgGs were purified from plasmas collected from the pediatric trial before and after immunization and pools of total IgGs were made. Another set of total IgGs was purified from U.S. adults immunized with AMA1 (US-total IgG. From these total IgGs, AMA1-specific and non-AMA1 IgGs were affinity purified and the functional activity of these IgGs was evaluated by GIA. Competition ELISA was performed with the U.S.-total IgG and non-AMA1 IgGs from malaria-exposed children. RESULTS: AMA1-specific IgGs from malaria-exposed children and U.S. vaccinees showed similar growth-inhibitory activity at the same concentrations. When mixed with U.S.-total IgG, non-AMA1 IgGs from children showed an interference effect in GIA. Interestingly, the interference effect was higher with non-AMA1 IgGs from higher titer pools. The non-AMA1 IgGs did not compete with anti-AMA1 antibody in U.S.-total IgG in the competition ELISA. CONCLUSION: Children living in a malaria endemic area have a fraction of IgGs that interferes with the biological activity of anti-AMA1 antibody as judged by GIA. While the mechanism of interference is not resolved in this study, these results suggest it is not caused by direct competition between non-AMA1 IgG and AMA1 protein. This study indicates that anti-malaria IgGs induced by natural exposure may interfere with the biological effect of antibody induced by an AMA1

  18. 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

  19. Extra-embryonic syndecan 2 regulates organ primordia migration and fibrillogenesis throughout the zebrafish embryo.

    Science.gov (United States)

    Arrington, Cammon B; Yost, H Joseph

    2009-09-01

    One of the first steps in zebrafish heart and gut organogenesis is the migration of bilateral primordia to the midline to form cardiac and gut tubes. The mechanisms that regulate this process are poorly understood. Here we show that the proteoglycan syndecan 2 (Sdc2) expressed in the extra-embryonic yolk syncytial layer (YSL) acts locally at the YSL-embryo interface to direct organ primordia migration, and is required for fibronectin and laminin matrix assembly throughout the embryo. Surprisingly, neither endogenous nor exogenous sdc2 expressed in embryonic cells can compensate for knockdown of sdc2 in the YSL, indicating that Sdc2 expressed in extra-embryonic tissues is functionally distinct from Sdc2 in embryonic cells. The effects of sdc2 knockdown in the YSL can be rescued by extra-embryonic Sdc2 lacking an extracellular proteolytic cleavage (shedding) site, but not by extra-embryonic Sdc2 lacking extracellular glycosaminoglycan (GAG) addition sites, suggesting that distinct GAG chains on extra-embryonic Sdc2 regulate extracellular matrix assembly, cell migration and epithelial morphogenesis of multiple organ systems throughout the embryo.

  20. High-throughput identification of small molecules that affect human embryonic vascular development

    Science.gov (United States)

    Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R.; Honório, Inês; de Vries, Margreet R.; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H. A.; Pereira, Carlos F.; Mercader, Nadia; Ferreira, Lino

    2017-01-01

    Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature. PMID:28348206

  1. Type 1 and 3 inositol trisphosphate receptors are required for extra-embryonic vascular development.

    Science.gov (United States)

    Uchida, Keiko; Nakazawa, Maki; Yamagishi, Chihiro; Mikoshiba, Katsuhiko; Yamagishi, Hiroyuki

    2016-10-01

    The embryonic-maternal interface of the placental labyrinth, allantois, and yolk sac are vital during embryogenesis; however, the precise mechanism underlying the vascularization of these structures remains unknown. Herein we focus on the role of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R), which are intracellular Ca(2+) release channels, in placentation. Double knockout (DKO) of type 1 and 3 IP3Rs (IP3R1 and IP3R3, respectively) in mice resulted in embryonic lethality around embryonic day (E) 11.5. Because IP3R1 and IP3R3 were co-expressed in endothelial cells in the labyrinth, allantois, and yolk sac, we investigated extra-embryonic vascular development in IP3R1- and IP3R3-DKO mice. The formation of chorionic plates and yolk sac vessels seemed dysregulated around the timing of the chorio-allantoic attachment, immediately followed by the disorganization of allantoic vessels, the decreased expression of the spongiotrophoblast cell marker Tpbpa and the growth retardation of the embryos in DKO mice. Fluorescent immunohistochemistry demonstrated downregulation of a vascular endothelial marker, CD31, in labyrinth embryonic vessels and poor elongation of extra-embryonic mesoderm into the labyrinth layer in DKO placenta, whereas the branching of the DKO chorionic trophoblast was initiated. In addition, allantoic and yolk sac vessels in extra-embryonic tissues were less remodeled in DKO mice. In vitro endothelial cord formation and migration activities of cultured vascular endothelial cells derived from human umbilical vein were downregulated under the inhibition of IP3R. Our results suggest that IP3R1 and IP3R3 are required for extra-embryonic vascularization in the placenta, allantois, and yolk sac. This is the first demonstration of the essential role of IP3/IP3Rs signaling in the development of the vasculature at the embryonic-maternal interface. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Save the mystery - staging specificity of Pina Bausch's dance theatre

    Directory of Open Access Journals (Sweden)

    Monika Roszak

    2009-01-01

    Full Text Available In the article are presented the fundamental determinants of the style of the theatre of Pina Bausch. Tanztheater Wuppertal performances delight with their totality and originality (stage design, costumes, the musical layer, textual layer. Montage, collage, acting based on improvisation - these are the means which are the pillars of Bausch's theatre. Tanztheater Wuppertal performances have reformed the modern ballet and created a new kind of performance based - apart from dance, movement and pantomime - on the realistic activities, routine behaviours, dialogues and singing.

  3. Stage-specific predictive models for breast cancer survivability.

    Science.gov (United States)

    Kate, Rohit J; Nadig, Ramya

    2017-01-01

    Survivability rates vary widely among various stages of breast cancer. Although machine learning models built in past to predict breast cancer survivability were given stage as one of the features, they were not trained or evaluated separately for each stage. To investigate whether there are differences in performance of machine learning models trained and evaluated across different stages for predicting breast cancer survivability. Using three different machine learning methods we built models to predict breast cancer survivability separately for each stage and compared them with the traditional joint models built for all the stages. We also evaluated the models separately for each stage and together for all the stages. Our results show that the most suitable model to predict survivability for a specific stage is the model trained for that particular stage. In our experiments, using additional examples of other stages during training did not help, in fact, it made it worse in some cases. The most important features for predicting survivability were also found to be different for different stages. By evaluating the models separately on different stages we found that the performance widely varied across them. We also demonstrate that evaluating predictive models for survivability on all the stages together, as was done in the past, is misleading because it overestimates performance. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. Cytomegalovirus Replicates in Differentiated but not in Undifferentiated Human Embryonal Carcinoma Cells

    Science.gov (United States)

    Gonczol, Eva; Andrews, Peter W.; Plotkin, Stanley A.

    1984-04-01

    To study the mode of action of human cytomegalovirus, an important teratogenic agent in human populations, the susceptibility of a pluripotent human embryonal carcinoma cell line to the virus was investigated. Viral antigens were not expressed nor was infectious virus produced by human embryonal carcinoma cells after infection, although the virus was able to penetrate these cells. In contrast, retinoic acid-induced differentiated derivatives of embryonal carcinoma cells were permissive for antigen expression and infectious virus production. Replication of human cytomegalovirus in human teratocarcinoma cells may therefore depend on cellular functions associated with differentiation.

