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Sample records for castaneum identifies embryonic

  1. A system to efficiently maintain embryonic lethal mutations in the flour beetle Tribolium castaneum.

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    Berghammer, A; Bucher, G; Maderspacher, F; Klingler, M

    1999-06-01

    Due to its small size, short life cycle, and easy maintenance, the flour beetle Tribolium castaneum is well suited for the genetic analysis of development. One drawback of Tribolium as a genetic system is, however, the difficulty of keeping embryonic lethal lines. Presently, only few lethal mutations can be kept as balanced stocks. Therefore, heterozygous carriers must be identified anew in every generation in order to maintain a recessive embryonic mutation. To alleviate this problem we have devised a block system that allows the simultaneous processing of many mutant lines or test crosses for visual inspection of larval cuticle phenotypes. Using this technique, one person can maintain about 100 embryonic lethal stocks, which makes feasible the thorough genetic analysis of embryogenesis in this species.

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

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

    2013-01-01

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

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

    Science.gov (United States)

    Fraga, Amanda; Ribeiro, Lupis; Lobato, Mariana; Santos, Vitória; Silva, José Roberto; Gomes, Helga; da Cunha Moraes, Jorge Luiz; de Souza Menezes, Jackson; de Oliveira, Carlos Jorge Logullo; Campos, Eldo; da Fonseca, Rodrigo Nunes

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Amanda Fraga

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

  5. Knickkopf and retroactive proteins are required for formation of laminar serosal procuticle during embryonic development of Tribolium castaneum.

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    Chaudhari, Sujata S; Noh, Mi Young; Moussian, Bernard; Specht, Charles A; Kramer, Karl J; Beeman, Richard W; Arakane, Yasuyuki; Muthukrishnan, Subbaratnam

    2015-05-01

    results of this study demonstrate that in addition to their essential roles in maintenance and organization of chitin in epidermal cuticle in larval and later stages of insect development, TcKnk and TcRtv also are required for egg hatch, chitin maintenance and laminar organization of both serosal and larval cuticle during embryonic development of T. castaneum.

  6. Live imaging of Tribolium castaneum embryonic development using light-sheet-based fluorescence microscopy.

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    Strobl, Frederic; Schmitz, Alexander; Stelzer, Ernst H K

    2015-10-01

    Tribolium castaneum has become an important insect model organism for evolutionary developmental biology, genetics and biotechnology. However, few protocols for live fluorescence imaging of Tribolium have been reported, and little image data is available. Here we provide a protocol for recording the development of Tribolium embryos with light-sheet-based fluorescence microscopy. The protocol can be completed in 4-7 d and provides procedural details for: embryo collection, microscope configuration, embryo preparation and mounting, noninvasive live imaging for up to 120 h along multiple directions, retrieval of the live embryo once imaging is completed, and image data processing, for which exemplary data is provided. Stringent quality control criteria for developmental biology studies are also discussed. Light-sheet-based fluorescence microscopy complements existing toolkits used to study Tribolium development, can be adapted to other insect species, and requires no advanced imaging or sample preparation skills.

  7. Odoriferous Defensive stink gland transcriptome to identify novel genes necessary for quinone synthesis in the red flour beetle, Tribolium castaneum.

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    Jianwei Li

    Full Text Available Chemical defense is one of the most important traits, which endow insects the ability to conquer a most diverse set of ecological environments. Chemical secretions are used for defense against anything from vertebrate or invertebrate predators to prokaryotic or eukaryotic parasites or food competitors. Tenebrionid beetles are especially prolific in this category, producing several varieties of substituted benzoquinone compounds. In order to get a better understanding of the genetic and molecular basis of defensive secretions, we performed RNA sequencing in a newly emerging insect model, the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae. To detect genes that are highly and specifically expressed in the odoriferous gland tissues that secret defensive chemical compounds, we compared them to a control tissue, the anterior abdomen. 511 genes were identified in different subtraction groups. Of these, 77 genes were functionally analyzed by RNA interference (RNAi to recognize induced gland alterations morphologically or changes in gland volatiles by gas chromatography-mass spectrometry. 29 genes (38% presented strong visible phenotypes, while 67 genes (87% showed alterations of at least one gland content. Three of these genes showing quinone-less (ql phenotypes - Tcas-ql VTGl; Tcas-ql ARSB; Tcas-ql MRP - were isolated, molecularly characterized, their expression identified in both types of the secretory glandular cells, and their function determined by quantification of all main components after RNAi. In addition, microbe inhibition assays revealed that a quinone-free status is unable to impede bacterial or fungal growth. Phylogenetic analyses of these three genes indicate that they have evolved independently and specifically for chemical defense in beetles.

  8. Neuropeptides of the beetle, Tenebrio molitor identified using MALDI-TOF mass spectrometry and deduced sequences from the Tribolium castaneum genome.

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    Weaver, Robert J; Audsley, Neil

    2008-02-01

    Four neuropeptides were identified from the brain and corpora cardiaca-corpora allata (CC-CA) of the mealworm beetle Tenebrio molitor using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and information derived from the genome of the red flour beetle, Tribolium castaneum. Leucomyosuppressin (a FLRFamide), previously associated with cockroaches, but also subsequently identified from honey bee seen as a prominent peptide in both brain and CC-CA of T.molitor. A coding sequence for this peptide is found in the genome of T. castaneum. In addition, three FXPRLamides (pyrokinins), provisionally Tenmo-PK-1, Tenmo-PK-2 and Tenmo-PK-3 (HVVNFTPRLamide, SPPFAPRLamide, HL(I)SPFSPRLamide) were identified in both CC-CA and brain of T. molitor, again on the basis of predicted occurrence or similarity in T. castaneum. The sequence of Tenmo-PK-2 is the same as the PK-2 of the cockroach, Periplaneta americana. Other peptides readily predicted from the genome of T. castaneum include two AKH/HrTH peptides (Trica-AKH-1; pELNFSTDWamide and Trica-AKH-2; pELNFTPNWamide), the second of which is identical to Pyrap-AKH, an AKH-related peptide (Trica AKH-L; pEVTFSRDWPamide), two CRF-related diuretic factors (Trica-DH 37 and Trica-DH 47), the latter identical to Tenmo-DH 47, a putative antidiuretic factor (Trica-ADFb; LYDDGSYKPHVYGF-OH), two sulfakinin-like peptides (Trica-SK-1; pETSDDY(SO(3))GHLRFamide, and Trica SK-2; GEEPFDDYGHMRFamide), a potential allatostatin-C (Trica-AS; pESRYRQCYFNPISCF-OH), six allatostatin-B/myoinhibitory peptides (Trica-AST-B-1,2,3,4,5 & 6; DWNKDLHIWamide, GWNNLHEGWamide, AWQSLQSGWamide, NWGQFHGGWamide, SKWDNFRGSWamide, EPAWSNLGIWamide), an allatotropin-like peptide (Trica-ATL; GIEALKYHNMDLGTARGYamide), four 'CAPA'-related peptides (Trica-CAPA-1,2,3,4; NKLASVYALTPSLRVamide, RIGKMVSFPRIamide, PGANSGGMWFGPRLamide, SENFTPWAYIILNGEAPIIREVHYSPRLamide), proctolin (RYLPT), a potential SIFamide (Trica-SIFa; TYRKPPFNGSIFamide), an

  9. Nuclear structures in Tribolium castaneum oocytes.

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    Bogolyubov, Dmitry S; Batalova, Florina M; Kiselyov, Artyom M; Stepanova, Irina S

    2013-10-01

    The first ultrastructural and immunomorphological characteristics of the karyosphere (karyosome) and extrachromosomal nuclear bodies in the red flour beetle, Tribolium castaneum, are presented. The karyosphere forms early in the diplotene stage of meiotic prophase by the gathering of all oocyte chromosomes in a limited nuclear volume. Using the BrUTP assay, T. castaneum oocyte chromosomes united in the karyosphere maintain their transcriptional activity until the end of oocyte growth. Hyperphosphorylated RNA polymerase II and basal transcription factors (TFIID and TFIIH) were detected in the perichromatin region of the karyosphere. The T. castaneum karyosphere has an extrachromosomal capsule that separates chromosomes from the rest of the nucleoplasm. Certain structural proteins (F-actin, lamin B) were found in the capsule. Unexpectedly, the karyosphere capsule in T. castaneum oocytes was found to be enriched in TMG-capped snRNAs, which suggests that the capsule is not only a structural support for the karyosphere, but may be involved in biogenesis of snRNPs. We also identified the counterparts of 'universal' extrachromosomal nuclear domains, Cajal bodies (CBs) and interchromatin granule clusters (IGCs). Nuclear bodies containing IGC marker protein SC35 display some features unusual for typical IGCs. SC35 domains in T. castaneum oocytes are predominantly fibrillar complex bodies that do not contain trimethyl guanosine (TMG)-capped small nuclear (sn) RNAs. Microinjections of 2'-O-methyl (U)22 probes into the oocytes allowed revealing poly(A)+ RNAs in these nuclear domains. Several proteins related to mRNA export (heterogeneous ribonucleoprotein core protein A1, export adapters Y14 and Aly and export receptor NXF1) were also detected there. We believe that unusual SC35 nuclear domains of T. castaneum oocytes are possibly involved in mRNP but not snRNP biogenesis.

  10. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

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    Li Zou

    2015-02-01

    Full Text Available We generated a RUNX2-yellow fluorescent protein (YFP reporter system to study osteogenic development from human embryonic stem cells (hESCs. Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development.

  11. Ago2 immunoprecipitation identifies predicted microRNAs in human embryonic stem cells and neural precursors.

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    Loyal A Goff

    Full Text Available BACKGROUND: MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation. METHODOLOGY/PRINCIPAL FINDINGS: SOLiD ultra-deep sequencing identified >10(7 unique small RNAs from human embryonic stem cells (hESC and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs. CONCLUSIONS/SIGNIFICANCE: Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation.

  12. Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage

    NARCIS (Netherlands)

    Amps, Katherine; Andrews, Peter W.; Anyfantis, George; Armstrong, Lyle; Avery, Stuart; Baharvand, Hossein; Baker, Julie; Baker, Duncan; Munoz, Maria B.; Beil, Stephen; Benvenisty, Nissim; Ben-Yosef, Dalit; Biancotti, Juan-Carlos; Bosman, Alexis; Brena, Romulo Martin; Brison, Daniel; Caisander, Gunilla; Camarasa, Maria V.; Chen, Jieming; Chiao, Eric; Choi, Young Min; Choo, Andre B. H.; Collins, Daniel; Colman, Alan; Crook, Jeremy M.; Daley, George Q.; Dalton, Anne; De Sousa, Paul A.; Denning, Chris; Downie, Janet; Dvorak, Petr; Montgomery, Karen D.; Feki, Anis; Ford, Angela; Fox, Victoria; Fraga, Ana M.; Frumkin, Tzvia; Ge, Lin; Gokhale, Paul J.; Golan-Lev, Tamar; Gourabi, Hamid; Gropp, Michal; Lu Guangxiu, [No Value; Hampl, Ales; Harron, Katie; Healy, Lyn; Herath, Wishva; Holm, Frida; Hovatta, Outi; Hyllner, Johan; Inamdar, Maneesha S.; Irwanto, Astrid Kresentia; Ishii, Tetsuya; Jaconi, Marisa; Jin, Ying; Kimber, Susan; Kiselev, Sergey; Knowles, Barbara B.; Kopper, Oded; Kukharenko, Valeri; Kuliev, Anver; Lagarkova, Maria A.; Laird, Peter W.; Lako, Majlinda; Laslett, Andrew L.; Lavon, Neta; Lee, Dong Ryul; Lee, Jeoung Eun; Li, Chunliang; Lim, Linda S.; Ludwig, Tenneille E.; Ma, Yu; Maltby, Edna; Mateizel, Ileana; Mayshar, Yoav; Mileikovsky, Maria; Minger, Stephen L.; Miyazaki, Takamichi; Moon, Shin Yong; Moore, Harry; Mummery, Christine; Nagy, Andras; Nakatsuji, Norio; Narwani, Kavita; Oh, Steve K. W.; Oh, Sun Kyung; Olson, Cia; Otonkoski, Timo; Pan, Fei; Park, In-Hyun; Pells, Steve; Pera, Martin F.; Pereira, Lygia V.; Qi, Ouyang; Raj, Grace Selva; Reubinoff, Benjamin; Robins, Alan; Robson, Paul; Rossant, Janet; Salekdeh, Ghasem H.; Schulz, Thomas C.; Sermon, Karen; Mohamed, Jameelah Sheik; Shen, Hui; Sherrer, Eric; Sidhu, Kuldip; Sivarajah, Shirani; Skottman, Heli; Spits, Claudia; Stacey, Glyn N.; Strehl, Raimund; Strelchenko, Nick; Suemori, Hirofumi; Sun, Bowen; Suuronen, Riitta; Takahashi, Kazutoshi; Tuuri, Timo; Venu, Parvathy; Verlinsky, Yuri; Ward-van Oostwaard, Dorien; Weisenberger, Daniel J.; Wu, Yue; Yamanaka, Shinya; Young, Lorraine; Zhou, Qi

    2011-01-01

    The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphi

  13. Microarray analysis of LTR retrotransposon silencing identifies Hdac1 as a regulator of retrotransposon expression in mouse embryonic stem cells.

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    Judith Reichmann

    Full Text Available Retrotransposons are highly prevalent in mammalian genomes due to their ability to amplify in pluripotent cells or developing germ cells. Host mechanisms that silence retrotransposons in germ cells and pluripotent cells are important for limiting the accumulation of the repetitive elements in the genome during evolution. However, although silencing of selected individual retrotransposons can be relatively well-studied, many mammalian retrotransposons are seldom analysed and their silencing in germ cells, pluripotent cells or somatic cells remains poorly understood. Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data. This computational approach to genome-wide retrotransposon expression has allowed us to identify the histone deacetylase Hdac1 as a component of the retrotransposon silencing machinery in mouse embryonic stem cells, and to determine the retrotransposon targets of Hdac1 in these cells. We also identify retrotransposons that are targets of other retrotransposon silencing mechanisms such as DNA methylation, Eset-mediated histone modification, and Ring1B/Eed-containing polycomb repressive complexes in mouse embryonic stem cells. Furthermore, our computational analysis of retrotransposon silencing suggests that multiple silencing mechanisms are independently targeted to retrotransposons in embryonic stem cells, that different genomic copies of the same retrotransposon can be differentially sensitive to these silencing mechanisms, and helps define retrotransposon sequence elements that are targeted by silencing machineries. Thus repeat annotation of gene expression microarray data suggests that a complex interplay between silencing mechanisms represses retrotransposon loci in germ cells and embryonic stem cells.

  14. Bridging the gap between postembryonic cell lineages and identified embryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster

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    Oliver Birkholz

    2015-03-01

    Full Text Available The clarification of complete cell lineages, which are produced by specific stem cells, is fundamental for understanding mechanisms, controlling the generation of cell diversity and patterning in an emerging tissue. In the developing Central Nervous System (CNS of Drosophila, neural stem cells (neuroblasts exhibit two periods of proliferation: During embryogenesis they produce primary lineages, which form the larval CNS. After a phase of mitotic quiescence, a subpopulation of them resumes proliferation in the larva to give rise to secondary lineages that build up the CNS of the adult fly. Within the ventral nerve cord (VNC detailed descriptions exist for both primary and secondary lineages. However, while primary lineages have been linked to identified neuroblasts, the assignment of secondary lineages has so far been hampered by technical limitations. Therefore, primary and secondary neural lineages co-existed as isolated model systems. Here we provide the missing link between the two systems for all lineages in the thoracic and abdominal neuromeres. Using the Flybow technique, embryonic neuroblasts were identified by their characteristic and unique lineages in the living embryo and their further development was traced into the late larval stage. This comprehensive analysis provides the first complete view of which embryonic neuroblasts are postembryonically reactivated along the anterior/posterior-axis of the VNC, and reveals the relationship between projection patterns of primary and secondary sublineages.

  15. Tribolium castaneum larval gut transcriptome and proteome: A resource for the study of the coleopteran gut.

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    Morris, Kaley; Lorenzen, Marcé D; Hiromasa, Yasuaki; Tomich, John M; Oppert, Cris; Elpidina, Elena N; Vinokurov, Konstantin; Jurat-Fuentes, Juan Luis; Fabrick, Jeff; Oppert, Brenda

    2009-08-01

    Tribolium castaneum is an important agricultural pest and an advanced genetic model for coleopteran insects. We have taken advantage of the recently acquired T. castaneum genome to identify T. castaneum genes and proteins in one of the more critical environmental interfaces of the insect, the larval alimentary tract. Genetic transcripts isolated from the T. castaneum larval gut were labeled and hybridized to a custom array containing oligonucleotides from predicted genes in the T. castaneum genome. Through a ranking procedure based on relative labeling intensity, we found that approximately 17.6% of the genes represented in the array were predicted to be highly expressed in gut tissue. Several genes were selected to compare relative expression levels in larval gut, head, or carcass tissues using quantitative real-time PCR, and expression levels were, with few exceptions, consistent with the gut rankings. In parallel with the microarrays, proteins extracted from the T. castaneum larval gut were subjected to proteomic analysis. Two-dimensional electrophoretic analysis combined with MALDI-TOF resulted in the identification of 37 of 88 selected protein samples. As an alternative strategy, one-dimensional electrophoretic separation of T. castaneum larval gut proteins followed by two-dimensional nano-HPLC and ESI-MS/MS resulted in the identification of 98 proteins. A comparison of the proteomic studies indicated that 16 proteins were commonly identified in both, whereas 80 proteins from the proteomic analyses corresponded to genes with gut rankings indicative of high expression in the microarray analysis. These data serve as a resource of T. castaneum transcripts and proteins in the larval gut and provide the basis for comparative transcriptomic and proteomic studies related to the gut of coleopteran insects.

  16. High-density polymer microarrays: identifying synthetic polymers that control human embryonic stem cell growth.

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    Hansen, Anne; Mjoseng, Heidi K; Zhang, Rong; Kalloudis, Michail; Koutsos, Vasileios; de Sousa, Paul A; Bradley, Mark

    2014-06-01

    The fabrication of high-density polymer microarray is described, allowing the simultaneous and efficient evaluation of more than 7000 different polymers in a single-cellular-based screen. These high-density polymer arrays are applied in the search for synthetic substrates for hESCs culture. Up-scaling of the identified hit polymers enables long-term cellular cultivation and promoted successful stem-cell maintenance.

  17. Ontogeny of identified cells from the median domain in the embryonic brain of the grasshopper Schistocerca gregaria.

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    Boyan, George; Posser, Susanne; Ludwig, Peter; Güntner, Michaela; Williams, Lez

    2004-04-01

    In this paper, we propose an ontogeny for previously identified cells from the median domain in the midline of the embryonic brain of the grasshopper Schistocerca gregaria. The so-called lateral cells (LCs) are characteristically located laterally within the median domain at its border with the protocerebral hemispheres. The LC occurs singly and can be identified in the early embryo on the basis of their expression of the cell surface lipocalin Lazarillo. Using immunocytochemical, dye injection, electron microscopical and histological methods, we show that these LC are neurons and derive as postmitotic cells directly from the epithelium of the median domain. Further, they and the other identified cells of the median domain such as the protocerebral commissure pioneers (PCP), co-express the Mes-3 antigen, consistent with a derivation from the mesectodermal germ layer of the embryo. Subsequent to axogenesis, electron microscopy reveals that these Mes-3-expressing LC fasciculate with the co-expressing PCPs within the developing protocerebral commissure. We present a model for the origin of all these cells based on histological data and bromodeoxyuridine incorporation. The model suggests a delamination of cells from the mesectoderm followed by a migration to their ultimate sites within the median domain.

  18. Functional analysis of C1 family cysteine peptidases in the larval gut of Tenebrio molitor and Tribolium castaneum

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    We studied protein digestion the tenebrionids Tenebrio molitor and Tribolium castaneum, pests of stored grains and grain products, to identify potential targets for biopesticide development. Tenebrionid larvae have highly compartmentalized guts, with primarily cysteine peptidases in the acidic anter...

  19. Network Analysis Identifies Crosstalk Interactions Governing TGF-β Signaling Dynamics during Endoderm Differentiation of Human Embryonic Stem Cells

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    Shibin Mathew

    2015-05-01

    Full Text Available The fate choice of human embryonic stem cells (hESCs is controlled by complex signaling milieu synthesized by diverse chemical factors in the growth media. Prevalence of crosstalks and interactions between parallel pathways renders any analysis probing the process of fate transition of hESCs elusive. This work presents an important step in the evaluation of network level interactions between signaling molecules controlling endoderm lineage specification from hESCs using a statistical network identification algorithm. Network analysis was performed on detailed signaling dynamics of key molecules from TGF-β/SMAD, PI3K/AKT and MAPK/ERK pathways under two common endoderm induction conditions. The results show the existence of significant crosstalk interactions during endoderm signaling and they identify differences in network connectivity between the induction conditions in the early and late phases of signaling dynamics. Predicted networks elucidate the significant effect of modulation of AKT mediated crosstalk leading to the success of PI3K inhibition in inducing efficient endoderm from hESCs in combination with TGF-β/SMAD signaling.

  20. Glutathione S-transferase (GST) genes in the red flour beetle, Tribolium castaneum, and comparative analysis with five additional insects.

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    Shi, Houxia; Pei, Lianghong; Gu, Shasha; Zhu, Shicheng; Wang, Yanyun; Zhang, Yi; Li, Bin

    2012-11-01

    Glutathione S-transferases are important detoxification enzymes involved in insecticide resistance. Sequencing the Tribolium castaneum genome provides an opportunity to investigate the structure, function, and evolution of GSTs on a genome-wide scale. Thirty-six putative cytosolic GSTs and 5 microsomal GSTs have been identified in T. castaneum. Furthermore, 40, 35, 13, 23, and 32 GSTs have been discovered the other insects, Drosophila, Anopheles, Apis, Bombyx, and Acyrthosiphon, respectively. Phylogenetic analyses reveal that insect-specific GSTs, Epsilon and Delta, are the largest species-specific expanded GSTs. In T. castaneum, most GSTs are tandemly arranged in three chromosomes. Particularly, Epsilon GSTs have an inverted long-fragment duplication in the genome. Other four widely distributed classes are highly conserved in all species. Given that GSTs specially expanded in Tribolium castaneum, these genes might help to resist poisonous chemical environments and produce resistance to kinds of different insecticides.