  5. Human embryonic stem cells hemangioblast express HLA-antigens

    Directory of Open Access Journals (Sweden)

    Min Wei-Ping

    2009-04-01

    Full Text Available Abstract Background It has been suggested that the initial differentiation of endothelial and hematopoietic cells during embryogenesis occurs from a common progenitor, called hemangioblast (hB. We hypothesized that these cells with dual hematopoietic/endothelial potential could be used in future regenerative medicine. Methods We used the two-step differentiation technology to generate bipotential blast cells from human embryonic stem cells (hES. This involved short differentiation in our in vitro EB system followed by differentiation in semisolid culture medium supplemented with mixture of cytokines. Results The occurrence of blast-colony-forming cells (BL-CFC during EB differentiation (day 0–6 was transient and peaked on day 3. The emergence of this event was associated with expression of mesoderm gene T, and inversely correlated with expression of endoderm gene FoxA2. Similarly, the highest BL-CFC number was associated with increase in expression of early hematopoietic/endothelial genes: CD34, CD31 and KDR. The derived colonies were composed of 30–50 blast cells on day 6 in culture. These cells had homogenous appearance in Wright-Giemsa stain, but to a different extent expressed markers of immature hematopoietic and endothelial cells (CD31, CD34, VE-cadherin, Flt-1 and mature differentiated cells (CD45, CD33, CD146. We found that some of them expressed fetal and embryonic globin genes. Interestingly, these cells expressed also HLA class I molecules, however at very low levels compared to endothelial and hematopoietic cells. The blast cells could be successfully differentiated to hematopoietic cells in a CFU assay. In these conditions, blast cells formed CFU-M colonies (63.4 ± 0.8% containing macrophages, BFU-E colonies (19.5 ± 3.5% containing nucleated red blood cells, and CFU-EM colonies (17.1 ± 2.7% composed of macrophages and nucleated erythrocytes. Cells of CFU-EM and BFU-E colonies expressed both ε – and γ- globin genes, but

  6. Cytomegalovirus induces abnormal chondrogenesis and osteogenesis during embryonic mandibular development

    Directory of Open Access Journals (Sweden)

    Bringas Pablo

    2008-03-01

    Full Text Available Abstract Background Human clinical studies and mouse models clearly demonstrate that cytomegalovirus (CMV disrupts normal organ and tissue development. Although CMV is one of the most common causes of major birth defects in humans, little is presently known about the mechanism(s underlying CMV-induced congenital malformations. Our prior studies have demonstrated that CMV infection of first branchial arch derivatives (salivary glands and teeth induced severely abnormal phenotypes and that CMV has a particular tropism for neural crest-derived mesenchyme (NCM. Since early embryos are barely susceptible to CMV infection, and the extant evidence suggests that the differentiation program needs to be well underway for embryonic tissues to be susceptible to viral infection and viral-induced pathology, the aim of this study was to determine if first branchial arch NCM cells are susceptible to mCMV infection prior to differentiation of NCM derivatives. Results E11 mouse mandibular processes (MANs were infected with mouse CMV (mCMV for up to 16 days in vitro. mCMV infection of undifferentiated embryonic mouse MANs induced micrognathia consequent to decreased Meckel's cartilage chondrogenesis and mandibular osteogenesis. Specifically, mCMV infection resulted in aberrant stromal cellularity, a smaller, misshapen Meckel's cartilage, and mandibular bone and condylar dysmorphogenesis. Analysis of viral distribution indicates that mCMV primarily infects NCM cells and derivatives. Initial localization studies indicate that mCMV infection changed the cell-specific expression of FN, NF-κB2, RelA, RelB, and Shh and Smad7 proteins. Conclusion Our results indicate that mCMV dysregulation of key signaling pathways in primarily NCM cells and their derivatives severely disrupts mandibular morphogenesis and skeletogenesis. The pathogenesis appears to be centered around the canonical and noncanonical NF-κB pathways, and there is unusual juxtaposition of abnormal stromal

  7. Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis

    NARCIS (Netherlands)

    Le Guen, Ludovic; Karpanen, Terhi; Schulte, Dörte; Harris, Nicole C; Koltowska, Katarzyna; Roukens, Guy; Bower, Neil I; van Impel, Andreas; Stacker, Steven A; Achen, Marc G; Schulte-Merker, Stefan; Hogan, Benjamin M

    The VEGFC/VEGFR3 signaling pathway is essential for lymphangiogenesis (the formation of lymphatic vessels from pre-existing vasculature) during embryonic development, tissue regeneration and tumor progression. The recently identified secreted protein CCBE1 is indispensible for lymphangiogenesis

  8. Correlation of body weight with hatchling blood glucose concentration and its relationship to embryonic survival.

    Science.gov (United States)

    Christensen, V L; Grimes, J L; Donaldson, W E; Lerner, S

    2000-12-01

    The negative correlation between selection for rapid growth and embryonic survival was investigated. Embryonic growth was assessed with hatchling weights of a closed population of commercial turkey breeders. Hatchling weights were highly significantly (P periods. High embryos grew faster than Low embryos with elevated organ glycogen concentrations. Organic acid analysis indicated elevated plasma alpha-ketoglutarate, urate, and beta-hydroxy butyrate concentrations, suggesting a greater reliance on gluconeogenesis for the High group. Posthatch growth was significantly positively correlated with hatchling blood glucose concentrations in toms but not in hens. Tom poults hatching with elevated glucose were heavier than low glucose hatch mates until 22 wk of age, but hen poults displayed no differences until 16 wk when High hens weighed less than Low hens. These data suggest that the negative correlation between rapid growth and embryonic survival is related to egg-shell conductance constants and embryonic energy metabolism.

  9. Regulated expression of transgenes in embryonic stem cell-derived neural cells.

    Science.gov (United States)

    Lorberbaum, David S; Gottlieb, David

    2011-02-01

    Discovery and characterization of gene promoters, enhancers and repressor binding elements is an important research area in neuroscience. Here, the suitability of embryonic stem cells and their neural derivatives as a model system for this research is investigated. Three neural transgenic constructs (from the Mnx1, Fabp7, and tuba1a genes) that have been validated in transgenic mice were inserted into embryonic stem cells as stable transgenes. These transgenic embryonic stem cells were differentiated into neural cultures and the pattern of transgene expression across a series of inducing conditions determined. The pattern of expression matched that predicted from transgenic mouse experiments for each of the three transgenes. The results show that embryonic stem cells and their neural derivatives comprise a promising model for investigating the mechanisms that control cell- and temporal-specific neural gene transcription. Copyright © 2010 Wiley-Liss, Inc.

  10. Femtosecond optical transfection as a tool for genetic manipulation of human embryonic stem cells

    Science.gov (United States)

    Torres-Mapa, M. L.; Gardner, J.; Bradburn, H.; King, J.; Dholakia, K.; Gunn-Moore, F.

    2013-03-01

    We demonstrate the use of femtosecond optical transfection for the genetic manipulation of human embryonic stem cells. Using a system with an SLM combined with a scanning mirror allows poration of both single-cell and colony-formed human embryonic stem cells in a rapid and targeted manner. In this work, we show successful transfection of plasmid DNA tagged with fluorescent reporters into human embryonic stem cells using three doses of focused femtosecond laser. A significant number of transfected cells retained their undifferentiated morphological feature of large nucleus with high nucleus to cytoplasmic ratio, 48h after photoporation. Furthermore, DNA constructs driven by different types of promoters were also successfully transfected into human embryonic stem cells using this technique.

  11. The embryonic nucleologenesis during inhibition of major transcriptional activity in bovine preimplantation embryos

    Czech Academy of Sciences Publication Activity Database

    Kovalská, M.; Hruška-Plocháň, Marian; Ostrup, O.; Adamkov, M.; Lehotský, J.; Strejček, F.; Statelová, M.; Mikušková, K.; Varga, I.; Petrovičová, I.

    2012-01-01

    Roč. 67, č. 4 (2012), s. 818-825 ISSN 0006-3088 Institutional support: RVO:67985904 Keywords : embryonic genome activation * nucleous * gene expression Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.506, year: 2012

  12. Restricted intra-embryonic origin of bona fide hematopoietic stem cells in the chicken

    NARCIS (Netherlands)

    Yvernogeau, Laurent; Robin, Catherine

    2017-01-01

    Hematopoietic stem cells (HSCs), which are responsible for blood cell production, are generated during embryonic development. Human and chicken embryos share features that position the chicken as a reliable and accessible alternative model to study developmental hematopoiesis. However, the existence

  13. Engineering human cell spheroids to model embryonic tissue fusion in vitro.

    Science.gov (United States)

    Epithelial-mesenchymal interactions drive embryonic fusion events during development and upon perturbation can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known abo...

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Data for human cell spheroid model of embryonic tissue fusion in vitro.

    Data.gov (United States)

    U.S. Environmental Protection Agency — Epithelial-mesenchymal interactions drive embryonic fusion events during development and upon perturbation can result in birth defects. Cleft palate and neural tube...

  16. Changes of DNA methylation level and spatial arrangement of primordial germ cells in embryonic day 15 to embryonic day 28 pig embryos

    DEFF Research Database (Denmark)

    Matzen, Sara Maj Hyldig; Østrup, Olga; Vejlsted, Morten

    2011-01-01

    The mammalian germline is generally assumed to undergo extensive epigenetic reprogramming during embryonic development, including a nearly complete erasure of DNA methylation. This assumption does, however, to large degree rely on data from mouse, and despite a well-grounded picture the general......-positive primordial germ cells (PGCs) compared with neighboring somatic cells in porcine embryos at Embryonic Day 15 (E15), E17, E20, E21, and E28. We show that, in agreement with the mouse model, a significantly lower level of DNA methylation was observed in the early migrating PGCs. This level...

  17. Transgenesis and nuclear transfer using porcine embryonic germ cells.

    Science.gov (United States)

    Ahn, Kwang Sung; Won, Ji Young; Heo, Soon Young; Kang, Jee Hyun; Yang, Hong Seok; Shim, Hosup

    2007-01-01

    Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). Porcine EG cell lines with capacities of both in vitro and in vivo differentiation have been established. Because EG cells can be cultured indefinitely in an undifferentiated state, they may be more suitable for nuclear donor cells in nuclear transfer (NT) than somatic cells that have limited lifespan in primary culture. Use of EG cells could be particularly advantageous to provide an inexhaustible source of transgenic cells for NT. In this study the efficiencies of transgenesis and NT using porcine fetal fibroblasts and EG cells were compared. The rate of development to the blastocyst stage was significantly higher in EG cell NT than somatic cell NT (94 of 518, 18.2% vs. 72 of 501, 14.4%). To investigate if EG cells can be used for transgenesis in pigs, green fluorescent protein (GFP) gene was introduced into porcine EG cells. Nuclear transfer embryos using transfected EG cells gave rise to blastocysts (29 of 137, 21.2%) expressing GFP based on observation under fluorescence microscope. The results obtained from the present study suggest that EG cell NT may have advantages over somatic cell NT, and transgenic pigs may be produced using EG cells.

  18. Frizzled 3 acts upstream of Alcam during embryonic eye development.

    Science.gov (United States)

    Seigfried, Franziska A; Cizelsky, Wiebke; Pfister, Astrid S; Dietmann, Petra; Walther, Paul; Kühl, Michael; Kühl, Susanne J

    2017-06-01

    Formation of a functional eye during vertebrate embryogenesis requires different processes such as cell differentiation, cell migration, cell-cell interactions as well as intracellular signalling processes. It was previously shown that the non-canonical Wnt receptor Frizzled 3 (Fzd3) is required for proper eye formation, however, the underlying mechanism is poorly understood. Here we demonstrate that loss of Fzd3 induces severe malformations of the developing eye and that this defect is phenocopied by loss of the activated leukocyte cell adhesion molecule (Alcam). Promoter analysis revealed the presence of a Fzd3 responsive element within the alcam promoter, which is responsible for alcam expression during anterior neural development. In-depth analysis identified the jun N-terminal protein kinase 1 (JNK1) and the transcription factor paired box 2 (Pax2) to be important for the activation of alcam expression. Altogether our study reveals that alcam is activated through non-canonical Wnt signalling during embryonic eye development in Xenopus laevis and shows that this pathway plays a similar role in different tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Induction of differentiation of murine embryonal carcinoma cells by ouabain

    International Nuclear Information System (INIS)

    Zimmerman, B.T.

    1986-01-01

    Embryonal carcinoma (EC) cells can be induced to differentiate by ouabain at concentrations which inhibit Na + , K + -ATPase activity as measured by inhibition of 86 Rb + uptake. Since the pharmacologic action of ouabain is thought to be specific, the authors investigated the role of Na + , K + -ATPase inhibition and specific metabolic consequences of this inhibition in the induction of EC differentiation, and explored whether this might be a common mode of action for a variety of structurally diverse inducers. The Na + , K + -ATPase maintains ionic gradients in cells. However, results of studies utilizing specific ionophores, channel blockers, and media deficient in specific components failed to demonstrate a consistent role for ion flux or concentration in the differentiation process. The Na + , K + -ATPase is a major consumer of ATP. They therefore examined the effect of Na + , K + -ATPase inhibition on the adenylate energy charge as measured by high performance liquid chromatography of adenylate nucleotides. Ouabain was found to significantly decrease the energy charge in sensitive cells suggesting a role for suppression of ATP turnover is triggering differentiation. However, direct inhibition of glycolysis also induced differentiation without decreasing the energy charge, suggesting that reduction of the energy charge is not a common mechanism for induction of differentiation of EC

  20. Histopathological and ultrastructural effects of Losartan on embryonic rat kidney.

    Science.gov (United States)

    Akil, Ipek; Inan, Sevinc; Gurcu, Beyhan; Nazikoglu, Aysegul; Ozbilgin, Kemal; Muftuoglu, Sevda

    2005-01-01

    The aim of our study was to investigate the histopathological, immunohistochemical and ultrastructural effects of Losartan (a selective angiotensin II type-1 receptor blocker) on renal development in rats. Twelve pregnant rats were divided into control and experimental groups. In the experimental group, Losartan (10 mg/kg/day) was given via nasogastric tube, between the sixth day of implantation and time of sacrifice on embryonic days 18 and 20. All formalin-fixed, paraffin wax-embedded renal tissue sections were stained with hematoxylin and eosin or labelled for binding of primary antibodies against transforming growth factor-beta (TGF-beta 1,-2,-3) using an avidin-biotin-peroxidase method. For electron microscopic examination, samples were fixed with glutaraldehyde and osmium tetroxide and embedded in araldite. Glomerular basement membrane (GBM) thickness was measured and compared using an unpaired t-test. Angiotensin II type-1 receptor antagonism by Losartan inhibited renal growth and delayed nephron maturation. Increased immunoreactivity of TGF-beta's was observed in developing nephron precursors and interstitial cells in the experimental group. Electron microscopical examination showed that thickening of the GBM was normal in the control group but an irregular thickening was seen in the experimental group (p < 0.001). It was also seen that epithelial cells of developing tubules underwent apoptosis in the experimental group. Thus, renal development in rats seems to depend on an intact renin-angiotensin system.

  1. Efficient differentiation of human embryonic stem cells to definitive endoderm.

    Science.gov (United States)

    D'Amour, Kevin A; Agulnick, Alan D; Eliazer, Susan; Kelly, Olivia G; Kroon, Evert; Baetge, Emmanuel E

    2005-12-01

    The potential of human embryonic stem (hES) cells to differentiate into cell types of a variety of organs has generated much excitement over the possible use of hES cells in therapeutic applications. Of great interest are organs derived from definitive endoderm, such as the pancreas. We have focused on directing hES cells to the definitive endoderm lineage as this step is a prerequisite for efficient differentiation to mature endoderm derivatives. Differentiation of hES cells in the presence of activin A and low serum produced cultures consisting of up to 80% definitive endoderm cells. This population was further enriched to near homogeneity using the cell-surface receptor CXCR4. The process of definitive endoderm formation in differentiating hES cell cultures includes an apparent epithelial-to-mesenchymal transition and a dynamic gene expression profile that are reminiscent of vertebrate gastrulation. These findings may facilitate the use of hES cells for therapeutic purposes and as in vitro models of development.