  1. Antifeedant Diterpenoids against Tribolium castaneum from the Stems and Twigs of Ceriops tagal (Rhizophoraceae

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    Zhi Wei Deng

    2011-07-01

    Full Text Available The screening of several Chinese mangrove plants for insecticidal principles showed that ethanol extract of Ceriops tagal stems and twigs possessed significant feeding deterrent activity against the red flour beetle, Tribolium castaneum (Family: Rhizophoraceae. From the ethanol extract, three feeding deterrent diterpenoids were isolated by bioassay-guided fractionation. The compounds were identified as tagalsin A, B, and H on the basis of their phytochemical and spectral data. Tagalsin A, B, and H exhibited strong feeding deterrent activity against T. castaneum adults with EC50 values of 375.3 ppm, 277.3 ppm, and 285.45 ppm, respectively.

  2. [Karyosphere capsule in Tribolium castaneum oocytes].

    Science.gov (United States)

    Batalova, F M; Bogoliubov, D S

    2013-01-01

    Structure and composition of the karyosphere (karyosome) capsule were studied in the oocytes of a laboratory insect, Tribolium castaneum, with the use of electron microscopy and immunoelectron cytochemistry. Basing on the study of nuclear structure dynamics, we distinguished 8 stages that characterize the period of oocyte growth. At the diplotene stage, T. castaneum oocyte chromosomes conjoin early into a compact karyosphere, but a significant chromatin condensation does not occur. The process of karyosphere formation is accompanied by the development of an extensive extrachromosome capsule surrounding chromatin. The capsule consists of a material of different morphological types. Significant molecular components of the T. castaneum karyosphere capsule are represented by the proteins of nuclear matrix including F-actin and lamin B. Besides the structural proteins, the Sm proteins of small nuclear (sn) RNPs and mature 2,2,7-trimethyl guanosine (TMG) 5'-capped snRNAs are revealed immunocytochemically in the karyosphere capsule. The obtained data can form a basis for further expansion of ideas on the functions of the karyosphere capsule as a specialized extrachromosomal nuclear domain of the oocytes. We believe that the T. castaneum karyosphere capsule plays not only a structural role, but may be involved directly in the processes related to gene expression.

  3. Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics

    Science.gov (United States)

    Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultur...

  4. A family of chemoreceptors in Tribolium castaneum (Tenebrionidae: Coleoptera.

    Directory of Open Access Journals (Sweden)

    Mohatmed Abdel-Latief

    Full Text Available Chemoperception in invertebrates is mediated by a family of G-protein-coupled receptors (GPCR. To date nothing is known about the molecular mechanisms of chemoperception in coleopteran species. Recently the genome of Tribolium castaneum was sequenced for use as a model species for the Coleoptera. Using blast searches analyses of the T. castaneum genome with previously predicted amino acid sequences of insect chemoreceptor genes, a putative chemoreceptor family consisting of 62 gustatory receptors (Grs and 26 olfactory receptors (Ors was identified. The receptors have seven transmembrane domains (7TMs and all belong to the GPCR receptor family. The expression of the T. castaneum chemoreceptor genes was investigated using quantification real- time RT-PCR and in situ whole mount RT-PCR analysis in the antennae, mouth parts, and prolegs of the adults and larvae. All of the predicted TcasGrs were expressed in the labium, maxillae, and prolegs of the adults but TcasGr13, 19, 28, 47, 62, 98, and 61 were not expressed in the prolegs. The TcasOrs were localized only in the antennae and not in any of the beetles gustatory organs with one exception; the TcasOr16 (like DmelOr83b, which was localized in the antennae, labium, and prolegs of the beetles. A group of six TcasGrs that presents a lineage with the sugar receptors subfamily in Drosophila melanogaster were localized in the lacinia of the Tribolium larvae. TcasGr1, 3, and 39, presented an ortholog to CO(2 receptors in D. melanogaster and Anopheles gambiae was recorded. Low expression of almost all of the predicted chemoreceptor genes was observed in the head tissues that contain the brains and suboesophageal ganglion (SOG. These findings demonstrate the identification of a chemoreceptor family in Tribolium, which is evolutionarily related to other insect species.

  5. Expression patterns of cysteine peptidase genes across the Tribolium castaneum life cycle provide clues to biological function.

    Science.gov (United States)

    Perkin, Lindsey; Elpidina, Elena N; Oppert, Brenda

    2016-01-01

    The red flour beetle, Tribolium castaneum, is a major agricultural pest responsible for considerable loss of stored grain and cereal products worldwide. T. castaneum larvae have a highly compartmentalized gut, with cysteine peptidases mostly in the acidic anterior part of the midgut that are critical to the early stages of food digestion. In previous studies, we described 26 putative cysteine peptidase genes in T. castaneum (types B, L, O, F, and K) located mostly on chromosomes 3, 7, 8, and 10. In the present study, we hypothesized that specific cysteine peptidase genes could be associated with digestive functions for food processing based on comparison of gene expression profiles in different developmental stages, feeding and non-feeding. RNA-Seq was used to determine the relative expression of cysteine peptidase genes among four major developmental stages (egg, larvae, pupae, and adult) of T. castaneum. We also compared cysteine peptidase genes in T. castaneum to those in other model insects and coleopteran pests. By combining transcriptome expression, phylogenetic comparisons, response to dietary inhibitors, and other existing data, we identified key cysteine peptidases that T. castaneum larvae and adults use for food digestion, and thus new potential targets for biologically-based control products.

  6. A Bmp Reporter Transgene Mouse Embryonic Stem Cell Model as a Tool to Identify and Characterize Chemical Teratogens.

    Science.gov (United States)

    Kugler, Josephine; Tharmann, Julian; Chuva de Sousa Lopes, Susana M; Kemler, Rolf; Luch, Andreas; Oelgeschläger, Michael

    2015-08-01

    Embryonic stem cells (ESCs) were first isolated from mouse embryos more than 30 years ago. They have proven invaluable not only in generating genetically modified mice that allow for analysis of gene function in tissue development and homeostasis but also as models for genetic disease. In addition, ESCs in vitro are finding inroads in pharmaceutical and toxicological testing, including the identification of teratogenic compounds. Here, we describe the use of a bone morphogenetic protein (Bmp)-reporter ESC line, isolated from a well-characterized transgenic mouse line, as a new tool for the identification of chemical teratogens. The Bmp-mediated expression of the green fluorescent protein enabled the quantification of dose- and time-dependent effects of valproic acid as well as retinoic acid. Significant effects were detectable at concentrations that were comparable to the ones observed in the classical embryonic stem cell test, despite the fact that the reporter gene is expressed in distinct cell types, including endothelial and endodermal cells. Thus these cells provide a valuable new tool for the identification and characterization of relevant mechanisms of embryonic toxicity.

  7. iBeetle-Base: a database for RNAi phenotypes in the red flour beetle Tribolium castaneum.

    Science.gov (United States)

    Dönitz, Jürgen; Schmitt-Engel, Christian; Grossmann, Daniela; Gerischer, Lizzy; Tech, Maike; Schoppmeier, Michael; Klingler, Martin; Bucher, Gregor

    2015-01-01

    The iBeetle-Base (http://ibeetle-base.uni-goettingen.de) makes available annotations of RNAi phenotypes, which were gathered in a large scale RNAi screen in the red flour beetle Tribolium castaneum (iBeetle screen). In addition, it provides access to sequence information and links for all Tribolium castaneum genes. The iBeetle-Base contains the annotations of phenotypes of several thousands of genes knocked down during embryonic and metamorphic epidermis and muscle development in addition to phenotypes linked to oogenesis and stink gland biology. The phenotypes are described according to the EQM (entity, quality, modifier) system using controlled vocabularies and the Tribolium morphological ontology (TrOn). Furthermore, images linked to the respective annotations are provided. The data are searchable either for specific phenotypes using a complex 'search for morphological defects' or a 'quick search' for gene names and IDs. The red flour beetle Tribolium castaneum has become an important model system for insect functional genetics and is a representative of the most species rich taxon, the Coleoptera, which comprise several devastating pests. It is used for studying insect typical development, the evolution of development and for research on metabolism and pest control. Besides Drosophila, Tribolium is the first insect model organism where large scale unbiased screens have been performed.

  8. Tribolium castaneum as a model for high-throughput RNAi screening.

    Science.gov (United States)

    Knorr, Eileen; Bingsohn, Linda; Kanost, Michael R; Vilcinskas, Andreas

    2013-01-01

    Coleopteran insects are a highly diverse and successful order, and many beetle species are significant agricultural pests. New biorational strategies for managing populations of beetles and other insect species are needed as pests develop resistance to chemical insecticides and Bt toxins. There is now an opportunity to use genome sequence data to identify genes that are essential for insect growth, development, or survival as new targets for designing control technology. This goal requires a method for high-throughput in vivo screening of thousands of genes to identify candidate genes that, when their expression is disrupted, have a phenotype that may be useful in insect pest control. Tribolium castaneum, the red flour beetle, is a model organism that offers considerable advantages for such screening, including ease of rearing in large numbers, a sequenced genome, and a strong, systemic RNAi response for specific depletion of gene transcripts. The RNAi effect in T. castaneum can be elicited in any tissue and any stage by the injection of dsRNA into the hemocoel, and injection of dsRNA into adult females can even be used to identify phenotypes in offspring. A pilot RNAi screen (iBeetle) is underway. Several T. castaneum genes with promising RNAi phenotypes for further development as mechanisms for plant protection have been identified. These include heat shock protein 90, chitin synthase, the segmentation gene hairy, and a matrix metalloprotease. Candidate genes identified in T. castaneum screens can then be tested in agricultural pest species (in which screening is not feasible), to evaluate their effectiveness for use in potential plant-based RNAi control strategies. Delivery of dsRNA expressed by genetically modified crops to the midgut of phytophagous insects is under investigation as a new tool for very specific protection of plants from insect pest species. The T. castaneum screening platform offers a system for discovery of candidate genes with high potential

  9. Screen for abnormal mitochondrial phenotypes in mouse embryonic stem cells identifies a model for succinyl-CoA ligase deficiency and mtDNA depletion

    Directory of Open Access Journals (Sweden)

    Taraka R. Donti

    2014-02-01

    Full Text Available Mutations in subunits of succinyl-CoA synthetase/ligase (SCS, a component of the citric acid cycle, are associated with mitochondrial encephalomyopathy, elevation of methylmalonic acid (MMA, and mitochondrial DNA (mtDNA depletion. A FACS-based retroviral-mediated gene trap mutagenesis screen in mouse embryonic stem (ES cells for abnormal mitochondrial phenotypes identified a gene trap allele of Sucla2 (Sucla2SAβgeo, which was used to generate transgenic mice. Sucla2 encodes the ADP-specific β-subunit isoform of SCS. Sucla2SAβgeo homozygotes exhibited recessive lethality, with most mutants dying late in gestation (e18.5. Mutant placenta and embryonic (e17.5 brain, heart and muscle showed varying degrees of mtDNA depletion (20–60%. However, there was no mtDNA depletion in mutant liver, where the gene is not normally expressed. Elevated levels of MMA were observed in embryonic brain. SCS-deficient mouse embryonic fibroblasts (MEFs demonstrated a 50% reduction in mtDNA content compared with wild-type MEFs. The mtDNA depletion resulted in reduced steady state levels of mtDNA encoded proteins and multiple respiratory chain deficiencies. mtDNA content could be restored by reintroduction of Sucla2. This mouse model of SCS deficiency and mtDNA depletion promises to provide insights into the pathogenesis of mitochondrial diseases with mtDNA depletion and into the biology of mtDNA maintenance. In addition, this report demonstrates the power of a genetic screen that combines gene trap mutagenesis and FACS analysis in mouse ES cells to identify mitochondrial phenotypes and to develop animal models of mitochondrial dysfunction.

  10. Methuselah-like genes affect development, stress resistance, lifespan and reproduction in Tribolium castaneum.

    Science.gov (United States)

    Li, Chengjun; Zhang, Yi; Yun, Xiaopei; Wang, Yanyun; Sang, Ming; Liu, Xing; Hu, Xingxing; Li, Bin

    2014-10-01

    Methuselah (Mth) is associated with lifespan, stress resistance and reproduction in Drosophila melanogaster, but Mth is not present in nondrosophiline insects. A number of methuselah-likes (mthls) have been identified in nondrosophiline insects, but it is unknown whether the functions of mth are shared by mthls or are divergent from them. Five mthls have been identified in Tribolium castaneum. Although they have different developmental expression patterns, they all enhance resistance to starvation. Only mthl1 and mthl2 enhance resistance to high temperature, whereas mthl4 and mthl5 negatively regulate oxidative stress in T. castaneum. Unlike in the fly with mth mutation, knockdown of mthls, except mthl3, shortens the lifespan of T. castaneum. Moreover, mthl1 and mthl2 are critical for Tribolium development. mthl1 plays important roles in larval and pupal development and adult eclosion, while mthl2 is required for eclosion. Moreover, mthl1 and mthl2 silencing reduces the fertility of T. castaneum, and mthl1 and mthl4 are also essential for embryo development. In conclusion, mthls have a significant effect on insect development, lifespan, stress resistance and reproduction. These results provide experimental evidence for functional divergence among mthls/mth and clues for the signal transduction of Mthls.

  11. Next generation sequencing based transcriptome analysis of septic-injury responsive genes in the beetle Tribolium castaneum.

    Science.gov (United States)

    Altincicek, Boran; Elashry, Abdelnaser; Guz, Nurper; Grundler, Florian M W; Vilcinskas, Andreas; Dehne, Heinz-Wilhelm

    2013-01-01

    Beetles (Coleoptera) are the most diverse animal group on earth and interact with numerous symbiotic or pathogenic microbes in their environments. The red flour beetle Tribolium castaneum is a genetically tractable model beetle species and its whole genome sequence has recently been determined. To advance our understanding of the molecular basis of beetle immunity here we analyzed the whole transcriptome of T. castaneum by high-throughput next generation sequencing technology. Here, we demonstrate that the Illumina/Solexa sequencing approach of cDNA samples from T. castaneum including over 9.7 million reads with 72 base pairs (bp) length (approximately 700 million bp sequence information with about 30× transcriptome coverage) confirms the expression of most predicted genes and enabled subsequent qualitative and quantitative transcriptome analysis. This approach recapitulates our recent quantitative real-time PCR studies of immune-challenged and naïve T. castaneum beetles, validating our approach. Furthermore, this sequencing analysis resulted in the identification of 73 differentially expressed genes upon immune-challenge with statistical significance by comparing expression data to calculated values derived by fitting to generalized linear models. We identified up regulation of diverse immune-related genes (e.g. Toll receptor, serine proteinases, DOPA decarboxylase and thaumatin) and of numerous genes encoding proteins with yet unknown functions. Of note, septic-injury resulted also in the elevated expression of genes encoding heat-shock proteins or cytochrome P450s supporting the view that there is crosstalk between immune and stress responses in T. castaneum. The present study provides a first comprehensive overview of septic-injury responsive genes in T. castaneum beetles. Identified genes advance our understanding of T. castaneum specific gene expression alteration upon immune-challenge in particular and may help to understand beetle immunity in general.

  12. Tcmof regulates larval/pupal development and female fecundity in red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Wang, Yanyun; Li, Chengjun; Sang, Ming; Li, Bin

    2015-02-01

    Males absent on the first (MOF) was originally identified as an essential component of the X chromosome dosage compensation system in Drosophila melanogaster, and is also a member of the MYST family of histone acetyltransferases. MOF has been extensively studied in D. melanogaster and mammals. However, whether MOF is involved in dosage compensation and/or other vital functions for newly emerging model insects such as Tribolium castaneum, is unclear. We cloned the mof from T. castaneum, named Tcmof. Phylogenetic analysis revealed that mof is highly conserved in eukaryotes but lost in birds. qPCR showed that Tcmof was most highly expressed in the early embryo stage and equally expressed in males and females. Treating larvae with ds-Tcmof led 79.1% of the insects to arrest during its eclosion; the remaining insects died either in the larval stage or immediately following eclosion. Treating pupae with the same construct eliminated the fertility of T. castaneum. This effect was rescued by reciprocal crosses with wild-type females, but not males. We infer that the mof gene is essential for larval/pupal development and female fertility in T. castaneum.

  13. A Soluble Pyrophosphatase Is Essential to Oogenesis and Is Required for Polyphosphate Metabolism in the Red Flour Beetle (Tribolium castaneum)

    Science.gov (United States)

    Carvalho, Klébea; Ribeiro, Lupis; Moraes, Jorge; da Silva, José Roberto; Costa, Evenilton P.; Souza-Menezes, Jackson; Logullo, Carlos; Nunes da Fonseca, Rodrigo; Campos, Eldo

    2015-01-01

    Polyphosphates have been found in all cell types examined to date and play diverse roles depending on the cell type. In eukaryotic organisms, polyphosphates have been mainly investigated in mammalian cells with few studies on insects. Some studies have demonstrated that a pyrophosphatase regulates polyphosphate metabolism, and most of them were performed on trypanosomatids. Here, we investigated the effects of sPPase gene knocked down in oogenesis and polyphosphate metabolism in the red flour beetle (Tribolium castaneum) A single sPPase gene was identified in insect genome and is maternally provided at the mRNA level and not restricted to any embryonic or extraembryonic region during embryogenesis. After injection of Tc-sPPase dsRNA, female survival was reduced to 15% of the control (dsNeo RNA), and egg laying was completely impaired. The morphological analysis by nuclear DAPI staining of the ovarioles in Tc-sPPase dsRNA-injected females showed that the ovariole number is diminished, degenerated oocytes can be observed, and germarium is reduced. The polyphosphate level was increased in cytoplasmic and nuclear fractions in Tc-sPPase RNAi; Concomitantly, the exopolyphosphatase activity decreased in both fractions. Altogether, these data suggest a role for sPPase in the regulation on polyphosphate metabolism in insects and provide evidence that Tc-sPPase is essential to oogenesis. PMID:25811926

  14. A Soluble Pyrophosphatase Is Essential to Oogenesis and Is Required for Polyphosphate Metabolism in the Red Flour Beetle (Tribolium castaneum

    Directory of Open Access Journals (Sweden)

    Klébea Carvalho

    2015-03-01

    Full Text Available Polyphosphates have been found in all cell types examined to date and play diverse roles depending on the cell type. In eukaryotic organisms, polyphosphates have been mainly investigated in mammalian cells with few studies on insects. Some studies have demonstrated that a pyrophosphatase regulates polyphosphate metabolism, and most of them were performed on trypanosomatids. Here, we investigated the effects of sPPase gene knocked down in oogenesis and polyphosphate metabolism in the red flour beetle (Tribolium castaneum. A single sPPase gene was identified in insect genome and is maternally provided at the mRNA level and not restricted to any embryonic or extraembryonic region during embryogenesis. After injection of Tc-sPPase dsRNA, female survival was reduced to 15% of the control (dsNeo RNA, and egg laying was completely impaired. The morphological analysis by nuclear DAPI staining of the ovarioles in Tc-sPPase dsRNA-injected females showed that the ovariole number is diminished, degenerated oocytes can be observed, and germarium is reduced. The polyphosphate level was increased in cytoplasmic and nuclear fractions in Tc-sPPase RNAi; Concomitantly, the exopolyphosphatase activity decreased in both fractions. Altogether, these data suggest a role for sPPase in the regulation on polyphosphate metabolism in insects and provide evidence that Tc-sPPase is essential to oogenesis.

  15. Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells.

    Science.gov (United States)

    Phanstiel, Doug; Brumbaugh, Justin; Berggren, W Travis; Conard, Kevin; Feng, Xuezhu; Levenstein, Mark E; McAlister, Graeme C; Thomson, James A; Coon, Joshua J

    2008-03-18

    Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally, antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails, but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here, we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally, we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For example, H4R3 methylation was observed only in the presence of H4K20 dimethylation; such context-specific patterning exemplifies the power of this technique.

  16. Expressional and functional analysis of CYP15A1, a juvenile hormone epoxidase, in the red flour beetle Tribolium castaneum.

    Science.gov (United States)

    Minakuchi, Chieka; Ishii, Fumika; Washidu, Yumiko; Ichikawa, Akio; Tanaka, Toshiharu; Miura, Ken; Shinoda, Tetsuro

    2015-09-01

    Juvenile hormone (JH) is synthesized and secreted by the corpora allata. In the final two steps of JH biosynthesis, farnesoic acid (FA) is converted to JH through methylation by JH acid O-methyltransferase (JHAMT) and epoxidation by the cytochrome P450 enzyme CYP15. In the present study, we identified a homolog of CYP15 from the red flour beetle Tribolium castaneum (TcCYP15A1), and analyzed its expression as well as its role in JH biosynthesis. Quantitative RT-PCR analysis showed that the level of TcCYP15A1 mRNA was high in the embryonic stage as well as in the middle of the final larval instar. In the embryonic stage, the transcript level of TcCYP15A1 started to increase 30h after egg laying (AEL), peaked 54-60h AEL, and was followed by an increase of TcJHAMT mRNA, suggesting that JH biosynthesis started at this time point. TcCYP15A1 mRNA was present, but not exclusively so in the larval corpora allata. The recombinant TcCYP15A1 protein epoxidized both FA and methyl farnesoate (MF) in highly stereo-specific manners. These results confirmed that TcCYP15A1 is involved in JH biosynthesis. The RNAi-mediated knockdown of TcCYP15A1 in the pre-final larval instar did not result in precocious metamorphosis to pupa, indicating that MF may exhibit JH-like activity in order to maintain the larval status. The double knockdown of TcJHAMT and TcCYP15A1 resulted in pupae and adults with shorter wings, suggesting that the precursors of JH, JH acid and MF, may be essential for wing expansion.