  2. Transcription pausing regulates mouse embryonic stem cell differentiation.

    Science.gov (United States)

    Tastemel, Melodi; Gogate, Aishwarya A; Malladi, Venkat S; Nguyen, Kim; Mitchell, Courtney; Banaszynski, Laura A; Bai, Xiaoying

    2017-12-01

    The pluripotency of embryonic stem cells (ESCs) relies on appropriate responsiveness to developmental cues. Promoter-proximal pausing of RNA polymerase II (Pol II) has been suggested to play a role in keeping genes poised for future activation. To identify the role of Pol II pausing in regulating ESC pluripotency, we have generated mouse ESCs carrying a mutation in the pause-inducing factor SPT5. Genomic studies reveal genome-wide reduction of paused Pol II caused by mutant SPT5 and further identify a tight correlation between pausing-mediated transcription effect and local chromatin environment. Functionally, this pausing-deficient SPT5 disrupts ESC differentiation upon removal of self-renewal signals. Thus, our study uncovers an important role of Pol II pausing in regulating ESC differentiation and suggests a model that Pol II pausing coordinates with epigenetic modification to influence transcription during mESC differentiation. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Common marmoset embryonic stem cell can differentiate into cardiomyocytes

    International Nuclear Information System (INIS)

    Chen Hao; Hattori, Fumiyuki; Murata, Mitsushige; Li Weizhen; Yuasa, Shinsuke; Onizuka, Takeshi; Shimoji, Kenichiro; Ohno, Yohei; Sasaki, Erika; Kimura, Kensuke; Hakuno, Daihiko

    2008-01-01

    Common marmoset monkeys have recently attracted much attention as a primate research model, and are preferred to rhesus and cynomolgus monkeys due to their small bodies, easy handling and efficient breeding. We recently reported the establishment of common marmoset embryonic stem cell (CMESC) lines that could differentiate into three germ layers. Here, we report that our CMESC can also differentiate into cardiomyocytes and investigated their characteristics. After induction, FOG-2 was expressed, followed by GATA4 and Tbx20, then Nkx2.5 and Tbx5. Spontaneous beating could be detected at days 12-15. Immunofluorescent staining and ultrastructural analyses revealed that they possessed characteristics typical of functional cardiomyocytes. They showed sinus node-like action potentials, and the beating rate was augmented by isoproterenol stimulation. The BrdU incorporation assay revealed that CMESC-derived cardiomyocytes retained a high proliferative potential for up to 24 weeks. We believe that CMESC-derived cardiomyocytes will advance preclinical studies in cardiovascular regenerative medicine

  4. 'Crook' pipettes: embryonic emigrations from agriculture to reproductive biomedicine.

    Science.gov (United States)

    Franklin, Sarah

    2007-06-01

    While cloning, stem cells, and regenerative medicine are often imagined in a futurial idiom--as expectations, hype, hope and promises--this article approaches the remaking of genealogy in such contexts from a historical route. Through a series of somewhat disparate historical connections linking Australian sheep to the development of clinical IVF and the cloning of Dolly at the Roslin Institute in Scotland in 1996, this article explores the linkages through which agriculture, embryology, and reproductive biomedicine are thickly intertwined. Key to this examination is not only the history of experimental sheep breeding, and its somewhat unexpectedly genealogical connections to (Australian) national identity ('wool in the veins'), but also the re-emergence of a distinctive frontier ethos in the context of assisted conception, and later human embryonic stem cell derivation. I have set this scene of genealogical interconnection against the criss-crossing traffic between Britain and Australia, and the wool trade, to emphasise the importance of global, as well as local, connections in the bloodlines of animals such as Dolly. In sum, this article examines the idea of the 'biological frontier' by exploring its histories as a means to offset the assumption that this frequently encountered idiom describes a future that is, or must be, by definition, unknown and unknowable.

  5. Mineralization and growth of cultured embryonic skeletal tissue in microgravity

    Science.gov (United States)

    Klement, B. J.; Spooner, B. S.

    1999-01-01

    Microgravity provides a unique environment in which to study normal and pathological phenomenon. Very few studies have been done to examine the effects of microgravity on developing skeletal tissue such as growth plate formation and maintenance, elongation of bone primordia, or the mineralization of growth plate cartilage. Embryonic mouse premetatarsal triads were cultured on three space shuttle flights to study cartilage growth, differentiation, and mineralization, in a microgravity environment. The premetatarsal triads that were cultured in microgravity all formed cartilage rods and grew in length. However, the premetatarsal cartilage rods cultured in microgravity grew less in length than the ground control cartilage rods. Terminal chondrocyte differentiation also occurred during culture in microgravity, as well as in the ground controls, and the matrix around the hypertrophied chondrocytes was capable of mineralizing in both groups. The same percentage of premetatarsals mineralized in the microgravity cultures as mineralized in the ground control cultures. In addition, the sizes of the mineralized areas between the two groups were very similar. However, the amount of 45Ca incorporated into the mineralized areas was significantly lower in the microgravity cultures, suggesting that the composition or density of the mineralized regions was compromised in microgravity. There was no significant difference in the amount of 45Ca liberated from prelabeled explants in microgravity or in the ground controls.

  6. A method for labeling vasculature in embryonic mice.

    Science.gov (United States)

    Bryson, Jerrod L; Coles, Mark C; Manley, Nancy R

    2011-10-07

    The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects. Previous studies have described techniques used to label the neonatal and adult thymus vasculature. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1-isolectin B₄) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-β to label thymic perivascular mesenchyme. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart.

  7. Organ culture and immunostaining of mouse embryonic kidneys.

    Science.gov (United States)

    Barak, Hila; Boyle, Scott C

    2011-01-01

    The study of organogenesis in mammals allows investigation of a wide variety of basic cell biological processes in the context of the intact organ. This has become especially important in the age of genetics, as the consequences of gene deletion or mutation in the mouse can be directly linked to human congenital abnormalities. The ability to culture some organs ex vivo during development has emerged as an important tool to understand how tissues are constructed and the signaling pathways that regulate these processes. It has been especially useful in organs that grow via branching morphogenic mechanisms, such as the lung and kidney. Here we demonstrate isolation, ex vivo growth, and fluorescent immunostaining of mouse embryonic day 12.5 (E12.5) kidneys. To demonstrate nephron formation using live imaging, we have isolated and cultured kidneys from mice carrying a green fluorescent protein (GFP) transgene driven by the Hes 1 promoter, which is expressed early in the developing nephron. We also provide a protocol for robust imaging of multiple kidney structures in the whole-mount setting. These techniques serve as a basic platform for the analysis of branching morphogenesis and nephron formation in genetic mouse models or in response to exogenous factors, such as agonists or inhibitors, which can be directly added to the culture medium.

  8. ECM modulated early kidney development in embryonic organ culture.

    Science.gov (United States)

    Sebinger, David D R; Ofenbauer, Andreas; Gruber, Petra; Malik, Susann; Werner, Carsten

    2013-09-01

    The use of exogenous signals is gaining importance in renal regenerative therapies. We wanted to explore the role of extracellular matrix (ECM) constituents on renal structure formation during renal organogenesis. We used a recently established organ culture setup to expose embryonic kidney rudiments directly to a large set of surface-immobilized or dissolved ECM molecules and growth factors. Organ culture was also performed on immobilized adult kidney ECM extracts and on reactive polymer films without any biomolecular components. The applied conditions resulted in distinct differences of organ phenotypes, underlining the multifaceted role of exogenous signals during kidney development. Specific ECM components, including collagen I and laminin, supported nephronal and tubular structure formation of the developing organ. ECM biopolymers, e.g. hyaluronic acid, were found to determine the fate of developing explants in a concentration- and molecular weight-dependent manner. The organ culture system used was an effective and robust means to identify exogenous signals that direct kidney development. This system can provide valuable insight for future regenerative therapies of kidney diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Normal embryonic stages of the Longnose Gar, Lepisosteus osseus

    Science.gov (United States)

    Long, Wilbur L; Ballard, William W

    2001-01-01

    Background Gaps exist in the modern literature that describes patterns of development in living groups of actinopterygian fishes. Relatively recent descriptions of development exist for the teleost fishes, bowfin, sturgeon, paddlefish and bichirs. Such literature dealing with the gars is to be found in older work, done approximately a century ago. The present study concerns the gars, of which the garpike, Lepisosteus osseus, is a representative example. Results The embryonic period of life of this fish is divided, as required for experimentation, into 34 stages, from fertilization to exhaustion of the yolk supply. Diagnostic structural characteristics are cited for each stage, and the rate of development is indicated. Conclusions Three features of development are especially noted that compare or contrast with other members of the Neopterygii, and with the Chondrostei. These are meroblastic cleavage, a well-defined yolk syncytial layer (ysl), and a pit at the posterodorsal edge of the blastoderm, which defines an overhanging dorsal lip. Meroblastic cleavage and the ysl in the garpike show an affinity to those character states in the teleosts, though not with Amia, the other neopterygian fish. The posterodorsal pit and dorsal lip are reminiscent of similar features in the Chondrostei. Lepisosteus is unique among the Neopterygii with respect to this character state. Such comparisons set the stage for a broader understanding of the mechanisms for development in these organisms, and of the evolutionary relationships between them. PMID:11319037

  10. Identification and expression analysis of zebrafish glypicans during embryonic development.

    Directory of Open Access Journals (Sweden)

    Mansi Gupta

    Full Text Available Heparan sulfate Proteoglycans (HSPG are ubiquitous molecules with indispensable functions in various biological processes. Glypicans are a family of HSPG's, characterized by a Gpi-anchor which directs them to the cell surface and/or extracellular matrix where they regulate growth factor signaling during development and disease. We report the identification and expression pattern of glypican genes from zebrafish. The zebrafish genome contains 10 glypican homologs, as opposed to six in mammals, which are highly conserved and are phylogenetically related to the mammalian genes. Some of the fish glypicans like Gpc1a, Gpc3, Gpc4, Gpc6a and Gpc6b show conserved synteny with their mammalian cognate genes. Many glypicans are expressed during the gastrulation stage, but their expression becomes more tissue specific and defined during somitogenesis stages, particularly in the developing central nervous system. Existence of multiple glypican orthologs in fish with diverse expression pattern suggests highly specialized and/or redundant function of these genes during embryonic development.

  11. Object Segmentation and Ground Truth in 3D Embryonic Imaging.

    Directory of Open Access Journals (Sweden)

    Bhavna Rajasekaran

    Full Text Available Many questions in developmental biology depend on measuring the position and movement of individual cells within developing embryos. Yet, tools that provide this data are often challenged by high cell density and their accuracy is difficult to measure. Here, we present a three-step procedure to address this problem. Step one is a novel segmentation algorithm based on image derivatives that, in combination with selective post-processing, reliably and automatically segments cell nuclei from images of densely packed tissue. Step two is a quantitative validation using synthetic images to ascertain the efficiency of the algorithm with respect to signal-to-noise ratio and object density. Finally, we propose an original method to generate reliable and experimentally faithful ground truth datasets: Sparse-dense dual-labeled embryo chimeras are used to unambiguously measure segmentation errors within experimental data. Together, the three steps outlined here establish a robust, iterative procedure to fine-tune image analysis algorithms and microscopy settings associated with embryonic 3D image data sets.

  12. Effects of Microgravity on Embryonic Quail Eye Development

    Science.gov (United States)

    Barrett, Joyce E.; Wells, Diane C.; Paulsen, Avelina Q.; Conrad, Gary W.

    1997-01-01

    Immunohistochemical methods were used to stain neurofilament protein in corneal nerves of Embryonic Day 16 (E16) quail eyes that had been fixed in 4% paraformaldehyde at room temperature for several months. Fixation was according to the methods used by the Mir 21/NASA 2 Avian Developmental Biology Flight Experiments for quail embryos incubated on the Mir Space Station. After fixation, corneas were pretreated to improve immunohistochemical visualization of neurofilaments. A sequential combination of three pretreatments [microwave heating in saline G, followed by extraction with sodium dodecyl sulfate (SDS) at 37 C, followed by digestion with hyaluronidase at 37 C], produced increased antibody staining of corneal nerve neurofilament proteins, compared with corneas subjected to no prior pretreatments. Darker nerve staining and increased numbers of fine branches were observed, together with lower background staining after such pretreatments. In contrast, use of any single pretreatment or pair of pretreatments resulted in only slight and inconsistent enhancement of nerve staining. Only the sequential combination of all three pretreatments resulted in consistently better nerve staining.

  13. Embryonic mouse pre-metatarsal development in organ culture

    Science.gov (United States)

    Klement, B. J.; Spooner, B. S.

    1993-01-01

    Embryonic mouse pre-metatarsals were removed from embryos at 13 days of gestation and cultured in a defined, serum-free medium for up to 15 days. By histological analysis, we observe that the cultured pre-metatarsal tissue undergoes a similar developmental profile as pre-metatarsals growing normally in vivo. The initial mesenchyme condensation regions undergo differentiation and morphogenesis to form distinct rods made up of cartilage tissue. A marker of this differentiation step is the synthesis of type II collagen. Metabolic labelling, pepsin digestion, SDS-PAGE, and autoradiography were used to demonstrate this protein when cartilage tissue is present in the cultures. After additional culture time, terminal chondrocyte differentiation and morphogenesis take place in specific regions of the cartilage rods to form bands of hypertrophied chondrocytes. One marker of this differentiation step is the synthesis of the enzyme alkaline phosphatase. We have measured the activity of this enzyme throughout the culture period and see a substantial increase at the time of terminal chondrocyte differentiation. Another feature of hypertrophied chondrocytes is that the matrix around the cells becomes calcified. Calcified matrix in our cultured pre-metatarsals was visualized by staining with alizarin red. By supplementing the defined culture medium with ITS, we observed that terminal chondrocyte differentiation took place in a shorter culture time. Supplementation of the medium with serum results in a similar acceleration of terminal differentiation, and, with additional culture time, an osteoid-like matrix forms around the central region of the rods.

  14. Smoc2 modulates embryonic myelopoiesis during zebrafish development.

    Science.gov (United States)

    Mommaerts, Hendrik; Esguerra, Camila V; Hartmann, Ursula; Luyten, Frank P; Tylzanowski, Przemko

    2014-11-01

    SMOC2 is a member of the BM-40 (SPARC) family of matricellular proteins, reported to influence signaling in the extracellular compartment. In mice, Smoc2 is expressed in many different tissues and was shown to enhance the response to angiogenic growth factors, mediate cell adhesion, keratinocyte migration, and metastasis. Additionally, SMOC2 is associated with vitiligo and craniofacial and dental defects. The function of Smoc2 during early zebrafish development has not been determined to date. In pregastrula zebrafish embryos, smoc2 is expressed ubiquitously. As development progresses, the expression pattern becomes more anteriorly restricted. At the onset of blood cell circulation, smoc2 morphants presented a mild ventralization of posterior structures. Molecular analysis of the smoc2 morphants indicated myelopoietic defects in the rostral blood islands during segmentation stages. Hemangioblast development and further specification of the myeloid progenitor cells were shown to be impaired. Additional experiments indicated that Bmp target genes were down-regulated in smoc2 morphants. Our findings reveal that Smoc2 is an essential player in the development of myeloid cells of the anterior lateral plate mesoderm during embryonic zebrafish development. Furthermore, our data show that Smoc2 affects the transcription of Bmp target genes without affecting initial dorsoventral patterning or mesoderm development. Copyright © 2014 Wiley Periodicals, Inc.

  15. Effects of Hypergravity on Statocyst Development in Embryonic Aplysia californica

    Science.gov (United States)

    Pedrozo, Hugo A.; Wiederhold, Michael L.

    1994-01-01

    Aplysia californica is a marine gastropod mollusc with bilaterally paired statocysts as gravity-reccptor organs. Data from three experiments in which embryonic Aplysia californica were exposed to 2 x g arc discussed. The experimental groups were exposed to excess gravity until hatching (9-12 day), whereas control groups were maintained at normal gravity. Body diameter was measured before exposure to 2 x g. Statocyst, statolith and body diameter were each determined for samples of 20 embryos from each group on successive days. Exposure to excess gravity led to an increase in body size. Statocyst size was not affected by exposure to 2 x g. Statolith size decreased with treatment as indicated by smaller statolith-to-body ratios observed in the 2 x g group in all three experiments. Mean statolith diameter was significantly smaller for the 2 x g group in Experiment 1 but not in Experiments 2 and 3. Defective statocysts, characterized by very small or no statoliths, were found in the 2 x g group in Experiments 1 and 2.