  17. Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics

    Energy Technology Data Exchange (ETDEWEB)

    Kleinstreuer, N.C., E-mail: kleinstreuer.nicole@epa.gov [NCCT, US EPA, RTP, NC 27711 (United States); Smith, A.M.; West, P.R.; Conard, K.R.; Fontaine, B.R. [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States); Weir-Hauptman, A.M. [Covance, Inc., Madison, WI 53704 (United States); Palmer, J.A. [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States); Knudsen, T.B.; Dix, D.J. [NCCT, US EPA, RTP, NC 27711 (United States); Donley, E.L.R. [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States); Cezar, G.G. [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States); University of Wisconsin-Madison, Madison, WI 53706 (United States)

    2011-11-15

    Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast Trade-Mark-Sign chemical screening and prioritization research project. Metabolites from hES cultures were evaluated for known and novel signatures that may be indicative of developmental toxicity. Significant fold changes in endogenous metabolites were detected for 83 putatively annotated mass features in response to the subset of ToxCast chemicals. The annotations were mapped to specific human metabolic pathways. This revealed strong effects on pathways for nicotinate and nicotinamide metabolism, pantothenate and CoA biosynthesis, glutathione metabolism, and arginine and proline metabolism pathways. Predictivity for adverse outcomes in mammalian prenatal developmental toxicity studies used ToxRefDB and other sources of information, including Stemina Biomarker Discovery's predictive DevTox Registered-Sign model trained on 23 pharmaceutical agents of known developmental toxicity and differing potency. The model initially predicted developmental toxicity from the blinded ToxCast compounds in concordance with animal data with 73% accuracy. Retraining the model with data from the unblinded test compounds at one concentration level increased the predictive accuracy for the remaining concentrations to 83%. These preliminary results on a 11-chemical subset of the ToxCast chemical library indicate that metabolomics analysis of the hES secretome provides information valuable for predictive modeling and mechanistic understanding of mammalian developmental toxicity. -- Highlights: Black-Right-Pointing-Pointer We tested 11 environmental compounds in a hESC metabolomics platform. Black-Right-Pointing-Pointer Significant changes in secreted small molecule metabolites were observed. Black-Right-Pointing-Pointer Perturbed mass features map to pathways critical for normal

  18. Anatomical localization and stereoisomeric composition of Tribolium castaneum aggregation pheromones

    Science.gov (United States)

    Lu, Yujie; Beeman, Richard W.; Campbell, James F.; Park, Yoonseong; Aikins, Michael J.; Mori, Kenji; Akasaka, Kazuaki; Tamogami, Shigeyuki; Phillips, Thomas W.

    2011-09-01

    We report that the abdominal epidermis and associated tissues are the predominant sources of male-produced pheromones in the red flour beetle, Tribolium castaneum and, for the first time, describe the stereoisomeric composition of the natural blend of isomers of the aggregation pheromone 4,8-dimethyldecanal (DMD) in this important pest species. Quantitative analyses via gas chromatography-mass spectrometry showed that the average amount of DMD released daily by single feeding males of T. castaneum was 878 ± 72 ng (SE). Analysis of different body parts identified the abdominal epidermis as the major source of aggregation pheromone; the thorax was a minor source, while no DMD was detectable in the head. No internal organs or obvious male-specific glands were associated with pheromone deposition. Complete separation of all four stereoisomers of DMD was achieved following oxidation to the corresponding acid, derivatization with (1 R, 2 R)- and (1 S, 2 S)-2-(anthracene-2,3-dicarboximido)cyclohexanol to diastereomeric esters, and their separation on reversed-phase high-performance liquid chromatography at -54°C. Analysis of the hexane eluate from Porapak-Q-collected volatiles from feeding males revealed the presence of all four isomers (4 R,8 R)/(4 R,8 S)/(4 S,8 R)/(4 S,8 S) at a ratio of approximately 4:4:1:1. A walking orientation bioassay in a wind tunnel with various blends of the four synthetic isomers further indicated that the attractive potency of the reconstituted natural blend of 4:4:1:1 was equivalent to that of the natural pheromone and greater than that of the 1:1 blend of (4 R,8 R)/(4 R,8 S) used in commercial lures.

  19. Systems biology approach identifies the kinase Csnk1a1 as a regulator of the DNA damage response in embryonic stem cells

    DEFF Research Database (Denmark)

    Carreras Puigvert, Jordi; von Stechow, Louise; Siddappa, Ramakrishnaiah

    2013-01-01

    In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference...... screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin. Networks derived from canonical pathways shared in all three data sets were implicated in DNA...... damage repair, cell cycle and survival, and differentiation. Experimental probing of these networks identified a mode of DNA damage-induced Wnt signaling that limited apoptosis. Silencing or deleting the p53 gene demonstrated that genotoxic stress elicited Wnt signaling in a p53-independent manner...

  20. Gene families of cuticular proteins analogous to peritrophins (CPAPs in Tribolium castaneum have diverse functions.

    Directory of Open Access Journals (Sweden)

    Sinu Jasrapuria

    Full Text Available The functional characterization of an entire class of 17 genes from the red flour beetle, Tribolium castaneum, which encode two families of Cuticular Proteins Analogous to Peritrophins (CPAPs has been carried out. CPAP genes in T. castaneum are expressed exclusively in cuticle-forming tissues and have been classified into two families, CPAP1 and CPAP3, based on whether the proteins contain either one (CPAP1, or three copies (CPAP3 of the chitin-binding domain, ChtBD2, with its six characteristically spaced cysteine residues. Individual members of the TcCPAP1 and TcCPAP3 gene families have distinct developmental patterns of expression. Many of these proteins serve essential and non-redundant functions in maintaining the structural integrity of the cuticle in different parts of the insect anatomy. Three genes of the TcCPAP1 family and five genes of the TcCPAP3 family are essential for insect development, molting, cuticle integrity, proper locomotion or fecundity. RNA interference (RNAi targeting TcCPAP1-C, TcCPAP1-H, TcCPAP1-J or TcCPAP3-C transcripts resulted in death at the pharate adult stage of development. RNAi for TcCPAP3-A1, TcCPAP3-B, TcCPAP3-D1 or TcCPAP3-D2 genes resulted in different developmental defects, including adult/embryonic mortality, abnormal elytra or hindwings, or an abnormal 'stiff-jointed' gait. These results provide experimental support for specialization in the functions of CPAP proteins in T. castaneum and a biological rationale for the conservation of CPAP orthologs in other orders of insects. This is the first comprehensive functional analysis of an entire class of cuticular proteins with one or more ChtBD2 domains in any insect species.

  1. Fine-scale analysis of parasite resistance genes in the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Zhong, Daibin; Pai, Aditi; Wang, Mei-Hui; Keech, Naomi; Yan, Guiyun

    2013-09-01

    Parasite infection impacts population dynamics through effects on fitness and fecundity of the individual host. In addition to the known roles of environmental factors, host susceptibility to parasites has a genetic basis that has not been well characterized. We previously mapped quantitative trait loci (QTL) for susceptibility to rat tapeworm (Hymenolepis diminuta) infection in Tribolium castaneum using dominant AFLP markers; however, the resistance genes were not identified. Here, we refined the QTL locations and increased the marker density in the QTL regions using new microsatellite markers, sequence-tagged site markers, and single-strand conformational polymorphism markers. Resistance QTL in three linkage groups (LG3, LG6, and LG8) were each mapped to intervals castaneum resistance to H. diminuta infection.

  2. Repellent Constituents of essential oil from Citrus wilsonii stem barks against Tribolium castaneum.

    Science.gov (United States)

    Wua, Yan; Chenb, Hai-Ping; Wei, Jian-Yu; Yang, Kai; Tian, Zhao-Fu; Li, Xiao-Lan; Wang, Ping-Juan; Wang, Cheng-Fang; Du, Shu-Shan; Cai, Qian

    2014-10-01

    The essential oil obtained from Citrus wilsonii Tanaka stem barks with hydrodistillation was investigated by GC-FID and GC-MS. The main components of the essential oil were identified to be nerol acetate (44.5%), nerol (13.6%), citronellyl propionate (13.5%) and α-terpineol (3.6%). Among them, the four active constituents, predicted with a bioactivity-test, were isolated and identified as nerolacetate, nerol, citronellyl propionate and α-terpineol. It was found that the essential oil of C. wilsonii stem barks possessed strong repellency (86% and 92%, respectively, at 78.6 nL/cm2, after 2 and 4 h treatment) against Tribolium castaneum adults. Repellency of the four active compounds was also determined. Nerolacetate, nerol, citronellyl propionate and α-terpineol were strongly repellent (100%, 100%, 90% and 96%, respectively, at 15.7 nL/cm2, after 2h treatment) against T. castaneum. Nerol exhibited the same level of repellency as the positive control, DEET. The results indicate that the essential oil of C. wilsonii stem barks and its active compounds have the potential to be developed as natural repellents for control of T. castaneum.

  3. Larval RNA interference in the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Linz, David M; Clark-Hachtel, Courtney M; Borràs-Castells, Ferran; Tomoyasu, Yoshinori

    2014-10-13

    The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle's body cavity. In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

  4. Identification and characterization of novel ER-based hsp90 gene in the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Zhang, Yi; Gu, Shasha; Li, Chengjun; Sang, Ming; Wu, Wei; Yun, Xiaopei; Hu, Xingxing; Li, Bin

    2014-09-01

    Heat-shock protein 90 (HSP90) is a highly conserved molecular chaperone found in all species except for Archaea, which is required not only for stress tolerance but also for normal development. Recently, it was reported that HSP83, one member of the cytosolic HSP90 family, contributes to oogenesis and responds to heat resistance in Tribolium castaneum. Here, a novel ER-based HSP90 gene, Tchsp90, has been identified in T. castaneum. Phylogenetic analysis showed that hsp90s and hsp83s evolved separately from a common ancestor but that hsp90s originated earlier. Quantitative real-time polymerase chain reaction illustrated that Tchsp90 is expressed in all developmental stages and is highly expressed at early pupa and late adult stages. Tchsp90 was upregulated in response to heat stress but not to cold stress. Laval RNAi revealed that Tchsp90 is important for larval/pupal development. Meanwhile, parental RNAi indicated that it completely inhibited female fecundity and partially inhibited male fertility once Tchsp90 was knocked down and that it will further shorten the lifespan of T. castaneum. These results suggest that Tchsp90 is essential for development, lifespan, and reproduction in T. castaneum in addition to its response to heat stress.

  5. Insecticidal properties of essential oils against Tribolium castaneum (Herbst) and their inhibitory effects on acetylcholinesterase and adenosine triphosphatases.

    Science.gov (United States)

    Abou-Taleb, Hamdy K; Mohamed, Magdy I E; Shawir, Mohamed S; Abdelgaleil, Samir A M

    2016-01-01

    Essential oils from 20 Egyptian plants were obtained by using hydrodistillation. The chemical composition of the isolated oils was identified by gas chromatograph/mass spectrometer. Fumigant and contact toxicities of the essential oils were evaluated against the adults of Tribolium castaneum. In fumigation assays, the oil of Origanum vulgare (LC50 = 9.97 mg/L air) displayed the highest toxicity towards the adults of T. castaneum. In contact assays, the oils of Artemisia monosperma (LC50 = 0.07 mg/cm(2)) and O. vulgare (LC50 = 0.07 mg/cm(2)) were the most potent toxicants against the adults of T. castaneum. Biochemical studies showed that the tested oils caused pronounced inhibition of acetylcholinesterase (AChE) and adenosine triphosphatases (ATPases) isolated from the larvae of T. castaneum. The oil Cupressus macrocarpa (IC50 = 12.3 mg/L) was the most potent inhibitor of AChE, while the oil of Calistemon viminals (IC50 = 4.4 mg/L) was the most potent inhibitor of ATPases.

  6. The expression and function of the achaete-scute genes in Tribolium castaneum reveals conservation and variation in neural pattern formation and cell fate specification

    Science.gov (United States)

    Wheeler, Scott R.; Carrico, Michelle L.; Wilson, Beth A.; Brown, Susan J.; Skeath, James B.

    2003-01-01

    The study of achaete-scute (ac/sc) genes has recently become a paradigm to understand the evolution and development of the arthropod nervous system. We describe the identification and characterization of the ac/sc genes in the coleopteran insect species Tribolium castaneum. We have identified two Tribolium ac/sc genes - achaete-scute homolog (Tc-ASH) a proneural gene and asense (Tc-ase) a neural precursor gene that reside in a gene complex. Focusing on the embryonic central nervous system we find that Tc-ASH is expressed in all neural precursors and the proneural clusters from which they segregate. Through RNAi and misexpression studies we show that Tc-ASH is necessary for neural precursor formation in Tribolium and sufficient for neural precursor formation in Drosophila. Comparison of the function of the Drosophila and Tribolium proneural ac/sc genes suggests that in the Drosophila lineage these genes have maintained their ancestral function in neural precursor formation and have acquired a new role in the fate specification of individual neural precursors. Furthermore, we find that Tc-ase is expressed in all neural precursors suggesting an important and conserved role for asense genes in insect nervous system development. Our analysis of the Tribolium ac/sc genes indicates significant plasticity in gene number, expression and function, and implicates these modifications in the evolution of arthropod neural development.

  7. Malpighian tubule development in the red flour beetle (Tribolium castaneum).

    Science.gov (United States)

    King, Benedict; Denholm, Barry

    2014-11-01

    Malpighian tubules (MpTs) are the major organ for excretion and osmoregulation in most insects. MpT development is characterised for Drosophila melanogaster, but not other species. We therefore do not know the extent to which the MpT developmental programme is conserved across insects. To redress this we provide a comprehensive description of MpT development in the beetle Tribolium castaneum (Coleoptera), a species separated from Drosophila by >315 million years. We identify similarities with Drosophila MpT development including: 1) the onset of morphological development, beginning when tubules bud from the gut and proliferate to increase organ size. 2) the tubule is shaped by convergent-extension movements and oriented cell divisions. 3) differentiated tip cells activate EGF-signalling in distal MpT cells through the ligand Spitz. 4) MpTs contain two main cell types - principal and stellate cells, differing in morphology and gene expression. We also describe development of the beetle cryptonephridial system, an adaptation for water conservation, which represents a major modification of the MpT ground plan characterised by intimate association between MpTs and rectum. This work establishes a new model to compare MpT development across insects, and provides a framework to help understand how an evolutionary novelty - the cryptonephridial system - arose during organ evolution.

  8. Oenocyte development in the red flour beetle Tribolium castaneum.

    Science.gov (United States)

    Burns, Kevin A; Gutzwiller, Lisa M; Tomoyasu, Yoshinori; Gebelein, Brian

    2012-04-01

    Oenocytes are a specialized cell type required for lipid processing, pheromone secretion, and developmental signaling. Their development has been well characterized in Drosophila melanogaster, but it remains unknown whether the developmental program is conserved in other insect species. In this study, we compare and contrast the specification and development of larval oenocytes between Drosophila and the red flour beetle, Tribolium castaneum. First, we identify several useful reagents to label larval oenocytes, including both a Tribolium GFP enhancer trap line and a simple flurophore-conjugated streptavidin staining method that recognizes oenocytes across insect species. Second, we use these tools to describe oenocyte development in Tribolium embryos, and our findings provide evidence for conserved roles of MAP kinase signaling as well as the Spalt, Engrailed, hepatocyte nuclear factor-4, and ventral veins lacking factors in producing abdominal-specific oenocyte cells. However, Tribolium embryos produce four times as many oenocytes per abdominal segment as Drosophila, and unlike in Drosophila, these cells rapidly downregulate the expression of the Spalt transcription factor. Thus, these results provide new insight into the molecular pathways regulating oenocyte specification across insect species.

  9. Characterisation and tissue distribution of the PISCF allatostatin receptor in the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Audsley, Neil; Vandersmissen, Hans Peter; Weaver, Robert; Dani, Paulina; Matthews, June; Down, Rachel; Vuerinckx, Kristel; Kim, Young-Joon; Vanden Broeck, Jozef

    2013-01-01

    peptide may have multiple roles in beetles. However, Trica-AS had no effect on the spontaneous contractions of the gut or ovaries of T. castaneum but this peptide did stimulate the release of proteases from the anterior midgut of another beetle, Tenebrio molitor. The activation of the Trica-AS receptor by Trica-ASTCC implies a physiological role for this peptide in beetles, which remains to be identified.

  10. Chemical composition, insecticidal and insect repellent activity of Schinus molle L. leaf and fruit essential oils against Trogoderma granarium and Tribolium castaneum.

    Science.gov (United States)

    Abdel-Sattar, Essam; Zaitoun, Ahmed A; Farag, Mohamed A; Gayed, Sabah H El; Harraz, Fathalla M H

    2010-02-01

    Fruit and leaf essential oils of Schinus molle showed insect repellent and insecticidal activity against Trogoderma granarium and Tribolium castaneum. In these oils, 65 components were identified by GC-MS analysis. Hydrocarbons dominated the oil composition with monoterpenes occurring in the largest amounts in fruits and leaves, 80.43 and 74.84%, respectively. p-Cymene was identified as a major component in both oils. The high yield and efficacy of S. molle essential oil against T. granarium and T. castaneum suggest that it may provide leads for active insecticidal agents.

  11. Co-motif discovery identifies an Esrrb-Sox2-DNA ternary complex as a mediator of transcriptional differences between mouse embryonic and epiblast stem cells.

    Science.gov (United States)

    Hutchins, Andrew Paul; Choo, Siew Hua; Mistri, Tapan Kumar; Rahmani, Mehran; Woon, Chow Thai; Ng, Calista Keow Leng; Jauch, Ralf; Robson, Paul

    2013-02-01

    Transcription factors (TF) often bind in heterodimeric complexes with each TF recognizing a specific neighboring cis element in the regulatory region of the genome. Comprehension of this DNA motif grammar is opaque, yet recent developments have allowed the interrogation of genome-wide TF binding sites. We reasoned that within this data novel motif grammars could be identified that controlled distinct biological programs. For this purpose, we developed a novel motif-discovery tool termed fexcom that systematically interrogates ChIP-seq data to discover spatially constrained TF-TF composite motifs occurring over short DNA distances. We applied this to the extensive ChIP-seq data available from mouse embryonic stem cells (ESCs). In addition to the well-known and most prevalent sox-oct motif, we also discovered a novel constrained spacer motif for Esrrb and Sox2 with a gap of between 2 and 8 bps that Essrb and Sox2 cobind in a selective fashion. Through the use of knockdown experiments, we argue that the Esrrb-Sox2 complex is an arbiter of gene expression differences between ESCs and epiblast stem cells (EpiSC). A number of genes downregulated upon dual Esrrb/Sox2 knockdown (e.g., Klf4, Klf5, Jam2, Pecam1) are similarly downregulated in the ESC to EpiSC transition and contain the esrrb-sox motif. The prototypical Esrrb-Sox2 target gene, containing an esrrb-sox element conserved throughout eutherian and metatherian mammals, is Nr0b1. Through positive regulation of this transcriptional repressor, we argue the Esrrb-Sox2 complex promotes the ESC state through inhibition of the EpiSC transcriptional program and the same trio may also function to maintain trophoblast stem cells.

  12. MicroRNA evolution, expression, and function during short germband development in Tribolium castaneum.

    Science.gov (United States)

    Ninova, Maria; Ronshaugen, Matthew; Griffiths-Jones, Sam

    2016-01-01

    MicroRNAs are well-established players in the development of multicellular animals. Most of our understanding of microRNA function in arthropod development comes from studies in Drosophila. Despite their advantages as model systems, the long germband embryogenesis of fruit flies is an evolutionary derived state restricted to several holometabolous insect lineages. MicroRNA evolution and expression across development in animals exhibiting the ancestral and more widespread short germband mode of embryogenesis has not been characterized. We sequenced small RNA libraries of oocytes and successive intervals covering the embryonic development of the short germband model organism, Tribolium castaneum. We analyzed the evolution and temporal expression of the microRNA complement and sequenced libraries of total RNA to investigate the relationships with microRNA target expression. We show microRNA maternal loading and sequence-specific 3' end nontemplate oligoadenylation of maternally deposited microRNAs that is conserved between Tribolium and Drosophila. We further uncover large clusters encoding multiple paralogs from several Tribolium-specific microRNA families expressed during a narrow interval of time immediately after the activation of zygotic transcription. These novel microRNAs, together with several early expressed conserved microRNAs, target a significant number of maternally deposited transcripts. Comparison with Drosophila shows that microRNA-mediated maternal transcript targeting is a conserved process in insects, but the number and sequences of microRNAs involved have diverged. The expression of fast-evolving and species-specific microRNAs in the early blastoderm of T. castaneum is consistent with previous findings in Drosophila and shows that the unique permissiveness for microRNA innovation at this stage is a conserved phenomenon.

  13. Tribolium castaneum Transformer-2 regulates sex determination and development in both males and females.

    Science.gov (United States)

    Shukla, Jayendra Nath; Palli, Subba Reddy

    2013-12-01

    Tribolium castaneum Transformer (TcTra) is essential for female sex determination and maintenance through the regulation of sex-specific splicing of doublesex (dsx) pre-mRNA. In females, TcTra also regulates the sex-specific splicing of its own pre-mRNA to ensure continuous production of functional Tra protein. Transformer protein is absent in males and hence dsx pre-mRNA is spliced in a default mode. The mechanisms by which males inhibit the production of functional Tra protein are not known. Here, we report on functional characterization of transformer-2 (tra-2) gene (an ortholog of Drosophila transformer-2) in T. castaneum. RNA interference-mediated knockdown in the expression of gene coding for tra-2 in female pupae or adults resulted in the production of male-specific isoform of dsx and both female and male isoforms of tra suggesting that Tra-2 is essential for the female-specific splicing of tra and dsx pre-mRNAs. Interestingly, knockdown of tra-2 in males did not affect the splicing of dsx but resulted in the production of both female and male isoforms of tra suggesting that Tra-2 suppresses female-specific splicing of tra pre-mRNA in males. This dual regulation of sex-specific splicing of tra pre-mRNA ensures a tight regulation of sex determination and maintenance. These data suggest a critical role for Tra-2 in suppression of female sex determination cascade in males. In addition, RNAi studies showed that Tra-2 is also required for successful embryonic and larval development in both sexes.

  14. The genome of the model beetle and pest Tribolium castaneum

    DEFF Research Database (Denmark)

    Denell, Robin; Gibbs, Richard; Muzny, Donna

    2008-01-01

    Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to inte...

  15. Non-invasive long-term fluorescence live imaging of Tribolium castaneum embryos.

    Science.gov (United States)

    Strobl, Frederic; Stelzer, Ernst H K

    2014-06-01

    Insect development has contributed significantly to our understanding of metazoan development. However, most information has been obtained by analyzing a single species, the fruit fly Drosophila melanogaster. Embryonic development of the red flour beetle Tribolium castaneum differs fundamentally from that of Drosophila in aspects such as short-germ development, embryonic leg development, extensive extra-embryonic membrane formation and non-involuted head development. Although Tribolium has become the second most important insect model organism, previous live imaging attempts have addressed only specific questions and no long-term live imaging data of Tribolium embryogenesis have been available. By combining light sheet-based fluorescence microscopy with a novel mounting method, we achieved complete, continuous and non-invasive fluorescence live imaging of Tribolium embryogenesis at high spatiotemporal resolution. The embryos survived the 2-day or longer imaging process, developed into adults and produced fertile progeny. Our data document all morphogenetic processes from the rearrangement of the uniform blastoderm to the onset of regular muscular movement in the same embryo and in four orientations, contributing significantly to the understanding of Tribolium development. Furthermore, we created a comprehensive chronological table of Tribolium embryogenesis, integrating most previous work and providing a reference for future studies. Based on our observations, we provide evidence that serosa window closure and serosa opening, although deferred by more than 1 day, are linked. All our long-term imaging datasets are available as a resource for the community. Tribolium is only the second insect species, after Drosophila, for which non-invasive long-term fluorescence live imaging has been achieved.