  16. Correlating cell behavior with tissue topology in embryonic epithelia.

    Directory of Open Access Journals (Sweden)

    Sebastian A Sandersius

    2011-04-01

    Full Text Available Measurements on embryonic epithelial tissues in a diverse range of organisms have shown that the statistics of cell neighbor numbers are universal in tissues where cell proliferation is the primary cell activity. Highly simplified non-spatial models of proliferation are claimed to accurately reproduce these statistics. Using a systematic critical analysis, we show that non-spatial models are not capable of robustly describing the universal statistics observed in proliferating epithelia, indicating strong spatial correlations between cells. Furthermore we show that spatial simulations using the Subcellular Element Model are able to robustly reproduce the universal histogram. In addition these simulations are able to unify ostensibly divergent experimental data in the literature. We also analyze cell neighbor statistics in early stages of chick embryo development in which cell behaviors other than proliferation are important. We find from experimental observation that cell neighbor statistics in the primitive streak region, where cell motility and ingression are also important, show a much broader distribution. A non-spatial Markov process model provides excellent agreement with this broader histogram indicating that cells in the primitive streak may have significantly weaker spatial correlations. These findings show that cell neighbor statistics provide a potentially useful signature of collective cell behavior.

  17. Monitoring developmental force distributions in reconstituted embryonic epithelia.

    Science.gov (United States)

    Przybyla, L; Lakins, J N; Sunyer, R; Trepat, X; Weaver, V M

    2016-02-01

    The way cells are organized within a tissue dictates how they sense and respond to extracellular signals, as cues are received and interpreted based on expression and organization of receptors, downstream signaling proteins, and transcription factors. Part of this microenvironmental context is the result of forces acting on the cell, including forces from other cells or from the cellular substrate or basement membrane. However, measuring forces exerted on and by cells is difficult, particularly in an in vivo context, and interpreting how forces affect downstream cellular processes poses an even greater challenge. Here, we present a simple method for monitoring and analyzing forces generated from cell collectives. We demonstrate the ability to generate traction force data from human embryonic stem cells grown in large organized epithelial sheets to determine the magnitude and organization of cell-ECM and cell-cell forces within a self-renewing colony. We show that this method can be used to measure forces in a dynamic hESC system and demonstrate the ability to map intracolony protein localization to force organization. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Col11a1 Regulates Bone Microarchitecture during Embryonic Development

    Directory of Open Access Journals (Sweden)

    Anthony Hafez

    2015-12-01

    Full Text Available Collagen XI alpha 1 (Col11a1 is an extracellular matrix molecule required for embryonic development with a role in both nucleating the formation of fibrils and regulating the diameter of heterotypic fibrils during collagen fibrillar assembly. Although found in many different tissues throughout the vertebrate body, Col11a1 plays an essential role in endochondral ossification. To further understand the function of Col11a1 in the process of bone formation, we compared skeletal mineralization in wild-type (WT mice and Col11a1-deficient mice using X-ray microtomography (micro-CT and histology. Changes in trabecular bone microstructure were observed and are presented here. Additionally, changes to the periosteal bone collar of developing long bones were observed and resulted in an increase in thickness in the case of Col11a1-deficient mice compared to WT littermates. Vertebral bodies were incompletely formed in the absence of Col11a1. The data demonstrate that Col11a1 depletion results in alteration to newly-formed bone and is consistent with a role for Col11a1 in mineralization. These findings indicate that expression of Col11a1 in the growth plate and perichondrium is essential for trabecular bone and bone collar formation during endochondral ossification. The observed changes to mineralized tissues further define the function of Col11a1.

  19. Ebf1 controls early cell differentiation in the embryonic striatum.

    Science.gov (United States)

    Garel, S; Marín, F; Grosschedl, R; Charnay, P

    1999-12-01

    Ebf1/Olf-1 belongs to a small multigene family encoding closely related helix-loop-helix transcription factors, which have been proposed to play a role in neuronal differentiation. Here we show that Ebf1 controls cell differentiation in the murine embryonic striatum, where it is the only gene of the family to be expressed. Ebf1 targeted disruption affects postmitotic cells that leave the subventricular zone (SVZ) en route to the mantle: they appear to be unable to downregulate genes normally restricted to the SVZ or to activate some mantle-specific genes. These downstream genes encode a variety of regulatory proteins including transcription factors and proteins involved in retinoid signalling as well as adhesion/guidance molecules. These early defects in the SVZ/mantle transition are followed by an increase in cell death, a dramatic reduction in size of the postnatal striatum and defects in navigation and fasciculation of thalamocortical fibres travelling through the striatum. Our data therefore show that Ebf1 plays an essential role in the acquisition of mantle cell molecular identity in the developing striatum and provide information on the genetic hierarchies that govern neuronal differentiation in the ventral telencephalon.

  20. Computer Simulation of Embryonic Systems: What can a ...

    Science.gov (United States)

    (1) Standard practice for assessing developmental toxicity is the observation of apical endpoints (intrauterine death, fetal growth retardation, structural malformations) in pregnant rats/rabbits following exposure during organogenesis. EPA’s computational toxicology research program (ToxCast) generated vast in vitro cellular and molecular effects data on >1858 chemicals in >600 high-throughput screening (HTS) assays. The diversity of assays has been increased for developmental toxicity with several HTS platforms, including the devTOX-quickPredict assay from Stemina Biomarker Discovery utilizing the human embryonic stem cell line (H9). Translating these HTS data into higher order-predictions of developmental toxicity is a significant challenge. Here, we address the application of computational systems models that recapitulate the kinematics of dynamical cell signaling networks (e.g., SHH, FGF, BMP, retinoids) in a CompuCell3D.org modeling environment. Examples include angiogenesis (angiodysplasia) and dysmorphogenesis. Being numerically responsive to perturbation, these models are amenable to data integration for systems Toxicology and Adverse Outcome Pathways (AOPs). The AOP simulation outputs predict potential phenotypes based on the in vitro HTS data ToxCast. A heuristic computational intelligence framework that recapitulates the kinematics of dynamical cell signaling networks in the embryo, together with the in vitro profiling data, produce quantitative pr

  1. Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

    Science.gov (United States)

    Epsztejn-Litman, Silvina; Cohen-Hadad, Yaara; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Levy-Lahad, Ephrat; Schonberger, Oshrat; Eldar-Geva, Talia; Zeligson, Sharon; Eiges, Rachel

    2015-01-01

    We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

  2. Retroviral transcriptional regulation and embryonic stem cells: war and peace.

    Science.gov (United States)

    Schlesinger, Sharon; Goff, Stephen P

    2015-03-01

    Retroviruses have evolved complex transcriptional enhancers and promoters that allow their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES cell pluripotency. This overlap is not coincidental; retrovirus-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps "noisy" control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Human embryonic and induced pluripotent stem cells in clinical trials.

    Science.gov (United States)

    Ilic, Dusko; Devito, Liani; Miere, Cristian; Codognotto, Stefano

    2015-01-01

    Human embryonic and induced pluripotent stem cells (hESC and hiPSC) have tremendous potential for clinical implementation. In spite of all hurdles and controversy, clinical trials in treatment of spinal cord injury, macular degeneration of retina, type 1 diabetes and heart failure are already ongoing. ClinicalTrials.gov database, International Clinical Trials Registry Platform, PubMed and press releases and websites of companies and institutions working on hESC- and iPSC-based cellular therapy. The initial results from multiple clinical trials demonstrate that hESC-based therapies are safe and promising. Are iPSC cells safe in the clinical application? Is there a room for both hESC and iPSC in the future clinical applications? Increasing number of new clinical trials. Development of hESC- and/or iPSC-based cellular therapy for other diseases. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Tension regulates myosin dynamics during Drosophila embryonic wound repair.