  16. MicroRNA transcriptome analysis identifies miR-365 as a novel negative regulator of cell proliferation in Zmpste24-deficient mouse embryonic fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Xing-dong [Institute of Aging Research, Guangdong Medical College, Xin Cheng Avenue 1#, Songshan Lake, Dongguan, Guangdong 523808 (China); Institute of Biochemistry & Molecular Biology, Guangdong Medical College, Zhanjiang 524023 (China); Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan 523808 (China); Institute of Laboratory Medicine, Guangdong Medical College, Dongguan, Guangdong 523808 (China); Jung, Hwa Jin [Departments of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461 (United States); Gombar, Saurabh [Departments of Systems Biology, Albert Einstein College of Medicine, Bronx, NY 10461 (United States); Park, Jung Yoon [Departments of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461 (United States); Zhang, Chun-long; Zheng, Huiling [Institute of Aging Research, Guangdong Medical College, Xin Cheng Avenue 1#, Songshan Lake, Dongguan, Guangdong 523808 (China); Institute of Biochemistry & Molecular Biology, Guangdong Medical College, Zhanjiang 524023 (China); Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan 523808 (China); Ruan, Jie; Li, Jiang-bin [Institute of Aging Research, Guangdong Medical College, Xin Cheng Avenue 1#, Songshan Lake, Dongguan, Guangdong 523808 (China); Institute of Biochemistry & Molecular Biology, Guangdong Medical College, Zhanjiang 524023 (China); Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan 523808 (China); Institute of Laboratory Medicine, Guangdong Medical College, Dongguan, Guangdong 523808 (China); Kaeberlein, Matt [Institute of Aging Research, Guangdong Medical College, Xin Cheng Avenue 1#, Songshan Lake, Dongguan, Guangdong 523808 (China); Department of Pathology, University of Washington, Seattle, WA 98195 (United States); and others

    2015-07-15

    Highlights: • A comprehensive miRNA transcriptome of MEFs from Zmpste24{sup −/−} and control mice. • Identification of miR-365 as a down-regulated miRNA in Zmpste24{sup −/−} MEFs. • Characterization of miR-365 as a modulator of cellular growth in part by targeting Rasd1. - Abstract: Zmpste24 is a metalloproteinase responsible for the posttranslational processing and cleavage of prelamin A into mature laminA. Zmpste24{sup −/−} mice display a range of progeroid phenotypes overlapping with mice expressing progerin, an altered version of lamin A associated with Hutchinson-Gilford progeria syndrome (HGPS). Increasing evidence has demonstrated that miRNAs contribute to the regulation of normal aging process, but their roles in progeroid disorders remain poorly understood. Here we report the miRNA transcriptomes of mouse embryonic fibroblasts (MEFs) established from wild type (WT) and Zmpste24{sup −/−} progeroid mice using a massively parallel sequencing technology. With data from 19.5 × 10{sup 6} reads from WT MEFs and 16.5 × 10{sup 6} reads from Zmpste24{sup −/−} MEFs, we discovered a total of 306 known miRNAs expressed in MEFs with a wide dynamic range of read counts ranging from 10 to over 1 million. A total of 8 miRNAs were found to be significantly down-regulated, with only 2 miRNAs upregulated, in Zmpste24{sup −/−} MEFs as compared to WT MEFs. Functional studies revealed that miR-365, a significantly down-regulated miRNA in Zmpste24{sup −/−} MEFs, modulates cellular growth phenotypes in MEFs. Overexpression of miR-365 in Zmpste24{sup −/−} MEFs increased cellular proliferation and decreased the percentage of SA-β-gal-positive cells, while inhibition of miR-365 function led to an increase of SA-β-gal-positive cells in WT MEFs. Furthermore, we identified Rasd1, a member of the Ras superfamily of small GTPases, as a functional target of miR-365. While expression of miR-365 suppressed Rasd1 3′ UTR luciferase-reporter activity

  17. Hox genes require homothorax and extradenticle for body wall identity specification but not for appendage identity specification during metamorphosis of Tribolium castaneum.

    Science.gov (United States)

    Smith, Frank W; Jockusch, Elizabeth L

    2014-11-01

    The establishment of segment identity is a key developmental process that allows for divergence along the anteroposterior body axis in arthropods. In Drosophila, the identity of a segment is determined by the complement of Hox genes it expresses. In many contexts, Hox transcription factors require the protein products of extradenticle (exd) and homothorax (hth) as cofactors to perform their identity specification functions. In holometabolous insects, segment identity may be specified twice, during embryogenesis and metamorphosis. To glean insight into the relationship between embryonic and metamorphic segmental identity specification, we have compared these processes in the flour beetle Tribolium castaneum, which develops ventral appendages during embryogenesis that later metamorphose into adult appendages with distinct morphologies. At metamorphosis, comparisons of RNAi phenotypes indicate that Hox genes function jointly with Tc-hth and Tc-exd to specify several region-specific aspects of the adult body wall. On the other hand, Hox genes specify appendage identities along the anteroposterior axis independently of Tc-hth/Tc-exd and Tc-hth/Tc-exd specify proximal vs. distal identity within appendages independently of Hox genes during this stage. During embryogenesis, Tc-hth and Tc-exd play a broad role in the segmentation process and are required for specification of body wall identities in the thorax; however, contrasting with results from other species, we did not obtain homeotic transformations of embryonic appendages in response to Tc-hth or Tc-exd RNAi. In general, the homeotic effects of interference with the function of Hox genes and Tc-hth/Tc-exd during metamorphosis did not match predictions based on embryonic roles of these genes. Comparing metamorphic patterning in T. castaneum to embryonic and post-embryonic development in hemimetabolous insects suggests that holometabolous metamorphosis combines patterning processes of both late embryogenesis and

  18. Comparative RNA-sequencing analysis of mthl1 functions and signal transductions in Tribolium castaneum.

    Science.gov (United States)

    Li, Chengjun; Wu, Wei; Sang, Ming; Liu, Xing; Hu, Xingxing; Yun, Xiaopei; Li, Bin

    2014-09-01

    Methuselah-like 1 of Tribolium castaneum (TcMthl1) has been reported to play crucial roles in development, lifespan, stress resistance and reproduction. However, the signaling system of TcMthl1 is unknown. Thus, we compare the transcriptome profile of RNAi treated larvae (ds-Tcmthl1) and control larvae of T. castaneum by RNA-sequencing, and obtained 14,613,514 sequence reads aligned with 13,533 genes; 812 differentially expressed genes (DEGs) were identified. These DEGs were classified into 47 GO functional groups, including such functions as the immune system process, the response to stimulus, the developmental process and reproduction. Interestingly, knock-down of Tcmthl1 suppressed both of Toll and IMD immunity pathways which most likely modulated the effects of Tcmthl1 on lifespan and stress resistance. Additionally, the DEGs encoding Blimp-1, Ftz-F1, E74 and Timeless may participate in the development and reproduction of ds-Tcmthl1 insects. The findings of these DEGs and pathways will provide valuable insight into TcMthl1 signaling and regulation system.

  19. Functional analysis of the ATP-binding cassette (ABC transporter gene family of Tribolium castaneum

    Directory of Open Access Journals (Sweden)

    Broehan Gunnar

    2013-01-01

    Full Text Available Abstract Background The ATP-binding cassette (ABC transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. Most are integral membrane proteins that transport a broad spectrum of substrates across lipid membranes. In insects, ABC transporters are of special interest because of their role in insecticide resistance. Results We have identified 73 ABC transporter genes in the genome of T. castaneum, which group into eight subfamilies (ABCA-H. This coleopteran ABC family is significantly larger than those reported for insects in other taxonomic groups. Phylogenetic analysis revealed that this increase is due to gene expansion within a single clade of subfamily ABCC. We performed an RNA interference (RNAi screen to study the function of ABC transporters during development. In ten cases, injection of double-stranded RNA (dsRNA into larvae caused developmental phenotypes, which included growth arrest and localized melanization, eye pigmentation defects, abnormal cuticle formation, egg-laying and egg-hatching defects, and mortality due to abortive molting and desiccation. Some of the ABC transporters we studied in closer detail to examine their role in lipid, ecdysteroid and eye pigment transport. Conclusions The results from our study provide new insights into the physiological function of ABC transporters in T. castaneum, and may help to establish new target sites for insect control.

  20. EFFECTS OF ESSENTIAL OIL FORMULATIONS ON THE ADULT INSECT TRIBOLIUM CASTANEUM (HERBST (COL., TENEBRIONIDAE

    Directory of Open Access Journals (Sweden)

    Aleksandra Popovic

    2013-06-01

    Full Text Available Stored product pests such as Tribolium castaneum ( Herbst, 1979 are a major problem. Adult insects were obtained from laboratory cultures maintained in the dark in incubators at 25 1C and 70 80percent r.h., reared on wheat flour and fed with flour disks containing a known concentration of essential oil of 9 plants. The chemical components of essential oil of 3 plants, collected on the area of Montenegro, were also identified using GC-MS analysis. The results of insecticidal effect of essential oils were discussed. Also, mortality rate of adult insects was tested. In this research, the essential oils of C. glandulosa which were rich in monoterpene alcohols carvacrol and contained ketonic component showed strong insecticidal and fumigant activity against adults of T. castaneum. Less toxic effect showed essential oils of Satureja montana which had a lower carvacrol and ketonic content. On the other hand, essential oils of Teucrium polium which did not contain ketonic component did not show any activity. Therefore, it was observed that essential oils of C. glandulosa with concentration of 1.14% showed powerful toxic and repellent effect, with very high mortality rate after 24h (56,67%.

  1. Diagnostic molecular markers for phosphine resistance in U.S. populations of Tribolium castaneum and Rhyzopertha dominica.

    Science.gov (United States)

    Chen, Zhaorigetu; Schlipalius, David; Opit, George; Subramanyam, Bhadriraju; Phillips, Thomas W

    2015-01-01

    Stored product beetles that are resistant to the fumigant pesticide phosphine (hydrogen phosphide) gas have been reported for more than 40 years in many places worldwide. Traditionally, determination of phosphine resistance in stored product beetles is based on a discriminating dose bioassay that can take up to two weeks to evaluate. We developed a diagnostic cleaved amplified polymorphic sequence method, CAPS, to detect individuals with alleles for strong resistance to phosphine in populations of the red flour beetle, Tribolium castaneum, and the lesser grain borer, Rhyzopertha dominica, according to a single nucleotide mutation in the dihydrolipoamide dehydrogenase (DLD) gene. We initially isolated and sequenced the DLD genes from susceptible and strongly resistant populations of both species. The corresponding amino acid sequences were then deduced. A single amino acid mutation in DLD in populations of T. castaneum and R. dominica with strong resistance was identified as P45S in T. castaneum and P49S in R. dominica, both collected from northern Oklahoma, USA. PCR products containing these mutations were digested by the restriction enzymes MboI and BstNI, which revealed presence or absence, respectively of the resistant (R) allele and allowed inference of genotypes with that allele. Seven populations of T. castaneum from Kansas were subjected to discriminating dose bioassays for the weak and strong resistance phenotypes. Application of CAPS to these seven populations confirmed the R allele was in high frequency in the strongly resistant populations, and was absent or at a lower frequency in populations with weak resistance, which suggests that these populations with a low frequency of the R allele have the potential for selection of the strong resistance phenotype. CAPS markers for strong phosphine resistance will help to detect and confirm resistant beetles and can facilitate resistance management actions against a given pest population.

  2. Diagnostic molecular markers for phosphine resistance in U.S. populations of Tribolium castaneum and Rhyzopertha dominica.

    Directory of Open Access Journals (Sweden)

    Zhaorigetu Chen

    Full Text Available Stored product beetles that are resistant to the fumigant pesticide phosphine (hydrogen phosphide gas have been reported for more than 40 years in many places worldwide. Traditionally, determination of phosphine resistance in stored product beetles is based on a discriminating dose bioassay that can take up to two weeks to evaluate. We developed a diagnostic cleaved amplified polymorphic sequence method, CAPS, to detect individuals with alleles for strong resistance to phosphine in populations of the red flour beetle, Tribolium castaneum, and the lesser grain borer, Rhyzopertha dominica, according to a single nucleotide mutation in the dihydrolipoamide dehydrogenase (DLD gene. We initially isolated and sequenced the DLD genes from susceptible and strongly resistant populations of both species. The corresponding amino acid sequences were then deduced. A single amino acid mutation in DLD in populations of T. castaneum and R. dominica with strong resistance was identified as P45S in T. castaneum and P49S in R. dominica, both collected from northern Oklahoma, USA. PCR products containing these mutations were digested by the restriction enzymes MboI and BstNI, which revealed presence or absence, respectively of the resistant (R allele and allowed inference of genotypes with that allele. Seven populations of T. castaneum from Kansas were subjected to discriminating dose bioassays for the weak and strong resistance phenotypes. Application of CAPS to these seven populations confirmed the R allele was in high frequency in the strongly resistant populations, and was absent or at a lower frequency in populations with weak resistance, which suggests that these populations with a low frequency of the R allele have the potential for selection of the strong resistance phenotype. CAPS markers for strong phosphine resistance will help to detect and confirm resistant beetles and can facilitate resistance management actions against a given pest population.

  3. Diagnostic Molecular Markers for Phosphine Resistance in U.S. Populations of Tribolium castaneum and Rhyzopertha dominica

    Science.gov (United States)

    Chen, Zhaorigetu; Schlipalius, David; Opit, George; Subramanyam, Bhadriraju; Phillips, Thomas W.

    2015-01-01

    Stored product beetles that are resistant to the fumigant pesticide phosphine (hydrogen phosphide) gas have been reported for more than 40 years in many places worldwide. Traditionally, determination of phosphine resistance in stored product beetles is based on a discriminating dose bioassay that can take up to two weeks to evaluate. We developed a diagnostic cleaved amplified polymorphic sequence method, CAPS, to detect individuals with alleles for strong resistance to phosphine in populations of the red flour beetle, Tribolium castaneum, and the lesser grain borer, Rhyzopertha dominica, according to a single nucleotide mutation in the dihydrolipoamide dehydrogenase (DLD) gene. We initially isolated and sequenced the DLD genes from susceptible and strongly resistant populations of both species. The corresponding amino acid sequences were then deduced. A single amino acid mutation in DLD in populations of T. castaneum and R. dominica with strong resistance was identified as P45S in T. castaneum and P49S in R. dominica, both collected from northern Oklahoma, USA. PCR products containing these mutations were digested by the restriction enzymes MboI and BstNI, which revealed presence or absence, respectively of the resistant (R) allele and allowed inference of genotypes with that allele. Seven populations of T. castaneum from Kansas were subjected to discriminating dose bioassays for the weak and strong resistance phenotypes. Application of CAPS to these seven populations confirmed the R allele was in high frequency in the strongly resistant populations, and was absent or at a lower frequency in populations with weak resistance, which suggests that these populations with a low frequency of the R allele have the potential for selection of the strong resistance phenotype. CAPS markers for strong phosphine resistance will help to detect and confirm resistant beetles and can facilitate resistance management actions against a given pest population. PMID:25826251

  4. BeetleBase: the model organism database for Tribolium castaneum

    OpenAIRE

    Wang, Liangjiang; Wang, Suzhi; Li, Yonghua; Paradesi, Martin S. R.; Brown, Susan J

    2006-01-01

    BeetleBase () is an integrated resource for the Tribolium research community. The red flour beetle (Tribolium castaneum) is an important model organism for genetics, developmental biology, toxicology and comparative genomics, the genome of which has recently been sequenced. BeetleBase is constructed to integrate the genomic sequence data with information about genes, mutants, genetic markers, expressed sequence tags and publications. BeetleBase uses the Chado data model and software component...

  5. UVB Radiation Delays Tribolium castaneum Metamorphosis by Influencing Ecdysteroid Metabolism.

    Science.gov (United States)

    Sang, Wen; Yu, Lin; He, Li; Ma, Wei-Hua; Zhu, Zhi-Hui; Zhu, Fen; Wang, Xiao-Ping; Lei, Chao-Liang

    2016-01-01

    Ultraviolet B (UVB) radiation is an important environmental factor. It is generally known that UVB exhibits high genotoxicity due to causing DNA damage, potentially leading to skin carcinogenesis and aging in mammals. However, little is known about the effects of UVB on the development and metamorphosis of insects, which are the most abundant terrestrial animals. In the present study, we performed dose-response analyses of the effects UVB irradiation on Tribolium castaneum metamorphosis, assessed the function of the T. castaneum prothoracicotropic hormone gene (Trcptth), and analyzed ecdysteroid pathway gene expression profile and ecdysterone titers post-UVB irradiation. The results showed that UVB not only caused death of T. castaneum larvae, but also delayed larval-pupal metamorphosis and reduced the size and emergence rate of pupae. In addition, we verified the function of Trcptth, which is responsible for regulating metamorphosis. It was also found that the expression profiles of Trcptth as well as ecdysteroidogenesis and response genes were influenced by UVB radiation. Therefore, a disturbance pulse of ecdysteroid may be involved in delaying development under exposure to irradiation. To our knowledge, this is the first report indicating that UVB can influence the metamorphosis of insects. This study will contribute to a better understanding of the impact of UVB on signaling mechanisms in insect metamorphosis.

  6. UVB Radiation Delays Tribolium castaneum Metamorphosis by Influencing Ecdysteroid Metabolism.

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    Wen Sang

    Full Text Available Ultraviolet B (UVB radiation is an important environmental factor. It is generally known that UVB exhibits high genotoxicity due to causing DNA damage, potentially leading to skin carcinogenesis and aging in mammals. However, little is known about the effects of UVB on the development and metamorphosis of insects, which are the most abundant terrestrial animals. In the present study, we performed dose-response analyses of the effects UVB irradiation on Tribolium castaneum metamorphosis, assessed the function of the T. castaneum prothoracicotropic hormone gene (Trcptth, and analyzed ecdysteroid pathway gene expression profile and ecdysterone titers post-UVB irradiation. The results showed that UVB not only caused death of T. castaneum larvae, but also delayed larval-pupal metamorphosis and reduced the size and emergence rate of pupae. In addition, we verified the function of Trcptth, which is responsible for regulating metamorphosis. It was also found that the expression profiles of Trcptth as well as ecdysteroidogenesis and response genes were influenced by UVB radiation. Therefore, a disturbance pulse of ecdysteroid may be involved in delaying development under exposure to irradiation. To our knowledge, this is the first report indicating that UVB can influence the metamorphosis of insects. This study will contribute to a better understanding of the impact of UVB on signaling mechanisms in insect metamorphosis.

  7. Genomics, transcriptomics, and peptidomics of neuropeptides and protein hormones in the red flour beetle Tribolium castaneum

    DEFF Research Database (Denmark)

    Li, Bin; Predel, Reinhard; Neupert, Susanne;

    2008-01-01

    Neuropeptides and protein hormones are ancient molecules that mediate cell-to-cell communication. The whole genome sequence from the red flour beetle Tribolium castaneum, along with those from other insect species, provides an opportunity to study the evolution of the genes encoding neuropeptide...... and protein hormones. We identified 41 of these genes in the Tribolium genome by using a combination of bioinformatic and peptidomic approaches. These genes encode >80 mature neuropeptides and protein hormones, 49 peptides of which were experimentally identified by peptidomics of the central nervous system...... with a sequenced genome. The presence of many additional osmoregulatory peptides in Tribolium agrees well with its ability to live in very dry surroundings. In contrast to these extra genes, there are at least nine neuropeptide genes missing in Tribolium, including the genes encoding the prepropeptides...

  8. Effects of Artemisia herba-alba essential oils on survival stored cereal pests: Tribolium castaneum (Herbst (Coleoptera, Tenebrionidae and Trogoderma granarium (Everst (Coleoptera, Dermestidae

    Directory of Open Access Journals (Sweden)

    Ben Slimane Badreddine

    2016-05-01

    Full Text Available Objective: To assess the chemical components and toxicity of Artemisia herba-alba (A. herbaalba essential oil against two major stored cereal pests, Tribolium castaneum (T. castaneum and Trogoderma granarium (T. granarium. Methods: Two bioassay actions were tasted: repellent and fumigant actions against adult and larvae, respectively, to assess the effect of A. herba-alba essential oil. Results: Gas chromatography-mass spectrometer analyses of the essential oil contained β-thujone (12.50%, α-thujone (8.78%, sabinyl acetate (8.56%, terpinene-4-ol (8.51%, α-terpineol (3.35%, 1,8-cineol (5.45%, γ-terpene (4.82%, camphor (4.52%, dimethylethylbenzene (3.93% and α-terpinene (3.35% as the major components. Fumigant toxicity tests showed that A. herba-alba oil was more toxic than T. granarium (LC50 = 2.09 mg/mL, LC90 = 4.12 mg/mL and T. castaneum (LC50 = 6.39 mg/mL, LC90 = 10.10 mg/mL. Conclusions: This study has highlighted a bioinsecticide activity of A. herba-alba against two insect pests of stored foodstuffs (T. castaneum and T. granarium. The Artemisia essential oil offers an interesting potential insecticide that could be studied more deeply to isolate and identify the active substances, to study their physiological impact on other insects

  9. Gene array analysis of neural crest cells identifies transcription factors necessary for direct conversion of embryonic fibroblasts into neural crest cells

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    Tsutomu Motohashi

    2016-03-01

    Full Text Available Neural crest cells (NC cells are multipotent cells that emerge from the edge of the neural folds and migrate throughout the developing embryo. Although the gene regulatory network for generation of NC cells has been elucidated in detail, it has not been revealed which of the factors in the network are pivotal to directing NC identity. In this study we analyzed the gene expression profile of a pure NC subpopulation isolated from Sox10-IRES-Venus mice and investigated whether these genes played a key role in the direct conversion of Sox10-IRES-Venus mouse embryonic fibroblasts (MEFs into NC cells. The comparative molecular profiles of NC cells and neural tube cells in 9.5-day embryos revealed genes including transcription factors selectively expressed in developing trunk NC cells. Among 25 NC cell-specific transcription factor genes tested, SOX10 and SOX9 were capable of converting MEFs into SOX10-positive (SOX10+ cells. The SOX10+ cells were then shown to differentiate into neurons, glial cells, smooth muscle cells, adipocytes and osteoblasts. These SOX10+ cells also showed limited self-renewal ability, suggesting that SOX10 and SOX9 directly converted MEFs into NC cells. Conversely, the remaining transcription factors, including well-known NC cell specifiers, were unable to convert MEFs into SOX10+ NC cells. These results suggest that SOX10 and SOX9 are the key factors necessary for the direct conversion of MEFs into NC cells.

  10. Walking Responses of Tribolium castaneum (Coleoptera: Tenebrionidae) to Its Aggregation Pheromone and Odors of Wheat Infestations.

    Science.gov (United States)

    Stevenson, B J; Cai, L; Faucher, C; Michie, M; Berna, A; Ren, Y; Anderson, A; Chyb, S; Xu, W

    2017-03-03

    The red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is a worldwide pest of stored grains. Using "Y"-tube olfactometry we studied the response of T. castaneum to odors from simulated wheat infestations containing conspecifics, and infestations containing the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), and the granary weevil Sitophilus granarius (L.) (Coleoptera: Curculionidae). Tribolium castaneum larvae were significantly attracted to odors from all three test species. Tribolium castaneum adults were attracted to grains infested by R. dominica and flour infested by T. castaneum but repelled from grains infested by S. granarius. Further behavioral analysis with pheromones showed that T. castaneum were significantly attracted to their aggregation pheromone, dimethyldecanal (DMD), but not to the R. dominica aggregation pheromone, a mixture of dominicalure 1 and 2. Female T. castaneum adults were attracted to ∼50-fold less DMD than larvae and 100-fold less than male adults, suggesting they are more sensitive to DMD. This study improves our understanding of T. castaneum behaviors to infested grain volatile compounds and pheromones, and may help develop new control methods for grain pest species.

  11. Gamma radiation effects on the reproductive capacity of Tribolium castaneum

    Energy Technology Data Exchange (ETDEWEB)

    Hu, T.; Tsai, L.T.; Fu, Y.K.

    1984-10-01

    Unmated pairs of Tribolium castaneum (Hbst.) were treated with gamma radiation from 8.2 to 65.6 Gy at 8.2-Gy increment and allowed to mate and oviposit. The number of larva per fertile pair and the percentage of sterility at each dosage were determined. The regression line for logarithm of average number of larva progeny per pair and a probit sterility curve were calculated. The average number of progeny per pair was reduced 10% by a dosage of 28.99 Gy. The probit sterility line indicated that 107 Gy in the sterilizing dosage for this species. 9 refs., 2 figs., 1 tab.

  12. A Tribolium castaneum whole-embryo culture protocol for studying the molecular mechanisms and morphogenetic movements involved in insect development.

    Science.gov (United States)

    Macaya, Constanza C; Saavedra, Patricio E; Cepeda, Rodrigo E; Nuñez, Viviana A; Sarrazin, Andres F

    2016-01-01

    The development of the red flour beetle Tribolium castaneum is more representative of arthropods than the evolutionarily derived fly, Drosophila melanogaster. Thus, Tribolium is becoming an emerging organism model for studying the evolution of the mechanisms that control embryonic development in arthropods. In this regard, diverse genetic and molecular tools are currently available for Tribolium, as well as imaging and embryonic techniques. Recently, we developed a method for culturing embryos in order to study specific stages during Tribolium development. In this report, we present a detailed and "easy-to-follow" protocol for embryo handling and dissection, extending the use of whole-embryo culture to functional analysis by performing in vivo pharmacological manipulations. This experimental accessibility allowed us to study the relevance of microtubules in axis elongation, using nocodazole and taxol drugs to interfere with microtubule networks, followed by length measurement analysis. Additionally, we demonstrated that embryo handling had no effect on the development of Tribolium embryos, and we checked viability after dissection and bisection and during incubation using propidium iodide. The embryo culture protocol we describe here can be applied to study diverse developmental processes in Tribolium. We expect that this protocol can be adapted and applied to other arthropods.

  13. Identifying MicroRNA and mRNA Expression Profiles in Embryonic Stem Cells Derived from Parthenogenetic, Androgenetic and Fertilized Blastocysts

    Institute of Scientific and Technical Information of China (English)

    Xiang-Shun Cui; Xing-Hui Shen; Shao-Chen Sun; Sun-Wha Cho; Young-Tae Heo; Yong-Kook Kang; Teruhiko Wakayama

    2013-01-01

    MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNA molecules that play a pivotal role m several cellular functions.In this study,miRNA and messenger RNA (mRNA) profiles were examined by Illumina microarray in mouse embryonic stem cells (ESCs) derived from parthenogenetic,androgenetic,and fertilized blastocysts.The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression.Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs (aESCs) and fertilized ESCs (fESCs),a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs (pESCs) and fESCs,and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs.In addition,a total of 575,5 and 376 miRNA-mRNA target pairs were observed in aESCs vs.fESCs,pESCs vs.fESCs,and aESCs vs.pESCs,respectively.Furthermore,15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR.Finally,transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs.Inhibition of miR-880 increased the expression of Peg3,Dyrklb,and Prrg2 mRNA,inhibition of miR-363 increased the expression of Nfat5 and Soatl mRNA,and inhibition of miR-883b-5p increased Nfat5,Tacstd2,and Ppapdcl mRNA.These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.

  14. Visualizing late insect embryogenesis: extraembryonic and mesodermal enhancer trap expression in the beetle Tribolium castaneum.

    Science.gov (United States)

    Koelzer, Stefan; Kölsch, Yvonne; Panfilio, Kristen A

    2014-01-01

    The beetle Tribolium castaneum has increasingly become a powerful model for comparative research on insect development. One recent resource is a collection of piggyBac transposon-based enhancer trap lines. Here, we provide a detailed analysis of three selected lines and demonstrate their value for investigations in the second half of embryogenesis, which has thus far lagged behind research on early stages. Two lines, G12424 and KT650, show enhanced green fluorescent protein (EGFP) expression throughout the extraembryonic serosal tissue and in a few discrete embryonic domains. Intriguingly, both lines show for the first time a degree of regionalization within the mature serosa. However, their expression profiles illuminate distinct aspects of serosal biology: G12424 tracks the tissue's rapid maturation while KT650 expression likely reflects ongoing physiological processes. The third line, G04609, is stably expressed in mesodermal domains, including segmental muscles and the heart. Genomic mapping followed by in situ hybridization for genes near to the G04609 insertion site suggests that the transposon has trapped enhancer information for the Tribolium orthologue of midline (Tc-mid). Altogether, our analyses provide the first live imaging, long-term characterizations of enhancer traps from this collection. We show that EGFP expression is readily detected, including in heterozygote crosses that permit the simultaneous visualization of multiple tissue types. The tissue specificity provides live, endogenous marker gene expression at key developmental stages that are inaccessible for whole mount staining. Furthermore, the nonlocalized EGFP in these lines illuminates both the nucleus and cytoplasm, providing cellular resolution for morphogenesis research on processes such as dorsal closure and heart formation. In future work, identification of regulatory regions driving these enhancer traps will deepen our understanding of late developmental control, including in the

  15. Visualizing late insect embryogenesis: extraembryonic and mesodermal enhancer trap expression in the beetle Tribolium castaneum.

    Directory of Open Access Journals (Sweden)

    Stefan Koelzer

    Full Text Available The beetle Tribolium castaneum has increasingly become a powerful model for comparative research on insect development. One recent resource is a collection of piggyBac transposon-based enhancer trap lines. Here, we provide a detailed analysis of three selected lines and demonstrate their value for investigations in the second half of embryogenesis, which has thus far lagged behind research on early stages. Two lines, G12424 and KT650, show enhanced green fluorescent protein (EGFP expression throughout the extraembryonic serosal tissue and in a few discrete embryonic domains. Intriguingly, both lines show for the first time a degree of regionalization within the mature serosa. However, their expression profiles illuminate distinct aspects of serosal biology: G12424 tracks the tissue's rapid maturation while KT650 expression likely reflects ongoing physiological processes. The third line, G04609, is stably expressed in mesodermal domains, including segmental muscles and the heart. Genomic mapping followed by in situ hybridization for genes near to the G04609 insertion site suggests that the transposon has trapped enhancer information for the Tribolium orthologue of midline (Tc-mid. Altogether, our analyses provide the first live imaging, long-term characterizations of enhancer traps from this collection. We show that EGFP expression is readily detected, including in heterozygote crosses that permit the simultaneous visualization of multiple tissue types. The tissue specificity provides live, endogenous marker gene expression at key developmental stages that are inaccessible for whole mount staining. Furthermore, the nonlocalized EGFP in these lines illuminates both the nucleus and cytoplasm, providing cellular resolution for morphogenesis research on processes such as dorsal closure and heart formation. In future work, identification of regulatory regions driving these enhancer traps will deepen our understanding of late developmental control

  16. Gene expression profiling of tumours derived from rasV12/E1A-transformed mouse embryonic fibroblasts to identify genes required for tumour development

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    Dagorn Jean

    2005-01-01

    Full Text Available Abstract Background In cancer, cellular transformation is followed by tumour development. Knowledge on the mechanisms of transformation, involving activation of proto-oncogenes and inactivation of tumour-suppressor genes has considerably improved whereas tumour development remains poorly understood. An interesting way of gaining information on tumour progression mechanisms would be to identify genes whose expression is altered during tumour formation. We used the Affymetrix-based DNA microarray technology to analyze gene expression profiles of tumours derived from rasV12/E1A-transformed mouse embryo fibroblasts in order to identify the genes that could be involved in tumour development. Results Among the 12,000 genes analyzed in this study, only 489 showed altered expression during tumour development, 213 being up-regulated and 276 down-regulated. The genes differentially expressed are involved in a variety of cellular functions, including control of transcription, regulation of mRNA maturation and processing, regulation of protein translation, activation of interferon-induced genes, intracellular signalling, apoptosis, cell growth, angiogenesis, cytoskeleton, cell-to-cell interaction, extracellular matrix formation, metabolism and production of secretory factors. Conclusions Some of the genes identified in this work, whose expression is altered upon rasV12/E1A transformation of MEFs, could be new cancer therapeutic targets.

  17. Pesticidal Evaluation of Manilkara zapota (L. against Tribolium castaneum (Herbst

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    Muhammad Abu Osman

    2014-02-01

    Full Text Available This paper presents the residual eff ects of Manilkara zapota (L. P. Royen plant against the red flour beetle, Tribolium castaneum (Herbst. Four doses, i.e., 1238.5, 619.25, 309.6 and 154.8 μg/cm2 of ethyl acetate extract of stem bark of Manilkara zapota were applied on larvae and adult beetles. The effectiveness of the plant extract was increased with the increase of exposure time and after 72 hrs of exposure, the maximum residual toxicity was observed with LD50 of 228.8, 281.1, 413.4, 423.7, 455.2, 498.7 and 526.5 μg/cm2 for first, second, third, fourth, fifth, sixth instar larvae and adults, respectively. The results of this study also demonstrated that the earlier instars were more sensitive to the extracts than those of late instars as well as adults.

  18. Pesticidal Evaluation of Manilkara zapota (L. against Tribolium castaneum (Herbst

    Directory of Open Access Journals (Sweden)

    Muhammad Abu Osman

    2011-06-01

    Full Text Available This paper presents the residual eff ects of Manilkara zapota (L. P. Royen plant against the red flour beetle, Tribolium castaneum (Herbst. Four doses, i.e., 1238.5, 619.25, 309.6 and 154.8 μg/cm2 of ethyl acetate extract of stem bark of Manilkara zapota were applied on larvae and adult beetles. The effectiveness of the plant extract was increased with the increase of exposure time and after 72 hrs of exposure, the maximum residual toxicity was observed with LD50 of 228.8, 281.1, 413.4, 423.7, 455.2, 498.7 and 526.5 μg/cm2 for first, second, third, fourth, fifth, sixth instar larvae and adults, respectively. The results of this study also demonstrated that the earlier instars were more sensitive to the extracts than those of late instars as well as adults.

  19. Differential preference of colored surface in Tribolium castaneum (Herbst

    Directory of Open Access Journals (Sweden)

    AMS Reza

    2006-07-01

    Full Text Available Insects show color preferences mostly to those which resembles the color of foliage, flower or even their hosts. In the present study observations were made to determine vision towards different colored surfaces in young (second instar, and advanced (fourth instar larvae, and adults of Tribolium castaneum (Herbst (Coleoptera: Tenebrionidae. The larva and adult beetles showed significant color preferences when given a choice between white (control and colored surfaces at 24- and 48-h exposures. The second instar larvae were more attracted by yellow and pink than green surfaces. The fourth instar larvae did not show any significant preference between white and colored surfaces at 24-h exposure, but avoided red and pink surfaces (P>0.05 and had a marginal choice for black (P<0.05. The adults avoided green significantly at both exposure times and pink at 48-h exposure, but was moderately attracted by black (P<0.05 at both exposure times.

  20. Effects of microwave radiation and conductive heating on Tribolium castaneum microstructure.

    Science.gov (United States)

    Lu, H H; Zhou, J C; Yan, D; Zhao, S M; Xiong, S B

    2011-01-01

    Microwave radiation and conductive heating were used to completely kill adult Tribolium castaneum (Coleoptera: Tenebrionidae) in wheat flour to protect the flour during storage without significantly effecting its quality. The microstructure of T. castaneum was analyzed to reveal the mechanisms leading to death under microwave and heat treatments. Microwave radiation and conductive heating had different effects on the microstructure of the cuticle of adult T. castaneum and on the ultrastructure of the cells of the epidermis, fat body, and midgut. Both treatments caused a large cavity to appear in the nucleus and the disappearance of mitochondria and the Golgi apparatus. After microwave treatment, there was little change in the surface microstructure but the epidermis was of uneven thickness and the four outer layers of the cuticle were thinner. Nuclear size was essentially unchanged, but fat body cells were fewer and coalesced together. In contrast, conductive heating led to a disordered arrangement of cells on the surface of T. castaneum and indistinct boundaries between layers of the cuticle. The nuclei were enlarged and the fat body cells noticeably fewer and indistinct with a scattered distribution. Thus, microwave treatment produced less severe effects on the surface microstructure and cellular ultrastructure of T. castaneum than did conductive heating. It is concluded that these cellular and surface changes were responsible for the death of T. castaneum.

  1. Identification of maternally-loaded RNA transcripts in unfertilized eggs of Tribolium castaneum

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    Preuss Kevin M

    2012-11-01

    Full Text Available Abstract Background Maternal RNAs play a critical role in early development. Variation in the diversity and levels of maternally derived gene transcripts may be central to the origin of phenotypic novelty -- a longstanding problem in evolution and development. By studying maternal transcriptomes within and between divergent species, a better understanding of the evolutionary forces acting on maternal RNA allocation is possible. Results We present the first maternal transcriptome of the red flour beetle, Tribolium castaneum. Using a tiled whole-genome microarray, we found that 58.2% of T. castaneum genes are maternally loaded into eggs. Comparison of known Drosophila melanogaster maternal genes to our results showed widespread conservation of maternal expression with T. castaneum. Additionally, we found that many genes previously reported as having sex or tissue specific expression in T. castaneum were also maternally loaded. Identification of such pleiotropy is vital for proper modeling and testing of evolutionary theory using empirical data. The microarray design also allowed the detection of 2315 and 4060 novel transcriptionally active regions greater in length than 100 bp in unfertilized and fertilized T. castaneum eggs, respectively. These transcriptionally active regions represent novel exons of potentially unknown genes for future study. Conclusions Our results lay a foundation for utilizing T. castaneum as a model for understanding the role of maternal genes in evolution.

  2. The embryonic genes Dkk3, Hoxd8, Hoxd9 and Tbx1 identify muscle types in a diet-independent and fiber-type unrelated way

    Directory of Open Access Journals (Sweden)

    Boekschoten Mark V

    2010-03-01

    , we conclude that genes important for embryogenesis identify mouse muscle types in a diet-independent and fiber type-unrelated manner.

  3. Effects ofArtemisia herba-alba essential oils on survival stored cereal pests:Tribolium castaneum (Herbst) (Coleoptera, Tenebrionidae) and Trogoderma granarium (Everst) (Coleoptera, Dermestidae)

    Institute of Scientific and Technical Information of China (English)

    Ben Slimane Badreddine; Mariem Baouindi

    2016-01-01

    Objective:To assess the chemical components and toxicity ofArtemisia herba-alba (A. herba-alba) essential oil against two major stored cereal pests,Tribolium castaneum (T. castaneum) andTrogoderma granarium(T. granarium). Methods:Two bioassay actions were tasted: repellent and fumigant actions against adult and larvae, respectively, to assess the effect ofA. herba-alba essential oil. Results: Gas chromatography-mass spectrometer analyses of the essential oil containedβ-thujone (12.50%),α-thujone (8.78%), sabinyl acetate (8.56%), terpinene-4-ol (8.51%),α-terpineol (3.35%), 1,8-cineol (5.45%),γ-terpene (4.82%), camphor (4.52%), dimethyl-ethylbenzene (3.93%) andα-terpinene (3.35%) as the major components. Fumigant toxicity tests showed thatA. herba-alba oil was more toxic thanT. granarium (LC50 = 2.09 mg/mL, LC90 = 4.12 mg/mL) andT. castaneum (LC50 = 6.39 mg/mL, LC90 = 10.10 mg/mL). Conclusions: This study has highlighted a bioinsecticide activity ofA. herba-alba against two insect pests of stored foodstuffs (T. castaneum andT. granarium). TheArtemisia essential oil offers an interesting potential insecticide that could be studied more deeply to isolate and identify the active substances, to study their physiological impact on other insects.

  4. Protein N-glycosylation and N-glycan trimming are required for postembryonic development of the pest beetle Tribolium castaneum

    Science.gov (United States)

    Walski, Tomasz; Van Damme, Els J. M.; Smargiasso, Nicolas; Christiaens, Olivier; De Pauw, Edwin; Smagghe, Guy

    2016-01-01

    In holometabolous insects the transition from larva to adult requires a complete body reorganization and relies on N-glycosylated proteins. N-glycosylation is an important posttranslational modification that influences protein activity but its impact on the metamorphosis has not been studied yet. Here we used the red flour beetle, Tribolium castaneum, to perform a first comprehensive study on the involvement of the protein N-glycosylation pathway in metamorphosis. The transcript levels for genes encoding N-glycan processing enzymes increased during later developmental stages and, in turn, transition from larva to adult coincided with an enrichment of more extensively modified paucimannose glycans, including fucosylated ones. Blockage of N-glycan attachment resulted in larval mortality, while RNAi of α-glucosidases involved in early N-glycan trimming and quality control disrupted the larva to pupa transition. Additionally, simultaneous knockdown of multiple genes responsible for N-glycan processing towards paucimannose structures revealed their novel roles in pupal appendage formation and adult eclosion. Our findings revealed that, next to hormonal control, insect post-embryonic development and metamorphosis depend on protein N-glycan attachment and efficient N-glycan processing. Consequently, disruption of these processes could be an effective new approach for insect control. PMID:27731363

  5. Structure of the Tribolium castaneum Telomerase Catalytic Subunit TERT

    Energy Technology Data Exchange (ETDEWEB)

    Gillis,A.; Schuller, A.; Skordalakes, E.

    2008-01-01

    A common hallmark of human cancers is the overexpression of telomerase, a ribonucleoprotein complex that is responsible for maintaining the length and integrity of chromosome ends. Telomere length deregulation and telomerase activation is an early, and perhaps necessary, step in cancer cell evolution. Here we present the high-resolution structure of the Tribolium castaneum catalytic subunit of telomerase, TERT. The protein consists of three highly conserved domains, organized into a ring-like structure that shares common features with retroviral reverse transcriptases, viral RNA polymerases and B-family DNA polymerases. Domain organization places motifs implicated in substrate binding and catalysis in the interior of the ring, which can accommodate seven to eight bases of double-stranded nucleic acid. Modelling of an RNA-DNA heteroduplex in the interior of this ring demonstrates a perfect fit between the protein and the nucleic acid substrate, and positions the 3'-end of the DNA primer at the active site of the enzyme, providing evidence for the formation of an active telomerase elongation complex.

  6. Toxicity of Boldo Peumus boldus Molina for Sitophilus zeamais Motschulsky and Tribolium castaneum Herbst Toxicidad del Boldo, Peumus boldus Molina, sobre Sitophilus zeamais Motschulsky y Tribolium castaneum Herbst

    Directory of Open Access Journals (Sweden)

    Margarita Ortiz U

    2012-09-01

    Full Text Available The maize weevil (Sitophilus zeamais Motschulsky and the red flour beetle (Tribolium castaneum Herbst are two key pests of stored-grain products worldwide. The insecticidal activity of boldo (Peumus boldus Molina powder, liquid ethanolic and hexanic extracts against S. zeamais and T. castaneum were evaluated under laboratory conditions. The evaluated variables were mortality, emergence of adult insects (F1, and grain weight loss. The experimental design was completely randomized. The mortality in S. zeamais was 100% even at the lowest powder concentration (0.5% w/w, whereas emergence of F1 adult insects was 0% and grain weight loss was El gorgojo del maíz (Sitophilus zeamais Motschulsky y el gorgojo castaño de la harina (Tribolium castaneum Herbst son plagas primarias de productos almacenados a nivel mundial. Se evaluó en laboratorio la actividad insecticida de polvo y extractos líquidos etanólicos y hexánicos del boldo (Peumus boldus Molina sobre S. zeamais y T. castaneum. Las variables evaluadas fueron mortalidad y emergencia de insectos adultos (F1 y pérdida de peso de los granos con un diseño experimental completamente al azar. La mortalidad en S. zeamais fue 100%, incluso con la concentración menor (0,5% p/p mientras que la emergencia de insectos adultos y la pérdida de peso de granos de maíz fue < 0,08%. Para T. castaneum sólo las concentraciones de 8 y 16% p/p de polvo causaron una mortalidad de 100%. Los extractos en agua, etanol, y hexano tuvieron un efecto insecticida de 100% en S. zeamais, mientras que en T. castaneum sólo el extracto en etanol alcanzó este valor. Por lo tanto, el polvo y los extractos evaluados de P. boldus presentan actividad insecticida contra S. zeamais y T. castaneum y son promisorios para utilizarse contra éstas y otras plagas de granos almacenados.

  7. Expression patterns of cysteine peptidase genes across the Tribolium castaneum life cycle provide clues to biological function

    Science.gov (United States)

    The red flour beetle, Tribolium castaneum, is a major agricultural pest responsible for considerable loss of stored grain and cereal products worldwide. T. castaneum larvae have a highly compartmentalized gut, with cysteine peptidases mostly in the acidic anterior part of the midgut. We have descri...