    Science.gov (United States)

    Kobb, Anna B; Zulueta-Coarasa, Teresa; Fernandez-Gonzalez, Rodrigo

    2017-02-15

    Embryos repair epithelial wounds rapidly in a process driven by collective cell movements. Upon wounding, actin and the molecular motor non-muscle myosin II are redistributed in the cells adjacent to the wound, forming a supracellular purse string around the lesion. Purse string contraction coordinates cell movements and drives rapid wound closure. By using fluorescence recovery after photobleaching in Drosophila embryos, we found that myosin turns over as the purse string contracts. Myosin turnover at the purse string was slower than in other actomyosin networks that had a lower level of contractility. Mathematical modelling suggested that myosin assembly and disassembly rates were both reduced by tension at the wound edge. We used laser ablation to show that tension at the purse string increased as wound closure progressed, and that the increase in tension was associated with reduced myosin turnover. Reducing purse string tension by laser-mediated severing resulted in increased turnover and loss of myosin. Finally, myosin motor activity was necessary for its stabilization around the wound and for rapid wound closure. Our results indicate that mechanical forces regulate myosin dynamics during embryonic wound repair. © 2017. Published by The Company of Biologists Ltd.

  5. The Ultrastructural Signature of Human Embryonic Stem Cells.

    Science.gov (United States)

    Underwood, Jean M; Becker, Klaus A; Stein, Gary S; Nickerson, Jeffrey A

    2017-04-01

    The epigenetics and molecular biology of human embryonic stem cells (hES cells) have received much more attention than their architecture. We present a more complete look at hES cells by electron microscopy, with a special emphasis on the architecture of the nucleus. We propose that there is an ultrastructural signature of pluripotent human cells. hES cell nuclei lack heterochromatin, including the peripheral heterochromatin, that is common in most somatic cell types. The absence of peripheral heterochromatin may be related to the absence of lamins A and C, proteins important for linking chromatin to the nuclear lamina and envelope. Lamins A and C expression and the development of peripheral heterochromatin were early steps in the development of embryoid bodies. While hES cell nuclei had abundant nuclear pores, they also had an abundance of nuclear pores in the cytoplasm in the form of annulate lamellae. These were not a residue of annulate lamellae from germ cells or the early embryos from which hES cells were derived. Subnuclear structures including nucleoli, interchromatin granule clusters, and Cajal bodies were observed in the nuclear interior. The architectural organization of human ES cell nuclei has important implications for cell structure-gene expression relationships and for the maintenance of pluripotency. J. Cell. Biochem. 118: 764-774, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. Molecular and Functional Characterization of Broccoli EMBRYONIC FLOWER 2 Genes

    Science.gov (United States)

    Chen, Long-Fang O.; Lin, Chun-Hung; Lai, Ying-Mi; Huang, Jia-Yuan; Sung, Zinmay Renee

    2012-01-01

    Polycomb group (PcG) proteins regulate major developmental processes in Arabidopsis. EMBRYONIC FLOWER 2 (EMF2), the VEFS domain-containing PcG gene, regulates diverse genetic pathways and is required for vegetative development and plant survival. Despite widespread EMF2-like sequences in plants, little is known about their function other than in Arabidopsis and rice. To study the role of EMF2 in broccoli (Brassica oleracea var. italica cv. Elegance) development, we identified two broccoli EMF2 (BoEMF2) genes with sequence homology to and a similar gene expression pattern to that in Arabidopsis (AtEMF2). Reducing their expression in broccoli resulted in aberrant phenotypes and gene expression patterns. BoEMF2 regulates genes involved in diverse developmental and stress programs similar to AtEMF2 in Arabidopsis. However, BoEMF2 differs from AtEMF2 in the regulation of flower organ identity, cell proliferation and elongation, and death-related genes, which may explain the distinct phenotypes. The expression of BoEMF2.1 in the Arabidopsis emf2 mutant (Rescued emf2) partially rescued the mutant phenotype and restored the gene expression pattern to that of the wild type. Many EMF2-mediated molecular and developmental functions are conserved in broccoli and Arabidopsis. Furthermore, the restored gene expression pattern in Rescued emf2 provides insights into the molecular basis of PcG-mediated growth and development. PMID:22537758

  7. Transcriptome coexpression map of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Mattson Mark P

    2006-05-01

    Full Text Available Abstract Background Human embryonic stem (ES cells hold great promise for medicine and science. The transcriptome of human ES cells has been studied in detail in recent years. However, no systematic analysis has yet addressed whether gene expression in human ES cells may be regulated in chromosomal domains, and no chromosomal domains of coexpression have been identified. Results We report the first transcriptome coexpression map of the human ES cell and the earliest stage of ES differentiation, the embryoid body (EB, for the analysis of how transcriptional regulation interacts with genomic structure during ES self-renewal and differentiation. We determined the gene expression profiles from multiple ES and EB samples and identified chromosomal domains showing coexpression of adjacent genes on the genome. The coexpression domains were not random, with significant enrichment in chromosomes 8, 11, 16, 17, 19, and Y in the ES state, and 6, 11, 17, 19 and 20 in the EB state. The domains were significantly associated with Giemsa-negative bands in EB, yet showed little correlation with known cytogenetic structures in ES cells. Different patterns of coexpression were revealed by comparative transcriptome mapping between ES and EB. Conclusion The findings and methods reported in this investigation advance our understanding of how genome organization affects gene expression in human ES cells and help to identify new mechanisms and pathways controlling ES self-renewal or differentiation.

  8. CITED1 Expression in Wilms' Tumor and Embryonic Kidney

    Directory of Open Access Journals (Sweden)

    Harold N. Lovvorn, III

    2007-07-01

    Full Text Available Wilms' tumors, or nephroblastomas, are thought to arise from abnormal postnatal retention and dysregulated differentiation of nephrogenic progenitor cells that originate as a condensed metanephric mesenchyme within embryonic kidneys. We have previously shown that the transcriptional regulator CITED1 (CBP/p300-interacting transactivators with glutamic acid [E]/aspartic acid [D]-rich C-terminal domain is expressed exclusively in these nephrogenic progenitor cells and is downregulated as they differentiate to form nephronic epithelia. In the current study, we show that CITED1 expression persists in blastemal cell populations of both experimental rat nephroblastomas and human Wilms' tumors, and that primary human Wilms' tumors presenting with disseminated disease show the highest level of CITED1 expression. Unlike the predominantly cytoplasmic subcellular localization of CITED1 in the normal developing kidney, CITED1 is clearly detectable in the nuclear compartment of Wilms' tumor blastema. These findings indicate that CITED1 is a marker of primitive blastema in Wilms' tumors and suggest that persistent expression and/or altered subcellular localization of CITED1 in the condensed metanephric mesenchyme could play a role in Wilms' tumor initiation and pathogenesis.

  9. Tracking the embryonic stem cell transition from ground state pluripotency.

    Science.gov (United States)

    Kalkan, Tüzer; Olova, Nelly; Roode, Mila; Mulas, Carla; Lee, Heather J; Nett, Isabelle; Marks, Hendrik; Walker, Rachael; Stunnenberg, Hendrik G; Lilley, Kathryn S; Nichols, Jennifer; Reik, Wolf; Bertone, Paul; Smith, Austin

    2017-04-01

    Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naïve pluripotency. Here, we examine the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naïve status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a distinct formative phase of pluripotency preparatory to lineage priming. © 2017. Published by The Company of Biologists Ltd.

  10. Probing embryonic tissue mechanics with laser hole drilling

    International Nuclear Information System (INIS)

    Ma Xiaoyan; Lynch, Holley E; Scully, Peter C; Hutson, M Shane

    2009-01-01

    We use laser hole drilling to assess the mechanics of an embryonic epithelium during development—in vivo and with subcellular resolution. We ablate a subcellular cylindrical hole clean through the epithelium and track the subsequent recoil of adjacent cells (on ms time scales). We investigate dorsal closure in the fruit fly with emphasis on apical constriction of amnioserosa cells. The mechanical behavior of this epithelium falls between that of a continuous sheet and a 2D cellular foam (a network of tensile interfaces). Tensile stress is carried both by cell–cell interfaces and by the cells' apical actin networks. Our results show that stress is slightly concentrated along interfaces (1.6-fold), but only in early closure. Furthermore, closure is marked by a decrease in the recoil power-law exponent, implying a transition to a more solid-like tissue. We use the site and stage dependence of the recoil kinetics to constrain how the cellular mechanics change during closure. We apply these results to test extant computational models

  11. Live imaging of mitosis in the developing mouse embryonic cortex.

    Science.gov (United States)

    Pilaz, Louis-Jan; Silver, Debra L

    2014-06-04

    Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-α-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

  12. Cytotoxic Effect Of Verapamil On Human Embryonic Kidney Cell Line

    Directory of Open Access Journals (Sweden)

    Jamil L Ahmad

    2015-08-01

    Full Text Available Introduction The link between long term use of verapamil and cancer development has been suggested in literature many years back. However there are numerous controversies surrounding this association with several epidemiological studies in the positive negative and non-association between verapamil and cancer development. Aim To investigate in mechanistic terms the link between chronic use of a calcium channel blocker verapamil and cancer development using human embryonic kidney HEK293 cell line. Method Trypan blue dye exclusion cell counting and 3-amp615314 5-Dimethylthiazol-2-ylamp61533-2 5-diphenyl-tetrazolium bromide MTT assays were used to determine the proliferative as well as cytotoxic effects of verapamil. Results Verapamil had a growth inhibitory rather than proliferative effect on HEK293 cells and the growth inhibition was found to be significant p0.05. Conclusion The long term use of verapamil is associated with cellular growth inhibition and this possibly explained the rationale behind its use as part of combination chemotherapy for some human cancers.

  13. Cadmium inhibits neurogenesis in zebrafish embryonic brain development

    International Nuclear Information System (INIS)

    Chow, Elly Suk Hen; Hui, Michelle Nga Yu; Lin Chunchi; Cheng Shukhan

    2008-01-01

    Cadmium is a non-essential heavy metal found abundantly in the environment. Children of women exposed to cadmium during pregnancy display lower motor and perceptual abilities. High cadmium body burden in children is also related to impaired intelligence and lowered school achievement. However, little is known about the molecular and cellular basis of developmental neurotoxicity in the sensitive early life stages of animals. In this study, we explore neurological deficits caused by cadmium during early embryonic stages in zebrafish by examining regionalization of the neural tube, pattern formation and cell fate determination, commitment of proneural genes and induction of neurogenesis. We show that cadmium-treated embryos developed a smaller head with unclear boundaries between the brain subdivisions, particularly in the mid-hindbrain region. Embryos display normal anterior to posterior regionalization; however, the commitment of neural progenitor cells was affected by cadmium. We observe prominent reductions in the expression of several proneuronal genes including ngn1 in cell clusters, zash1a in the developing optic tectum, and zash1b in the telencephalon and tectum. Cadmium-treated embryos also have fewer differentiated neurons and glia in the facial sensory ganglia as indicated by decreased zn-12 expression. Also, a lower transcription level of neurogenic genes, ngn1 and neuroD, is observed in neurons. Our data suggest that cadmium-induced neurotoxicity can be caused by impaired neurogenesis, resulting in markedly reduced neuronal differentiation and axonogenesis

  14. Human embryonic stem cell therapies for neurodegenerative diseases.

    Science.gov (United States)

    Tomaskovic-Crook, Eva; Crook, Jeremy M

    2011-06-01

    There is a renewed enthusiasm for the clinical translation of human embryonic stem (hES) cells. This is abetted by putative clinically-compliant strategies for hES cell maintenance and directed differentiation, greater understanding of and accessibility to cells through formal cell registries and centralized cell banking for distribution, the revised US government policy on funding hES cell research, and paradoxically the discovery of induced pluripotent stem (iPS) cells. Additionally, as we consider the constraints (practical and fiscal) of delivering cell therapies for global healthcare, the more efficient and economical application of allogeneic vs autologous treatments will bolster the clinical entry of hES cell derivatives. Neurodegenerative disorders such as Parkinson's disease are primary candidates for hES cell therapy, although there are significant hurdles to be overcome. The present review considers key advances and challenges to translating hES cells into novel therapies for neurodegenerative diseases, with special consideration given to Parkinson's disease and Alzheimer's disease. Importantly, despite the focus on degenerative brain disorders and hES cells, many of the issues canvassed by this review are relevant to systemic application of hES cells and other pluripotent stem cells such as iPS cells.

  15. Epigenetic stability, adaptability, and reversibility in human embryonic stem cells.

    Science.gov (United States)

    Tompkins, Joshua D; Hall, Christine; Chen, Vincent Chang-yi; Li, Arthur Xuejun; Wu, Xiwei; Hsu, David; Couture, Larry A; Riggs, Arthur D

    2012-07-31

    The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for additional passaging. However, upon reversion to the original feeder-based culture conditions, numerous transcription changes are not reversible. Similarly, although the majority of DNA methylation changes are reversible, highlighting the plasticity of DNA methylation, a few are persistent. Collectively, this indicates these cells harbor a memory of culture history. For culture-induced DNA methylation changes, we also note an intriguing correlation: hypomethylation of regions 500-2440 bp upstream of promoters correlates with decreased expression, opposite to that commonly seen at promoter-proximal regions. Lastly, changes in regulation of G-coupled protein receptor pathways provide a partial explanation for many of the unique transcriptional changes observed during hESC adaptation and reverse adaptation.

  16. Embryonic Stem Cells in Development and Regenerative Medicine.

    Science.gov (United States)

    Doğan, Ayşegül

    2018-02-21

    After progressive improvement in embryonic stem (ES) cell field, several studies have been conducted to explore the usage of ES cells in regenerative medicine. Unlimited self renewal and pluripoteny properties, combined with encouraging preclinical trials, remark that ES cell technology might be promising for clinical practice. ES cells, which can form three germ layers in vitro, are potential candidates to study development at the cellular and molecular level. Understanding the cell fate decision and differentiation processes during development might enable generating functional progenitor cells for tissue restoration. Progression in gene modifications and tissue engineering technology has facilitated the derivation of desired cells for therapy. Success in differentiation protocols and identification the regulatory pathways simplify the research for clinical applications. Although there are established protocols for cell differentiation in vitro and promising preclinical studies in vivo, many challenges need to be adressed before clinical translation. In this review, ES cells are discussed as a model of development in vitro and as a potential candidate for regenerative medicine. This review also dissusses current challenges for ES cell based therapy.

  17. Expression cloning of camelid nanobodies specific for Xenopus embryonic antigens.

    Directory of Open Access Journals (Sweden)

    Keiji Itoh

    Full Text Available Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies, which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.

  18. Coupled Global and Targeted Proteomics of Human Embryonic Stem Cells during Induced Differentiation*S⃞

    OpenAIRE

    Yocum, Anastasia K.; Gratsch, Theresa E.; Leff, Nancy; Strahler, John R.; Hunter, Christie L.; Walker, Angela K.; Michailidis, George; Omenn, Gilbert S.; O'Shea, K. Sue; Andrews, Philip C.

    2008-01-01

    Elucidating the complex combinations of growth factors and signaling molecules that maintain pluripotency or, alternatively, promote the controlled differentiation of human embryonic stem cells (hESCs) has important implications for the fundamental understanding of human development, devising cell replacement therapies, and cancer cell biology. hESCs are commonly grown on irradiated mouse embryonic fibroblasts (MEFs) or in conditioned medium from MEFs. These culture conditions interfere with ...

  19. Human embryonic stem cells have enhanced repair of multiple forms of DNA damage

    DEFF Research Database (Denmark)

    Maynard, Scott; Swistowska, Anna Maria; Lee, Jae Wan

    2008-01-01

    fibroblasts (WI-38, hs27) and, with the exception of UV-C damage, HeLa cells. Microarray gene expression analysis showed that mRNA levels of several DNA repair genes are elevated in human embryonic stem cells compared with their differentiated forms (embryoid bodies). These data suggest that genomic...... maintenance pathways are enhanced in human embryonic stem cells, relative to differentiated human cells....

  20. Progressing a human embryonic stem-cell-based regenerative medicine therapy towards the clinic

    OpenAIRE

    Whiting, Paul; Kerby, Julie; Coffey, Peter; da Cruz, Lyndon; McKernan, Ruth

    2015-01-01

    Since the first publication of the derivation of human embryonic stem cells in 1998, there has been hope and expectation that this technology will lead to a wave of regenerative medicine therapies with the potential to revolutionize our approach to managing certain diseases. Despite significant resources in this direction, the path to the clinic for an embryonic stem-cell-based regenerative medicine therapy has not proven straightforward, though in the past few years progress has been made. H...