  8. BIOACTIVITY OF 1,8-CINEOLE AGAINST RED FLOUR BEETLE TRIBOLIUM CASTANEUM (HERBST

    Directory of Open Access Journals (Sweden)

    Anita Liška

    2011-06-01

    Full Text Available Red flour beetle Tribolium castaneum (Herbst is a major pest of stored products. The aim of this study was to assess the potential fumigant effects of 1,8-cineole, essential oil component, on the T. castaneum pupae. The compound was tested in 6 doses; in two treatments (fumigation without grain and with wheat grain, exposed for 48 h, in 4 repetitions, for each gender. The compound 1,8-cineole had lethal effect on the treated pupae at both genders and in the both treatments. Total proportion of the normally developed beetles was decreased. In addition, 1,8-cineole had also a growth regulator effect, producing adultoids and deformed units, with males more susceptible. In the treatment with the grain there were significant lower dead pupae, normally developed live male beetles and also deformed female units in the stage 2. In general, compound 1,8-cineole has multiple effect against T. castaneum in pupal stage.

  9. Loss of function of the yellow-e gene causes dehydration-induced mortality of adult Tribolium castaneum.

    Science.gov (United States)

    Noh, Mi Young; Kramer, Karl J; Muthukrishnan, Subbaratnam; Beeman, Richard W; Kanost, Michael R; Arakane, Yasuyuki

    2015-03-15

    Yellow protein (dopachrome conversion enzyme, DCE) is involved in the melanin biosynthetic pathway that significantly accelerates pigmentation reactions in insects. Recent studies have suggested that the insect yellow genes represent a rapidly evolving gene family generating functionally diverse paralogs, but the exact physiological functions of several yellow genes are still not understood. To study the function(s) of one of the yellow genes, yellow-e (TcY-e), in the red flour beetle, Tribolium castaneum, we performed real-time PCR to analyze its developmental and tissue-specific expression, and utilized immunohistochemistry to identify the localization of the TcY-e protein in adult cuticle. Injection of double-stranded RNA for TcY-e (dsTcY-e) into late instar larvae had no effect on larval-pupal molting or pupal development. The pupal cuticle, including that lining the setae, gin traps and urogomphi, underwent normal tanning. Adult cuticle tanning including that of the head, mandibles and legs viewed through the translucent pupal cuticle was initiated on schedule (pupal days 4-5), indicating that TcY-e is not required for pupal or pharate adult cuticle pigmentation in T. castaneum. The subsequent pupal-adult molt, however, was adversely affected. Although pupal cuticle apolysis and slippage were evident, some of the adults (~25%) were unable to shed their exuvium and died entrapped in their pupal cuticle. In addition, the resulting adults rapidly became highly desiccated. Interestingly, both the failure of the pupal-adult molt and desiccation-induced mortality were prevented by maintaining the dsTcY-e-treated insects at 100% relative humidity (rh). However, when the high humidity-rescued adults were removed from 100% rh and transferred to 50% rh, they rapidly dehydrated and died, whereas untreated beetles thrived throughout development at 50% rh. We also observed that the body color of the high humidity-rescued dsTcY-e-adults was slightly darker than that of

  10. Transcription profiling of immune genes during parasite infection in susceptible and resistant strains of the flour beetles (Tribolium castaneum).

    Science.gov (United States)

    Zhong, Daibin; Wang, Mei-Hui; Pai, Aditi; Yan, Guiyun

    2013-05-01

    The flour beetle, Tribolium castaneum, is an intermediate host for the tapeworm Hymenolepis diminuta and has become an important genetic model to explore immune responses to parasite infection in insect hosts. The present study examined the immune responses to tapeworm infection in resistant (TIW1) and susceptible (cSM) strains of the red flour beetle, T. castaneum, using real-time quantitative reverse transcription PCR on 29 immunity-related genes that exhibit antimicrobial properties. Thirteen of the 29 genes showed constitutive differences in expression between the two strains. Fourteen to fifteen of the 29 genes exhibited significant differences in transcription levels when beetles were challenged with tapeworm parasite in the resistant and susceptible strains. Nine genes (GNBP3, cSPH2, lysozyme4, defensin1, PGRP-SA, defensin2, coleoptericin1, attacin2 and serpin29) in cSM and 13 genes (lysozyme2, proPO1, GNBP3, cSPH2, lysozyme4, defensin1, PGRP-SA, defensin2, coleoptericin1, attacin2, proPO2/3, PGRP-LE and PGRP-SB) in TIW1 were up-regulated by infections or showed parasite infection-induced expression. Seven genes (attacin2, coleoptericin1, defensin1, defensin2, lysozyme2, PGRP-SA and PGRP-SB) were more than 10 folds higher in the resistant TIW1 strain than in the susceptible cSM strain after exposure to tapeworm parasites. This study demonstrated the effects of genetic background, the transcription profile to parasite infection, and identified the immunity-related genes that were significantly regulated by the infection of tapeworms in Tribolium beetles.

  11. Embryonic Stem Cell Markers

    Directory of Open Access Journals (Sweden)

    Lan Ma

    2012-05-01

    Full Text Available Embryonic stem cell (ESC markers are molecules specifically expressed in ES cells. Understanding of the functions of these markers is critical for characterization and elucidation for the mechanism of ESC pluripotent maintenance and self-renewal, therefore helping to accelerate the clinical application of ES cells. Unfortunately, different cell types can share single or sometimes multiple markers; thus the main obstacle in the clinical application of ESC is to purify ES cells from other types of cells, especially tumor cells. Currently, the marker-based flow cytometry (FCM technique and magnetic cell sorting (MACS are the most effective cell isolating methods, and a detailed maker list will help to initially identify, as well as isolate ESCs using these methods. In the current review, we discuss a wide range of cell surface and generic molecular markers that are indicative of the undifferentiated ESCs. Other types of molecules, such as lectins and peptides, which bind to ESC via affinity and specificity, are also summarized. In addition, we review several markers that overlap with tumor stem cells (TSCs, which suggest that uncertainty still exists regarding the benefits of using these markers alone or in various combinations when identifying and isolating cells.

  12. Susceptibility of Tribolium castaneum (Coleoptera: Tenebrionidae) and Trogoderma inclusum (Coleoptera: Dermestidae) to cold temperatures

    Science.gov (United States)

    Studies were conducted by exposing different life stages of Tribolium castaneum (Herbst), the red flour beetle, and Trogoderma variabile (Ballion), the warehouse beetle, for different time intervals to -18°C. Assessments were made of direct mortality to eggs, larvae, and adults, and eventual adult e...

  13. Effects of methoprene and synergized pyrethrin aerosol applications on Tribolium castaneum (Herbst) populations

    Science.gov (United States)

    Experiments were performed to investigate the effects of horizontal transfer of the insect growth regulator (IGR) methoprene on confined populations of Tribolium castaneum (Herbst) either with or without hidden refugia. Multiple applications were made with the IGR alone or combined with synergized p...

  14. Direct and indirect genetic effects in life-history traits of flour beetles (Tribolium castaneum)

    NARCIS (Netherlands)

    Ellen, Esther D.; Peeters, Katrijn; Verhoeven, Merel; Gols, Rieta; Harvey, Jeffrey A.; Wade, Michael J.; Dicke, Marcel; Bijma, Piter

    2016-01-01

    Indirect genetic effects (IGEs) are the basis of social interactions among conspecifics, and can affect genetic variation of nonsocial and social traits. We used flour beetles (Tribolium castaneum) of two phenotypically distinguishable populations to estimate genetic (co)variances and the effect of

  15. Direct and indirect genetic effects in life-history traits of flour beetles (Tribolium castaneum)

    NARCIS (Netherlands)

    Ellen, E.D.; Peeters, Katrijn; Verhoeven, Merel; Gols, Rieta; Harvey, J.A.; Wade, M.J.; Dicke, Marcel; Bijma, Piter

    2016-01-01

    Indirect genetic effects (IGEs) are the basis of social interactions among conspecifics, and can affect genetic variation of nonsocial and social traits. We used flour beetles (Tribolium castaneum) of two phenotypically distinguishable populations to estimate genetic (co)variances and the effect

  16. Mechanisms for horizontal transfer of methoprene from treated to untreated Tribolium castaneum (Herbst)

    Science.gov (United States)

    Experiments were performed to determine the relative impact of different mechanisms of horizontal transfer of methoprene by Tribolium castaneum (Herbst), the red flour beetle. Insects exposed to 5 methoprene treated developmental stages (late-stage larvae, pupae, or adults) resulted in 100% mortalit...

  17. Morpho-functional characterization and esterase patterns of the midgut of Tribolium castaneum Herbst, 1797 (Coleoptera: Tenebrionidae) parasitized by Gregarina cuneata (Apicomplexa: Eugregarinidae).

    Science.gov (United States)

    Gigliolli, Adriana A Sinópolis; Lapenta, Ana Silva; Ruvolo-Takasusuki, Maria Claudia Colla; Abrahão, Josielle; Conte, Hélio

    2015-09-01

    Tribolium castaneum (Coleoptera: Tenebrionidae) is a common pest of stored grains and byproducts and is normally infected by Gregarina cuneata (Apicomplexa: Eugregarinidae). The life cycle of this parasite includes the sporozoite, trophozoite, gamont, gametocyte, and oocyst stages, which occur between the epithelium and lumen of the host's midgut. This study aims to describe the morphofunctional alterations in the midgut and determine the esterase patterns in T. castaneum when parasitized by gregarines. To achieve this purpose, midguts of adult insects were isolated, processed, and analysed using light and electron microscopy. We determined total protein content, amylase activity, and the expression and related activities of the esterases by using polyacrylamide gel electrophoresis (PAGE). The midgut of T. castaneum is formed by digestive, regenerative, and endocrine cells. The effects of parasitism on the digestive cells are severe, because the gregarines remain attached to these cells to absorb all the nutrients they need throughout their development. In these cells, the most common alterations observed include expansion and fragmentation of the rough endoplasmic reticulum, development of the smooth endoplasmic reticulum, changes in mitochondrial cristae, cytoplasmic vacuolization, formation of myelin structures, spherites, large intercellular spaces, autophagic vesicles, expansion of the basal labyrinth, and cytoplasmic protrusions. Deposits of glycogen granules were also observed. Amylase activity was reduced in parasitized insects. Regenerative cells were found in disorganized crypts and did not differentiate into new cells, thus, compromising the restoration of the damaged epithelium. Though few morphological alterations were observed in the endocrine cells, results suggest that the synthesis and/or release of hormones might be impaired. Nine esterases (EST-1 to 9) were identified in the midgut of T. castaneum and were expressed in varying levels in response

  18. The roles of thermal transient receptor potential channels in thermotactic behavior and in thermal acclimation in the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Kim, Hong Geun; Margolies, David; Park, Yoonseong

    2015-05-01

    To survive in variable or fluctuating temperature, organisms should show appropriate behavioral and physiological responses which must be mediated through properly attuned thermal sensory mechanisms. Transient receptor potential channels (TRPs) are a family of cation channels a number of which, called thermo-TRPs, are known to function as thermosensors. We investigated the potential role of thermo-TPRs that have been previously identified in the fruit fly, Drosophila melanogaster, in thermotaxis and thermal acclimation in the red flour beetle, Tribolium castaneum. Phylogenetic analysis of the trp genes showed generally one-to-one orthology between those in D. melanogaster and in T. castaneum, although there are putative gene-losses in two TRP subfamilies of D. melanogaster. With RNA interference (RNAi) of T. castaneum thermo-TRP candidates painless, pyrexia and trpA1, we measured thermal avoidance behavior. RNAi of trpA1 resulted in reduced avoidance of high temperatures, 39 and 42 °C. We also measured the effects of RNAi on heat-induced knockout and death under a short exposure to high temperature (1min at 52 °C) either with or without a 10-min acclimation period at 42 °C. Relatively short exposure to high temperature was enough to induce high temperature thermal acclimation. RNAi of trpA1 led to faster knockout at 52 °C. RNAi of painless showed lower recovery rates from heat-induced knockout after thermal acclimation, and RNAi of pyrexia showed lower long-term survivorship without thermal acclimation. Therefore, we concluded that trpA1 is important in high temperature sensing and also in enhanced tolerance to high-temperature induced knockout; painless plays a role in rapid acclimation to high temperature; and pyrexia functions in protecting beetles from acute heat stress without acclimation.

  19. A Major Facilitator Superfamily protein encoded by TcMucK gene is not required for cuticle pigmentation, growth and development in Tribolium castaneum.

    Science.gov (United States)

    Mun, Seulgi; Noh, Mi Young; Osanai-Futahashi, Mizuko; Muthukrishnan, Subbaratnam; Kramer, Karl J; Arakane, Yasuyuki

    2014-06-01

    Insect cuticle pigmentation and sclerotization (tanning) are vital physiological processes for insect growth, development and survival. We have previously identified several colorless precursor molecules as well as enzymes involved in their biosynthesis and processing to yield the mature intensely colored body cuticle pigments. A recent study indicated that the Bombyx mori (silkmoth) gene, BmMucK, which encodes a protein orthologous to a Culex pipiens quiquefasciatus (Southern house mosquito) cis,cis, muconate transporter, is a member of the "Major Facilitator Superfamily" (MFS) of transporter proteins and is associated with the appearance of pigmented body segments of naturally occurring body color mutants of B. mori. While RNA interference of the BmMucK gene failed to result in any observable phenotype, RNAi using a dsRNA for an orthologous gene from the red flour beetle, Tribolium castaneum, was reported to result in molting defects and darkening of the cuticle and some body parts, leading to the suggestion that orthologs of MucK genes may differ in their functions among insects. To verify the role and essentiality of the ortholog of this gene in development and body pigmentation function in T. castaneum we obtained cDNAs for the orthologous gene (TcMucK) from RNA isolated from the GA-1 wild-type strain of T. castaneum. The sequence of a 1524 nucleotides-long cDNA for TcMucK which encodes the putatively full-length protein, was assembled from two overlapping RT-PCR fragments and the expression profile of this gene during development was analyzed by real-time PCR. This cDNA encodes a 55.8 kDa protein consisting of 507 amino acid residues and includes 11 putative transmembrane segments. Transcripts of TcMucK were detected throughout all of the developmental stages analyzed. The function of this gene was explored by injection of two different double-stranded RNAs targeting different regions of the TcMucK gene (dsTcMucKs) into young larvae to down

  20. Evaluación de la acción insecticida de la rapanona sobre Tribolium castaneum (Herbst

    Directory of Open Access Journals (Sweden)

    Chavez Germán

    1992-11-01

    Full Text Available Experiments were carried out to detect and estimate the insect-killing effect of rapanone on the flour beetle  T.castaneum. Two kinds ofessays were made: by ingestion and by contacto It was proved that rapanone has insecticide activity on T. castaneum. Relatively high LD50 were determined, in comprarison
    with those of conventional commercial insecticides. An inverse proportionality, between doses of rapanone and the mean productivity, was also found; adose of 7000 mg rapanone per kg standard culture medium is enough to exterminate, almost completely, a population of T. castaneum in one generation.
    Se realizaron experimentos para detectar y cuantificar efectos insecticidas de la rapa nona sobre el escarabajo de la harina, Tribolium castaneum. Las pruebas fueron de dos clases: por ingestión y por contacto. Se comprobó que la rapanona tiene propiedades insecticidas sobre T. castaneum. Las toxicidades agudas, medidas en términos de DL50 fueron relativamente altas comparadas con insecticidas sintéticos comerciales. También se encontró una proporcionalidad inversa entre dosis de rapanona y productividad promedio; una dosis de 7000 mg de rapanona por kilo de alimento, es suficiente para exterminar casi del todo una poblaci6n de T.castaneum en una sola generación.

  1. Genomic and gene regulatory signatures of cryptozoic adaptation: Loss of blue sensitive photoreceptors through expansion of long wavelength-opsin expression in the red flour beetle Tribolium castaneum

    Directory of Open Access Journals (Sweden)

    Cook Tiffany A

    2007-12-01

    Full Text Available Abstract Background Recent genome sequence analysis in the red flour beetle Tribolium castaneum indicated that this highly crepuscular animal encodes only two single opsin paralogs: a UV-opsin and a long wavelength (LW-opsin; however, these animals do not encode a blue (B-opsin as most other insects. Here, we studied the spatial regulation of the Tribolium single LW- and UV-opsin gene paralogs in comparison to that of the five opsin paralogs in the retina of Drosophila melanogaster. Results In situ hybridization analysis reveals that the Tribolium retina, in contrast with other insect retinas, constitutes a homogenous field of ommatidia that have seven LW-opsin expressing photoreceptors and one UV-/LW-opsin co-expressing photoreceptor per eye unit. This pattern is consistent with the loss of photoreceptors sensitive to blue wavelengths. It also identifies Tribolium as the first example of a species in insects that co-expresses two different opsins across the entire retina in violation of the widely observed "one receptor rule" of sensory cells. Conclusion Broader studies of opsin evolution in darkling beetles and other coleopteran groups have the potential to pinpoint the permissive and adaptive forces that played a role in the evolution of vision in Tribolium castaneum.

  2. A genome-wide inventory of neurohormone GPCRs in the red flour beetle Tribolium castaneum

    DEFF Research Database (Denmark)

    Hauser, Frank; Cazzamali, Giuseppe; Williamson, Michael

    2008-01-01

    Insect neurohormones (biogenic amines, neuropeptides, and protein hormones) and their G protein-coupled receptors (GPCRs) play a central role in the control of behavior, reproduction, development, feeding and many other physiological processes. The recent completion of several insect genome...... projects has enabled us to obtain a complete inventory of neurohormone GPCRs in these insects and, by a comparative genomics approach, to analyze the evolution of these proteins. The red flour beetle Tribolium castaneum is the latest addition to the list of insects with a sequenced genome and the first...... coleopteran (beetle) to be sequenced. Coleoptera is the largest insect order and about 30% of all animal species living on earth are coleopterans. Some coleopterans are severe agricultural pests, which is also true for T. castaneum, a global pest for stored grain and other dried commodities for human...

  3. The sperm ultrastructure and spermiogenesis of Tribolium castaneum (Coleoptera: Tenebrionidae) with evidence of cyst degeneration.

    Science.gov (United States)

    Dias, Glenda; Lino-Neto, José; Mercati, David; Dallai, Romano

    2015-06-01

    Previous studies on the spermatogenesis of tenebrionid beetles showed the unusual formation of two antiparallel sperm bundles per cyst. In this work we reported this feature also in Tribolium castaneum using light and transmission electron microscopy. The sperm structure of T. castaneum, similar to other tenebrionids, consists of a three-layered acrosome, an elongated nucleus and a flagellum with a 9+9+2 axoneme, two accessory bodies and two asymmetric mitochondrial derivatives. The presence of two antiparallel sperm bundles per cyst also in Meloidae and Rhipiphoridae suggests that it is a strong trait synapomorphic for Tenebrionoidea. The huge degeneration of whole sperm cells in several cysts of testes during spermiogenesis is also described.

  4. Efficacy of aerosol applications of methoprene and synergized pyrethrin against Tribolium castaneum adults and eggs.

    Science.gov (United States)

    Tucker, Angela M; Campbell, James F; Arthur, Frank H; Zhu, Kun Yan

    2014-06-01

    Experiments were performed to determine the efficacy of a single aerosol application of the insecticides methoprene and piperonyl butoxide-synergized pyrethrin, alone or in combination, and the insecticide carrier, Isopar M, against Tribolium castaneum (Herbst), the red flour beetle. The initial test exposed adults to insecticide treatments and placed male/female pairs in flour. All adults exposed to synergized pyrethrin were knocked down for at least 24 h after exposure but they recovered. High adult survival and similar average numbers of living F1 progeny were produced regardless of treatment exposure. In a separate test, insecticide treatments were directly applied to newly laid eggs, which resulted in the suppression of egg hatch. Synergized pyrethrin was the most effective insecticide (P castaneum egg hatch and development to the adult stage.

  5. Phosphine resistance in Tribolium castaneum and Rhyzopertha dominica from stored wheat in Oklahoma.

    Science.gov (United States)

    Opit, G P; Phillips, T W; Aikins, M J; Hasan, M M

    2012-08-01

    Phosphine gas, or hydrogen phosphide (PH3), is the most common insecticide applied to durable stored products worldwide and is routinely used in the United States for treatment of bulk-stored cereal grains and other durable stored products. Research from the late 1980s revealed low frequencies of resistance to various residual grain protectant insecticides and to phosphine in grain insect species collected in Oklahoma. The present work, which used the same previously established discriminating dose bioassays for phosphine toxicity as in the earlier study, evaluated adults of nine different populations of red flour beetle, Tribolium castaneum (Herbst), and five populations of lesser grain borer, Rhyzopertha dominica (F.) collected from different geographic locations in Oklahoma. One additional population for each species was a laboratory susceptible strain. Discriminating dose assays determined eight out of the nine T. castaneum populations, and all five populations of R. dominica, contained phosphine-resistant individuals, and highest resistance frequencies were 94 and 98%, respectively. Dose-response bioassays and logit analyses determined that LC99 values were approximately 3 ppm for susceptible and 377 ppm for resistant T. castaneum, and approximately 2 ppm for susceptible and 3,430 ppm for resistant R. dominica. The most resistant T. castaneum population was 119-fold more resistant than the susceptible strain and the most resistant R. dominica population was over 1,500-fold more resistant. Results suggest a substantial increase in phosphine resistance in these major stored-wheat pests in the past 21 yr, and these levels of resistance to phosphine approach those reported for other stored-grain pest species in other countries.

  6. Copulatory courtship and cryptic female choice in red flour beetles Tribolium castaneum.

    OpenAIRE

    Edvardsson, M; Arnqvist, G.

    2000-01-01

    Males of many animal species engage in courtship behaviours during and after copulation that appear to be solely aimed at stimulating the female. It has been suggested that these behaviours have evolved by cryptic female choice, whereby females are thought to impose biases on male postmating paternity success. Males of the red flour beetle Tribolium castaneum rub the lateral edges of the females' elytra with their tarsi during copulation. We manipulated female perception of this behaviour by ...

  7. First evidence of DNA methylation in insect Tribolium castaneum: environmental regulation of DNA methylation within heterochromatin.

    Science.gov (United States)

    Feliciello, Isidoro; Parazajder, Josip; Akrap, Ivana; Ugarković, Durđica

    2013-05-01

    DNA methylation has been studied in many eukaryotic organisms, in particular vertebrates, and was implicated in developmental and phenotypic variations. Little is known about the role of DNA methylation in invertebrates, although insects are considered as excellent models for studying the evolution of DNA methylation. In the red flour beetle, Tribolium castaneum (Tenebrionidae, Coleoptera), no evidence of DNA methylation has been found till now. In this paper, a cytosine methylation in Tribolium castaneum embryos was detected by methylation sensitive restriction endonucleases and immuno-dot blot assay. DNA methylation in embryos is followed by a global demethylation in larvae, pupae and adults. DNA demethylation seems to proceed actively through 5-hydroxymethylcytosine, most probably by the action of TET enzyme. Bisulfite sequencing of a highly abundant satellite DNA located in pericentromeric heterochromatin revealed similar profile of cytosine methylation in adults and embryos. Cytosine methylation was not only restricted to CpG sites but was found at CpA, CpT and CpC sites. In addition, complete cytosine demethylation of heterochromatic satellite DNA was induced by heat stress. The results reveal existence of DNA methylation cycling in T. castaneum ranging from strong overall cytosine methylation in embryos to a weak DNA methylation in other developmental stages. Nevertheless, DNA methylation is preserved within heterochromatin during development, indicating its role in heterochromatin formation and maintenance. It is, however, strongly affected by heat stress, suggesting a role for DNA methylation in heterochromatin structure modulation during heat stress response.

  8. Microbiota plays a role in oral immune priming in Tribolium castaneum

    Directory of Open Access Journals (Sweden)

    Momir eFuto

    2016-01-01

    Full Text Available Animals are inhabited by a diverse community of microorganisms. The relevance of such microbiota is increasingly being recognised in a broad spectrum of species, ranging from sponges to primates, revealing various beneficial roles that microbes can play. The red flour beetle Tribolium castaneum represents a well-established experimental model organism for studying questions in ecology and evolution, however, the relevance of its microbial community is still largely unknown. T. castaneum larvae orally exposed to inactivated bacterial components of the entomopathogen Bacillus thuringiensis bv. tenebrionis showed increased survival upon a subsequent challenge with spores of this bacterium. To investigate whether T. castaneum microbiota plays a role in this phenomenon, we established a protocol for raising microbe-free larvae and subsequently tested whether they differ in their ability to mount such a priming response. Here we demonstrate that larvae with significantly lowered microbial loads, show decreased survival upon secondary challenge with B. thuringiensis bv. tenebrionis spores, compared to animals which were allowed to regain their microbiota before priming. Although the exact mechanism of oral immune priming is unclear, we here suggest that microbiota plays a crucial role in oral immune priming in this species.

  9. Concentration dependent toxicokinetics of copper in the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae).

    Science.gov (United States)

    Bednarska, Agnieszka J; Stępień, Katarzyna

    2015-11-01

    To predict internal metal concentrations in animals under specific environmental exposures, the relationship between the exposure concentrations and values of toxicokinetic parameters must be known. At high exposure levels, the availability of carriers transporting metal ions through cellular membranes may become limited, thereby decreasing the assimilation rates (k A ). Furthermore, increased metal concentrations in food may result in greater damage to the gut and reduce the assimilation efficiency and/or increase the elimination rate (k E ). Therefore, k A should decrease and k E should increase with increasing metal concentrations. In fact, our study on Tribolium castaneum exposed to Cu at 500, 1000, 2000 and 4000 mg kg(-1) of dry flour showed that with increasing Cu concentrations, k A decreased from 0.0042 day(-1) at 500 mg kg(-1) to 0.0026 day(-1) at 4000 mg kg(-1) in females and from 0.0029 to 0.001 day(-1) in males and k E increased from 0.027 to 0.064 day(-1) and from 0.018 to 0.04 day(-1) in females and males, respectively. Significant differences in k A between the sexes were observed at 2000 and 4000 mg kg(-1), whereas significant differences between treatments were found for k A in males. Copper was efficiently regulated by T. castaneum: an eightfold increase in exposure concentrations resulted in only a ca. twofold increase in the internal concentration. No Cu effect on the respiratory metabolism of T. castaneum was found.

  10. JH modulates a cellular immunity of Tribolium castaneum in a Met-independent manner.

    Science.gov (United States)

    Hepat, Rahul; Kim, Yonggyun

    2014-04-01

    Juvenile hormone (JH) regulates diverse physiological processes in insects during entire developmental stages. Especially, the identification of Methoprene-tolerant (Met), a JH nuclear receptor, allows us to better understand molecular actions of JH to control gene expressions related with metamorphosis. However, several physiological processes including cellular immune response and some molecular actions of JH have been suspected to be mediated via its non-genomic actions. To prove its non-genomic action, JH nuclear signals were suppressed by RNA interference (RNAi) of Met or its downstream gene, Krüppel homolog 1 (Kr-h1), in the red flour beetle, Tribolium castaneum. These RNAi-treated larvae failed to undergo a normal development and suffered precocious metamorphosis. Hemocytes of T. castaneum exhibited their spreading behavior on extracellular matrix and nodule formation in response to bacterial challenge. When the larvae were treated with either RNAi of Met or Kr-h1, the hemocytes of the treated larvae were responsive to JH without any significant difference with those of control larvae. These results suggest that the response of hemocytes to JH is not mediated by its nuclear signal. On the other hand, the JH modulation of hemocyte behaviors of T. castaneum was significantly influenced by membrane and cytosolic protein activities, in which ethoxyzolamide (a specific inhibitor of carbonic anhydrase), calphostin C (a specific inhibitor of protein kinase C) or ouabain (a specific inhibitor of Na(+)-K(+) ATPase) significantly suppressed the responsiveness of hemocytes to JH.

  11. 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

  12. Innexin7a forms junctions that stabilize the basal membrane during cellularization of the blastoderm in Tribolium castaneum.

    Science.gov (United States)

    van der Zee, Maurijn; Benton, Matthew A; Vazquez-Faci, Tania; Lamers, Gerda E M; Jacobs, Chris G C; Rabouille, Catherine

    2015-06-15

    In insects, the fertilized egg undergoes a series of rapid nuclear divisions before the syncytial blastoderm starts to cellularize. Cellularization has been extensively studied in Drosophila melanogaster, but its thick columnar blastoderm is unusual among insects. We therefore set out to describe cellularization in the beetle Tribolium castaneum, the embryos of which exhibit a thin blastoderm of cuboidal cells, like most insects. Using immunohistochemistry, live imaging and transmission electron microscopy, we describe several striking differences to cellularization in Drosophila, including the formation of junctions between the forming basal membrane and the yolk plasmalemma. To identify the nature of this novel junction, we used the parental RNAi technique for a small-scale screen of junction proteins. We find that maternal knockdown of Tribolium innexin7a (Tc-inx7a), an ortholog of the Drosophila gap junction gene Innexin 7, leads to failure of cellularization. In Inx7a-depleted eggs, the invaginated plasma membrane retracts when basal cell closure normally begins. Furthermore, transiently expressed tagged Inx7a localizes to the nascent basal membrane of the forming cells in wild-type eggs. We propose that Inx7a forms the newly identified junctions that stabilize the forming basal membrane and enable basal cell closure. We put forward Tribolium as a model for studying a more ancestral mode of cellularization in insects.

  13. Scaffolding for Three-Dimensional Embryonic Vasculogenesis

    Science.gov (United States)

    Kraehenbuehl, Thomas P.; Aday, Sezin; Ferreira, Lino S.

    Biomaterial scaffolds have great potential to support efficient vascular differentiation of embryonic stem cells. Vascular cell fate-specific biochemical and biophysical cues have been identified and incorporated into three-dimensional (3D) biomaterials to efficiently direct embryonic vasculogenesis. The resulting vascular-like tissue can be used for regenerative medicine applications, further elucidation of biophysical and biochemical cues governing vasculogenesis, and drug discovery. In this chapter, we give an overview on the following: (1) developmental cues for directed differentiation of human embryonic stem cells (hESCs) into vascular cells, (2) 3D vascular differentiation in embryoid bodies (EBs), (3) preparation of 3D scaffolds for the vascular differentiation of hESCs, and (4) the most significant studies combining scaffolding and hESCs for development of vascular-like tissue.

  14. 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...

  15. The ABCs of eye color in Tribolium castaneum: orthologs of the Drosophila white, scarlet, and brown Genes.

    Science.gov (United States)

    Grubbs, Nathaniel; Haas, Sue; Beeman, Richard W; Lorenzen, Marcé D

    2015-03-01

    In Drosophila melanogaster, each of the three paralogous ABC transporters, White, Scarlet and Brown, is required for normal pigmentation of the compound eye. We have cloned the three orthologous genes from the beetle Tribolium castaneum. Conceptual translations of Tribolium white (Tcw), scarlet (Tcst), and brown (Tcbw) are 51, 48, and 32% identical to their respective Drosophila counterparts. We have identified loss-of-eye-pigment strains that bear mutations in Tcw and Tcst: the Tcw gene in the ivory (i) strain carries a single-base transversion, which leads to an E → D amino-acid substitution in the highly conserved Walker B motif, while the Tcst gene in the pearl (p) strain has a deletion resulting in incorporation of a premature stop codon. In light of these findings, the mutant strains i and p are herein renamed white(ivory) (w(i)) and scarlet(pearl) (st(p)), respectively. In addition, RNA inhibition of Tcw and Tcst recapitulates the mutant phenotypes, confirming the roles of these genes in normal eye pigmentation, while RNA interference of Tcbw provides further evidence that it has no role in eye pigmentation in Tribolium. We also consider the evolutionary implications of our findings.

  16. Capture of Tribolium castaneum and Tribolium confusum (Coleoptera: Tenebrionidae) in Floor Traps: The Effect of Previous Captures.

    Science.gov (United States)

    Athanassiou, Christos G; Kavallieratos, Nickolas G; Campbell, James F

    2016-02-01

    The impact of prior captures on the trapping performance of floor traps was evaluated for the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), and the confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae), in laboratory conditions. The effect of trap seeding, adding adults of the same or different species, was evaluated in order to determine possible effects of prior captures in the trap on each species' behavioral responses. The presence of seeded beetles of the same species resulted in an increase in beetle captures for both T. castaneum and T. confusum, but when traps were seeded with the opposite species, there was no increase in beetle captures for either species, and for T. castaneum overall captures in both seeded and unseeded traps was reduced. Overall, T. castaneum tended to have greater captures than T. confusum regardless of the treatment. When the two species were released together, this negated the increased response to seeded traps observed in the single-species treatments. These findings suggest the potential that the presence of beetles in a trap may be influencing the response of beetles in a nearby trap and that T. castaneum and T. confusum when they occur together may influence each other's response to traps.

  17. Temporal expression of CD184(CXCR4) and CD171(L1CAM) identifies distinct early developmental stages of human retinal ganglion cells in embryonic stem cell derived retina.

    Science.gov (United States)

    Aparicio, J G; Hopp, H; Choi, A; Mandayam Comar, J; Liao, V C; Harutyunyan, N; Lee, T C

    2016-11-17

    Human retinal ganglion cells (RGCs) derived from pluripotent stem cells (PSCs) have anticipated value for human disease study, drug screening, and therapeutic applications; however, their full potential remains underdeveloped. To characterize RGCs in human embryonic stem cell (hESC) derived retinal organoids we examined RGC markers and surface antigen expression and made comparisons to human fetal retina. RGCs in both tissues exhibited CD184 and CD171 expression and distinct expression patterns of the RGC markers BRN3 and RBPMS. The retinal progenitor cells (RPCs) of retinal organoids expressed CD184, consistent with its expression in the neuroblastic layer in fetal retina. In retinal organoids CD184 expression was enhanced in RGC competent RPCs and high CD184 expression was retained on post-mitotic RGC precursors; CD171 was detected on maturing RGCs. The differential expression timing of CD184 and CD171 permits identification and enrichment of RGCs from retinal organoids at differing maturation states from committed progenitors to differentiating neurons. These observations will facilitate molecular characterization of PSC-derived RGCs during differentiation, critical knowledge for establishing the veracity of these in vitro produced cells. Furthermore, observations made in the retinal organoid model closely parallel those in human fetal retina further validating use of retinal organoid to model early retinal development.

  18. A cell line derived from the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae).

    Science.gov (United States)

    Goodman, Cynthia L; Stanley, David; Ringbauer, Joseph A; Beeman, Richard W; Silver, Kristopher; Park, Yoonseong

    2012-08-01

    The red flour beetle, Tribolium castaneum, is a model organism for agricultural and medical research and its complete genome is sequenced. We established a continuously replicating T. castaneum cell line to complement existing physiological, genetic, and genomic research tools. We set up trial cell cultures from egg, pupa, and adult stages as tissue sources and incubated them in six separate cell culture media to determine the optimal combination of tissue source and medium for cell replication. Our most promising culture was generated by co-culturing adult (∼75 %) and pupal tissues in EX-CELL 420 medium containing 9 % FBS. Our new cell culture is designated BCIRL-TcA-CLG1 (TcA) and it has been subcultured more than 90 times. Amplification of genomic DNA with species-specific primers yielded DNA fragments of the expected sizes and with sequences identical to those from the published Tribolium genome. Additionally, we characterized this line using DNA fingerprinting (DAF-PCR) and compared it with three other coleopteran cell lines and its conspecific pupae to confirm identity. Its doubling time is 155.2 hr. Early passages consisted of attached cells and vesicles in suspension, whereas later passages consisted primarily of attached, spherical cells. Similar to other established cell lines, the ploidy of TcA cells was variable, ranging from 20 chromosomes/cell (diploid) to above 30 chromosomes/cell. TcA cells withstood incubation at 40°C for 1 h with no decrease in viability. We recorded increased levels of one heat shock protein (43 kDa) and of the hsp68a transcript following exposure to 40°C. Taken together, this represents the first report of a continuously replicating T. castaneum cell line. We expect the BCIRL-TcA-CLG1 line will become a useful tool in Tribolium research.

  19. Transovarial Effect of Novaluron on Tribolium castaneum (Coleoptera: Tenebrionidae) After Termination of Direct Contact.

    Science.gov (United States)

    Trostanetsky, A; Kostyukovsky, M; Quinn, E

    2015-01-01

    The insect growth regulator novaluron (Rimon 10 EC, Makhteshim-Agan Ltd, Israel) is used against many field pests on corn, vegetables, orchards, forests, and cotton plantations. Previously, we studied various effects of novaluron on stored grain pests. Termination in Tribolium castaneum (Herbst) eggs hatching after treating adults with novaluron and following restoration after adult transfer to untreated media was observed. The objective of this study was to investigate the restoration of T. castaneum egg hatch following transfer of adults from treated media to untreated favorable and unfavorable media. The time needed for hatching restoration of 50% of eggs laid by adults transferred from novaluron (1 ppm) treated flour to untreated flour (RT50) was 2.7 d. RT50 for those transferred to untreated wheat grain was 4.1 d. RT90 in flour was 3.6 d, in grain--6.1 d. Varieties of RTs in grain and in flour with nonoverlapping confidence intervals indicate that RTs were significantly different. Delay of eggs hatching restoration for adults transferred from treated flour to unfavorable media (Petri dishes with limited amount of flour, lying of eggs not detected) was observed. RT50 in flour was 2.1 d and RT90--3.1 d, while RT50 in the unfavorable media was 3.4 d and RT90 6.5 d. Delayed effect of egg hatching restoration after adult transfer to unfavorable media provides evidence of the significant role of insect physiological state in novaluron excretion and (or) degradation by T. castaneum females.

  20. Models to predict mortality of Tribolium castaneum (Coleoptera: Tenebrionidae) exposed to elevated temperatures during structural heat treatments.

    Science.gov (United States)

    Jian, Fuji; Subramanyam, Bhadriraju; Jayas, Digvir S; White, Noel D G

    2013-10-01

    Novel thermal death models were developed with certain assumptions, and these models were validated by using actual heat treatment data collected under laboratory conditions at constant temperatures over time and in commercial food-processing facilities where temperatures were dynamically changing over time. The predicted mortalities of both young larvae and adults of the red flour beetle, Tribolium castaneum (Herbst), were within 92-99% of actual measured insect mortalities. There was good concordance between predicted and observed mortalities of young larvae and adults of T. castaneum exposed to constant temperatures in laboratory growth chambers and at variable temperatures during structural heat treatments of commercial food-processing facilities. The models developed in this study can be used to determine effectiveness of structural heat treatments in killing young larvae and adults of T. castaneum and for characterizing insect thermotolerance.

  1. Tribolium castaneum Apolipophorin-III acts as an immune response protein against Bacillus thuringiensis Cry3Ba toxic activity.

    Science.gov (United States)

    Contreras, Estefanía; Rausell, Carolina; Real, M Dolores

    2013-07-01

    In this study, a 2.1-fold Apolipophorin-III mRNA up-regulation was found in Tribolium castaneum larvae challenged with Bacillus thuringiensis Cry3Ba spore-crystal mixture. Knockdown of Apolipophorin-III by RNAi resulted in increased T. castaneum larvae susceptibility following Cry3Ba spore-crystal treatment, demonstrating Apolipophorin-III involvement in insect defense against B. thuringiensis. We showed that Apolipophorin-III participates in T. castaneum immune response to B. thuringiensis activating the prophenoloxidase cascade since: (i) phenoloxidase activity significantly increased after Cry3Ba spore-crystal treatment compared to untreated or Cry1Ac spore-crystal treated larvae and (ii) phenoloxidase activity in Cry3Ba spore-crystal treated Apolipophorin-III silenced larvae was 71±14% lower than that of non-silenced intoxicated larvae.

  2. Chemical Composition and Toxicity against Sitophilus zeamais and Tribolium castaneum of the Essential Oil of Murraya exotica Aerial Parts

    Directory of Open Access Journals (Sweden)

    Zhi Long Liu

    2010-08-01

    Full Text Available In our screening program for new agrochemicals from Chinese medicinal herbs, Murraya exotica was found to possess insecticidal activity against the maize weevil, Sitophilus zeamais and red flour beetle, Tribolium castaneum. The essential oil of aerial parts of M. exotica was obtained by hydrodistillation and investigated by GC and GC-MS. The main components of M. exotica essential oil were spathulenol (17.7%, a-pinene (13.3%, caryophyllene oxide (8.6%, and a-caryophyllene (7.3%. Essential oil of M. exotica possessed fumigant toxicity against S. zeamais and T. castaneum adults with LC50 values of 8.29 and 6.84 mg/L, respectively. The essential oils also show contact toxicity against S. zeamais and T. castaneum adults with LD50 values of 11.41 and 20.94 mg/adult, respectively.

  3. Evaluation of reference genes for quantitative polymerase chain reaction across life cycle stages and tissue types of Tribolium castaneum.

    Science.gov (United States)

    Toutges, Michelle J; Hartzer, Kris; Lord, Jeffrey; Oppert, Brenda

    2010-08-25

    The genome of the genetic model for coleopteran insects, Tribolium castaneum, is now available for downstream applications. To facilitate gene expression studies in T. castaneum, genes were evaluated for suitability as normalizers in comparisons across tissues and/or developmental stages. In less diverse samples, such as comparisons within developmental stages or tissue only, normalizers for mRNA were more stable and consistent. Overall, the genes for ribosomal proteins rps6, rpl13a, rps3, and rps18 were the most stable normalizers for broad scale gene expression analysis in T. castaneum. However, their stability ranking was dependent upon the instrument as well as the analysis program. These data emphasize the need to optimize normalizers used in all real-time polymerase chain reaction experiments specifically for the experimental conditions and thermocycler and to carefully evaluate data generated by computational algorithms.

  4. Interaction of starvation and gamma radiation on the fecundity of tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

    Energy Technology Data Exchange (ETDEWEB)

    Khattak, S.U.K.; Shafique, M. (Nuclear Inst. for Agriculture and Biology, Faisalabad (Pakistan))

    Starved and unstarved beetles of Tribolium castaneum (Herbst) were irradiated with low doses of gamma radiation viz:-4, 6, 8, 12 krad and their fecundity was studied up to 8 days under controlled laboratory conditions. It was recorded that fecundity decreased immediately following irradiation, but the subsequent recovery was dose dependent. The rate of oviposition increased with an increased period of starvation. Pre-irradiation starved beetles were less fecunditive than the post-irradiation ones. Irradiated individuals, though, laid eggs, but none of the eggs hatched at 8 and 12 krad.

  5. Effect of gamma radiation on the eggs of Tribolium castaneum (Herbst)

    Energy Technology Data Exchange (ETDEWEB)

    Mehta, V.K.; Sethi, G.R. (Indian Agricultural Research Inst., New Delhi. Nuclear Research Lab.)

    1984-09-01

    Studies on the susceptibility of eggs of Tribolium castaneum (Herbst) to gamma radiation revealed that the radiosensitivity of eggs decreased with the increasing age of the embryo and the development of the embryo was arrested if the eggs were irradiated in the early stage of development (24-48 hr old). However, when 72-96 hr old eggs were irradiated, comparatively higher dose was required to render them nonviable. Mortality of the eggs of the same age group was directly related to the radiation dose. Survival of the insect to the adult stage was prevented by exposure to a dose of 8 Krad or more, irrespective of the age of the eggs.

  6. Bioactivity of Indonesian mahogany, Toona sureni (Blume (Meliaceae, against the red flour beetle, Tribolium castaneum (Coleoptera, Tenebrionidae

    Directory of Open Access Journals (Sweden)

    Sahana Parvin

    2012-09-01

    Full Text Available Bioactivity of Indonesian mahogany, Toona sureni (Blume (Meliaceae, against the red flour beetle, Tribolium castaneum (Coleoptera, Tenebrionidae. The insecticidal activity of Toona sureni (Blume Merr. was evaluated considering repellency, mortality and progeny production of F1 adults of Tribolium castaneum (Herbst, 1797 (Coleoptera, Tenebrionidae. Dried extract of seeds of T. sureni was dissolved in acetone to prepare solution of various concentrations (0.5, 1.0, 2.5 and 5.0%. To test for repellency, the insects were exposed to treated filter paper. Mortality of larvae, pupae and adults was evaluated by the treatment of spraying the insects with different concentrations of T. sureni extract. Residual effect of the extract was also evaluated considering the production of progeny of F1 adults. The highest repellency (93.30% of T. castaneum occurred at the highest concentration (5.0% suspension of T. sureni; while the lowest (0.0% repellency occurred at 0.5% suspension after 1 day of treatment. The highest mortality against adults (86.71%, larvae (88.32% and pupae (85% occurred at 5% suspension at 8 days after application. There was a negative correlation between the concentrations of T. sureni and the production of F1 adult's progeny of T. castaneum. The highest number of progeny (147 of T. castaneum occurred in the control at 7 days after treatment; and the lowest number of progeny (43 occurred at 5.0% concentration in 1 day after treatment. The results show that T. sureni is toxic to T. castaneum and has the potential to control all stages of this insect in stored wheat.

  7. Effect of cold water extracts of Acacia modesta Wall. and Glycyrrhiza glabra Linn. on Tribolium castaneum and Lemna minor.

    Science.gov (United States)

    Nazeefullah, Sayed; Dastagir, Ghulam; Ahmad, Bashir

    2014-03-01

    The aim of the present study was to introduce an alternative way for insects control through biodegradable plants materials. The different cold water extracts dilutions of Acacia modesta and Glycyrrhiza glabra were tested against Tribolium castaneum. The extracts dilutions of both plants caused mortality of the Tribolium castaneum. ANOVA revealed that dilutions and plants were highly significant. The interaction between plants and dilutions was also significant at P Glycyrrhiza glabra extracts significantly inhibited the growth of Lemna minor. ANOVA showed that dilutions of both plants extracts were significant at P < 0.05.

  8. Bioactivity of Indonesian mahogany, Toona sureni (Blume (Meliaceae, against the red flour beetle, Tribolium castaneum (Coleoptera, Tenebrionidae Bioatividade do mogno da Indonésia, Toona sureni (Blume (Meliaceae, contra o besouro-das-farinhas, Tribolium castaneum (Coleoptera, Tenebrionidae

    Directory of Open Access Journals (Sweden)

    Sahana Parvin

    2012-09-01

    Full Text Available Bioactivity of Indonesian mahogany, Toona sureni (Blume (Meliaceae, against the red flour beetle, Tribolium castaneum (Coleoptera, Tenebrionidae. The insecticidal activity of Toona sureni (Blume Merr. was evaluated considering repellency, mortality and progeny production of F1 adults of Tribolium castaneum (Herbst, 1797 (Coleoptera, Tenebrionidae. Dried extract of seeds of T. sureni was dissolved in acetone to prepare solution of various concentrations (0.5, 1.0, 2.5 and 5.0%. To test for repellency, the insects were exposed to treated filter paper. Mortality of larvae, pupae and adults was evaluated by the treatment of spraying the insects with different concentrations of T. sureni extract. Residual effect of the extract was also evaluated considering the production of progeny of F1 adults. The highest repellency (93.30% of T. castaneum occurred at the highest concentration (5.0% suspension of T. sureni; while the lowest (0.0% repellency occurred at 0.5% suspension after 1 day of treatment. The highest mortality against adults (86.71%, larvae (88.32% and pupae (85% occurred at 5% suspension at 8 days after application. There was a negative correlation between the concentrations of T. sureni and the production of F1 adult's progeny of T. castaneum. The highest number of progeny (147 of T. castaneum occurred in the control at 7 days after treatment; and the lowest number of progeny (43 occurred at 5.0% concentration in 1 day after treatment. The results show that T. sureni is toxic to T. castaneum and has the potential to control all stages of this insect in stored wheat.Bioatividade do mogno da Indonésia, Toona sureni (Blume (Meliaceae, contra o besouro-das-farinhas, Tribolium castaneum (Coleoptera, Tenebrionidae. A atividade inseticida de Toona sureni (Blume Merr. foi avaliada considerando repelência, mortalidade e a produção de progênie de adultos F1 de Tribolium castaneum (Herbst, 1797 (Coleoptera, Tenebrionidae. Extrato seco de sementes

  9. 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.

  10. Differentiation of neuron-like cells from mouse parthenogenetic embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    Xingrong Yan; Liwen Li; Fulin Chen; Yanhong Yang; Wei Liu; Wenxin Geng; Huichong Du; Jihong Cui; Xin Xie; Jinlian Hua; Shumin Yu

    2013-01-01

    Parthenogenetic embryonic stem cells have pluripotent differentiation potentials, akin to fertilized embryo-derived embryonic stem cells. The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells. Before differentiation, karyotype analysis was performed, with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells. Sex chromosomes were identified as XX. Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene, Oct4, at both the mRNA and protein levels, indicating pluripotent differentiation potential of the two embryonic stem cell subtypes. Embryonic stem cells were induced with retinoic acid to form embryoid bodies, and then dispersed into single cells. Single cells were differentiated in N2 differentiation medium for 9 days. Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin, βIII-tubulin and myelin basic protein. Quantitative real-time PCR found expression of neurogenesis related genes (Sox-1, Nestin, GABA, Pax6, Zic5 and Pitx1) in both types of embryonic stem cells, and Oct4 expression was significantly decreased. Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells. Thus, our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.

  11. Identification and evolution of two insulin receptor genes involved in Tribolium castaneum development and reproduction.

    Science.gov (United States)

    Sang, Ming; Li, Chengjun; Wu, Wei; Li, Bin

    2016-07-10

    The insulin and insulin-like signaling (IIS) pathway exists in a wide range of organisms from mammals to invertebrates and regulates several vital physiological functions. A phylogenetic analysis have indicated that insulin receptors have been duplicated at least twice among vertebrates, whereas only one duplication occurred in insects before the differentiation of Coleoptera, Hymenoptera, and Hemiptera. Thus, we cloned two putative insulin receptor genes, T.cas-ir1 and T.cas-ir2, from T. castaneum and determined that T.cas-ir1 is most strongly expressed during the late adult and early pupal stages, whereas T.cas-ir2 is most strongly expressed during the late larval stage. We found that larval RNAi against T.cas-ir1 and T.cas-ir2 causes 100% and 42.0% insect death, respectively, and that parental RNAi against T.cas-ir1 and T.cas-ir2 leads to 100% and 33.3% reductions in beetle fecundity, respectively. The hatching rate of ds-ir2 insects was 66.2%. Moreover, RNAi against these two genes increased the expression of the pkc, foxo, jnk, cdc42, ikk, and mekk genes but decreased erk gene expression. Despite these similarities, these two genes act via distinct regulatory pathways. These results indicate that these two receptors have functionally diverged with respect to the development and reproduction of T. castaneum, even though they retain some common regulatory signaling pathways.

  12. Longevity, growth rate and related traits among strains of Tribolium castaneum.

    Science.gov (United States)

    Soliman, M H; Lints, F A

    1975-01-01

    Longevity of eight laboratory strains of the flour beetle Tribolium castaneum, with various geographic backgrounds, was studied under constant laboratory conditions of 33 degrees C and 70% relative humidity in standard medium (95% whole wheat flour and 5% dried yeast) during a period of 227 days starting from the egg stage. The eggs were collected from the same parents, first a few days after emergence and afterwards at intervals of 13, 9, 10 and 11 days. Mean survival time (MST) was found to be strain-specified. It ranges from 128.6 days for KJ (Kyoto, Japan) to 174.2 days for ES (Edinburgh, Scotland). MST was highly correlated with the percentage of adults alive after 227 days, which did not change the ranking order of strain longevity. Parental age had no effect on longevity. The mean adult longevity of the strains was correlated with the available data on adult weight, growth rate, viability and productivity. There was no relationship between adult weight and longevity. LIfe span was found to depend on growth rate (measured as 13-day larval weight), percent viability (from 13-day larvae to adulthood) and productivity. Developmental time was also found to influence adult life span within certain limits (two extreme strains deviated). The data suggest that ageing and death in T. castaneum is under genetic control and support the idea that ageing, allied to development, is genetically controlled.

  13. Pharmacological and signalling properties of a D2-like dopamine receptor (Dop3) in Tribolium castaneum.

    Science.gov (United States)

    Verlinden, Heleen; Vleugels, Rut; Verdonck, Rik; Urlacher, Elodie; Vanden Broeck, Jozef; Mercer, Alison

    2015-01-01

    Dopamine is an important neurotransmitter in the central nervous system of vertebrates and invertebrates. Despite their evolutionary distance, striking parallels exist between deuterostomian and protostomian dopaminergic systems. In both, signalling is achieved via a complement of functionally distinct dopamine receptors. In this study, we investigated the sequence, pharmacology and tissue distribution of a D2-like dopamine receptor from the red flour beetle Tribolium castaneum (TricaDop3) and compared it with related G protein-coupled receptors in other invertebrate species. The TricaDop3 receptor-encoding cDNA shows considerable sequence similarity with members of the Dop3 receptor class. Real time qRT-PCR showed high expression in both the central brain and the optic lobes, consistent with the role of dopamine as neurotransmitter. Activation of TricaDop3 expressed in mammalian cells increased intracellular Ca(2+) signalling and decreased NKH-477 (a forskolin analogue)-stimulated cyclic AMP levels in a dose-dependent manner. We studied the pharmacological profile of the TricaDop3 receptor and demonstrated that the synthetic vertebrate dopamine receptor agonists, 2 - amino- 6,7 - dihydroxy - 1,2,3,4 - tetrahydronaphthalene hydrobromide (6,7-ADTN) and bromocriptine acted as agonists. Methysergide was the most potent of the antagonists tested and showed competitive inhibition in the presence of dopamine. This study offers important information on the Dop3 receptor from Tribolium castaneum that will facilitate functional analyses of dopamine receptors in insects and other invertebrates.

  14. Cryptic female choice during spermatophore transfer in Tribolium castaneum (Coleoptera: Tenebrionidae).

    Science.gov (United States)

    Fedina, Tatyana Y

    2007-01-01

    Sexual selection in both males and females promotes traits and behaviors that allow control over paternity when female mates with multiple males. Nonetheless, mechanisms of cryptic female choice have been consistently overlooked, due to traditional focus on sperm competition as well as difficulty in distinguishing male vs. female influence over processes occurring during and after mating. The first part of this study describes morphology and transformation of Tribolium castaneum spermatophores inferred from dissecting females immediately after normal or interrupted copulations. T. castaneum males are found to transfer spermatophores as an invaginated tube that everts inside the female bursa and which is filled with sperm during copulation. This sequence of events makes it feasible for females to control the sperm quantity transferred in each spermatophore. Through manipulation of the male phenotypic quality (by starvation) and manipulation of female control over sperm transfer (by killing a subset of females), the second part of this study examines whether females use control over transferred sperm quantity as a cryptic choice mechanism. Fed males transferred significantly more sperm per spermatophore than starved males but only when mating with live females. These results suggest an active differentiation by live females against starved males and provide an evidence for the proposed cryptic female choice mechanism.

  15. Effects of flour and milling debris on efficacy of beta-cyfluthrin for control of Tribolium castaneum

    Science.gov (United States)

    Laboratory and field studies were conducted to determine if accumulation of a flour food source or milling debris affected residual efficacy of beta-cyfluthrin for control of Tribolium castaneum (Herbst), the red flour beetle. In the laboratory study, the high label rate of 20 mg active ingredient (...

  16. Contact toxicity of deltamethrin against Tribolium castaneum (Coleoptera: Tenebrionidae), Sitophilus oryzae (Coleoptera: Curculionidae), and Rhyzopertha dominica (Coleoptera: Bostrichidae) adults

    Science.gov (United States)

    This study was conducted to evaluate deltamethrin-incorporated ZeroFly® Storage Bags for efficacy against stored-product insect pests. We evaluated response to deltamethrin concentrations for adults of three stored-product insects, Tribolium castaneum (Herbst), Sitophilus oryzae (L.), and Rhyzoperth...

  17. Properties and applications of embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Mouse embryonic stem (ES) cells are pluripotent cells derived from the early embryo and can be propagated stably in undifferentiated state in vitro. They retain the ability to differentiate into all cell types found in the embryonic and adult body in vivo, and can be induced to differentiate into many cell types under appropriate culture conditions in vitro. Using these properties, people have set up various differentiated systems of many cell types and tissues in vitro. Through analysis of these systems, one can identify novel bioactive factors and reveal mechanisms of cell differentiation and organogenesis. ES cell-derived differentiated cells can also be applied to cell transplantation therapy. In addition, we summarized the features and potential applications of human ES cells.

  18. 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

  19. Temporal regulation of embryonic M-phases.

    Science.gov (United States)

    Kubiak, Jacek Z; Bazile, Franck; Pascal, Aude; Richard-Parpaillon, Laurent; Polanski, Zbigniew; Ciemerych, Maria A; Chesnel, Franck

    2008-01-01

    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 other candidates

  20. Chemical composition of four essential oils from Eupatorium spp. Biological activities toward Tribolium castaneum (Coleoptera: Tenebrionidae

    Directory of Open Access Journals (Sweden)

    Hugo G. LANCELLE

    2009-01-01

    Full Text Available Se evaluaron las propiedades tóxicas y repelentes de los aceites esenciales de cuatro especies del género Eupatorium (Asteraceae: E. buniifolium Hook. et Arn, E. inulaefolium Kunth, E. arnottii Baker y E. viscidum Hook. & Arn, en diferentes concentraciones frente a adultos de Tribolium castaneum Herbst. Los aceites esenciales se aislaron de las partes aéreas de las plantas, mediante técnicas de hidrodestilación y se analizaron por los métodos GC-FID y GC-MS. Los ensayos de toxicidad por contacto demostraron que todos los aceites fueron tóxicos y la mortalidad fue, en todos los casos, dependiente de la dosis. El aceite esencial de E. buniifolium presentó la mayor actividad repelente.

  1. Genome-wide mapping of conserved microRNAs and their host transcripts in Tribolium castaneum

    Institute of Scientific and Technical Information of China (English)

    Qibin Luo; Qing Zhou; Xiaomin Yu; Hongbin Lin; Songnian Hu; Jun Yu

    2008-01-01

    MicroRNAs (miRNAs) are endogenous 22-nt RNAs, which play important regulatory roles by post-transcriptional gene silencing. A computational strategy has been developed for the identification of conserved miRNAs based on features of known metazoan miRNAs in red flour beetle (Tribolium castaneum), which is regarded as one of the major laboratory models of arthropods. Among 118 putative miRNAs, 47% and 53% of the predicted miRNAs from the red flour beetle are harbored by known protein-coding genes (intronic) and genes located outside (intergenic miRNA), respectively. There are 31 intronic miRNAs in the same transcriptional orientation as the host genes, which may share RNA polymerase Ⅱ and spliceosomal machinery with their host genes for their biogenesis. A hypothetical feedback model has been proposed based on the analysis of the relationship between intronic miRNAs and their host genes in the development of red flour beetle.

  2. Satellite DNA Modulates Gene Expression in the Beetle Tribolium castaneum after Heat Stress.

    Science.gov (United States)

    Feliciello, Isidoro; Akrap, Ivana; Ugarković, Đurđica

    2015-08-01

    Non-coding repetitive DNAs have been proposed to perform a gene regulatory role, however for tandemly repeated satellite DNA no such role was defined until now. Here we provide the first evidence for a role of satellite DNA in the modulation of gene expression under specific environmental conditions. The major satellite DNA TCAST1 in the beetle Tribolium castaneum is preferentially located within pericentromeric heterochromatin but is also dispersed as single repeats or short arrays in the vicinity of protein-coding genes within euchromatin. Our results show enhanced suppression of activity of TCAST1-associated genes and slower recovery of their activity after long-term heat stress relative to the same genes without associated TCAST1 satellite DNA elements. The level of gene suppression is not influenced by the distance of TCAST1 elements from the associated genes up to 40 kb from the genes' transcription start sites, but it does depend on the copy number of TCAST1 repeats within an element, being stronger for the higher number of copies. The enhanced gene suppression correlates with the enrichment of the repressive histone marks H3K9me2/3 at dispersed TCAST1 elements and their flanking regions as well as with increased expression of TCAST1 satellite DNA. The results reveal transient, RNAi based heterochromatin formation at dispersed TCAST1 repeats and their proximal regions as a mechanism responsible for enhanced silencing of TCAST1-associated genes. Differences in the pattern of distribution of TCAST1 elements contribute to gene expression diversity among T. castaneum strains after long-term heat stress and might have an impact on adaptation to different environmental conditions.

  3. Satellite DNA Modulates Gene Expression in the Beetle Tribolium castaneum after Heat Stress.

    Directory of Open Access Journals (Sweden)

    Isidoro Feliciello

    2015-08-01

    Full Text Available Non-coding repetitive DNAs have been proposed to perform a gene regulatory role, however for tandemly repeated satellite DNA no such role was defined until now. Here we provide the first evidence for a role of satellite DNA in the modulation of gene expression under specific environmental conditions. The major satellite DNA TCAST1 in the beetle Tribolium castaneum is preferentially located within pericentromeric heterochromatin but is also dispersed as single repeats or short arrays in the vicinity of protein-coding genes within euchromatin. Our results show enhanced suppression of activity of TCAST1-associated genes and slower recovery of their activity after long-term heat stress relative to the same genes without associated TCAST1 satellite DNA elements. The level of gene suppression is not influenced by the distance of TCAST1 elements from the associated genes up to 40 kb from the genes' transcription start sites, but it does depend on the copy number of TCAST1 repeats within an element, being stronger for the higher number of copies. The enhanced gene suppression correlates with the enrichment of the repressive histone marks H3K9me2/3 at dispersed TCAST1 elements and their flanking regions as well as with increased expression of TCAST1 satellite DNA. The results reveal transient, RNAi based heterochromatin formation at dispersed TCAST1 repeats and their proximal regions as a mechanism responsible for enhanced silencing of TCAST1-associated genes. Differences in the pattern of distribution of TCAST1 elements contribute to gene expression diversity among T. castaneum strains after long-term heat stress and might have an impact on adaptation to different environmental conditions.

  4. Response of body size and developmental time of Tribolium castaneum to constant versus fluctuating thermal conditions.

    Science.gov (United States)

    Małek, D; Drobniak, S; Gozdek, A; Pawlik, K; Kramarz, P

    2015-07-01

    Temperature has profound effects on biological functions at all levels of organization. In ectotherms, body size is usually negatively correlated with ambient temperature during development, a phenomenon known as The Temperature-Size Rule (TSR). However, a growing number of studies have indicated that temperature fluctuations have a large influence on life history traits and the implications of such fluctuations for the TSR are unknown. Our study investigated the effect of different constant and fluctuating temperatures on the body mass and development time of red flour beetles (Tribolium castaneum Herbst, 1797); we also examined whether the sexes differed in their responses to thermal conditions. We exposed the progeny of half-sib families of a T. castaneum laboratory strain to one of four temperature regimes: constant 30°C, constant 25°C, fluctuating with a daily mean of 30°C, or fluctuating with a daily mean of 25°C. Sex-specific development time and body mass at emergence were determined. Beetles developed the fastest and had the greatest body mass upon emergence when they were exposed to a constant temperature of 30°C. This pattern was reversed when beetles experienced a constant temperature of 25°C: slowest development and lowest body mass upon emergence were observed. Fluctuations changed those effects significantly - impact of temperature on development time was smaller, while differences in body mass disappeared completely. Our results do not fit TSR predictions. Furthermore, regardless of the temperature regime, females acquired more mass, while there were no differences between sexes in development time to eclosion. This finding fails to support one of the explanations for smaller male size: that selection favors the early emergence of males. We found no evidence of genotype × environment interactions for selected set of traits.

  5. Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae.

    Science.gov (United States)

    Gorman, Maureen J; Sullivan, Lucinda I; Nguyen, Thi D T; Dai, Huaien; Arakane, Yasuyuki; Dittmer, Neal T; Syed, Lateef U; Li, Jun; Hua, Duy H; Kanost, Michael R

    2012-03-01

    Laccase-2 is a highly conserved multicopper oxidase that functions in insect cuticle pigmentation and tanning. In many species, alternative splicing gives rise to two laccase-2 isoforms. A comparison of laccase-2 sequences from three orders of insects revealed eleven positions at which there are conserved differences between the A and B isoforms. Homology modeling suggested that these eleven residues are not part of the substrate binding pocket. To determine whether the isoforms have different kinetic properties, we compared the activity of laccase-2 isoforms from Tribolium castaneum and Anopheles gambiae. We partially purified the four laccases as recombinant enzymes and analyzed their ability to oxidize a range of laccase substrates. The predicted endogenous substrates tested were dopamine, N-acetyldopamine (NADA), N-β-alanyldopamine (NBAD) and dopa, which were detected in T. castaneum previously and in A. gambiae as part of this study. Two additional diphenols (catechol and hydroquinone) and one non-phenolic substrate (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) were also tested. We observed no major differences in substrate specificity between the A and B isoforms. Dopamine, NADA and NBAD were oxidized with catalytic efficiencies ranging from 51 to 550 min⁻¹ mM⁻¹. These results support the hypothesis that dopamine, NADA and NBAD are endogenous substrates for both isoforms of laccase-2. Catalytic efficiencies associated with dopa oxidation were low, ranging from 8 to 30 min⁻¹ mM⁻¹; in comparison, insect tyrosinase oxidized dopa with a catalytic efficiency of 201 min⁻¹ mM⁻¹. We found that dopa had the highest redox potential of the four endogenous substrates, and this property of dopa may explain its poor oxidation by laccase-2. We conclude that laccase-2 splice isoforms are likely to oxidize the same substrates in vivo, and additional experiments will be required to discover any isoform-specific functions.

  6. Informing tendon tissue engineering with embryonic development

    Science.gov (United States)

    Glass, Zachary A.; Schiele, Nathan R.; Kuo, Catherine K.

    2014-01-01

    Tendon is a strong connective tissue that transduces muscle-generated forces into skeletal motion. In fulfilling this role, tendons are subjected to repeated mechanical loading and high stress, which may result in injury. Tissue engineering with stem cells offers the potential to replace injured/damaged tissue with healthy, new living tissue. Critical to tendon tissue engineering is the induction and guidance of stem cells towards the tendon phenotype. Typical strategies have relied on adult tissue homeostatic and healing factors to influence stem cell differentiation, but have yet to achieve tissue regeneration. A novel paradigm is to use embryonic developmental factors as cues to promote tendon regeneration. Embryonic tendon progenitor cell differentiation in vivo is regulated by a combination of mechanical and chemical factors. We propose that these cues will guide stem cells to recapitulate critical aspects of tenogenesis and effectively direct the cells to differentiate and regenerate new tendon. Here, we review recent efforts to identify mechanical and chemical factors of embryonic tendon development to guide stem/progenitor cell differentiation toward new tendon formation, and discuss the role this work may have in the future of tendon tissue engineering. PMID:24484642

  7. Informing tendon tissue engineering with embryonic development.

    Science.gov (United States)

    Glass, Zachary A; Schiele, Nathan R; Kuo, Catherine K

    2014-06-27

    Tendon is a strong connective tissue that transduces muscle-generated forces into skeletal motion. In fulfilling this role, tendons are subjected to repeated mechanical loading and high stress, which may result in injury. Tissue engineering with stem cells offers the potential to replace injured/damaged tissue with healthy, new living tissue. Critical to tendon tissue engineering is the induction and guidance of stem cells towards the tendon phenotype. Typical strategies have relied on adult tissue homeostatic and healing factors to influence stem cell differentiation, but have yet to achieve tissue regeneration. A novel paradigm is to use embryonic developmental factors as cues to promote tendon regeneration. Embryonic tendon progenitor cell differentiation in vivo is regulated by a combination of mechanical and chemical factors. We propose that these cues will guide stem cells to recapitulate critical aspects of tenogenesis and effectively direct the cells to differentiate and regenerate new tendon. Here, we review recent efforts to identify mechanical and chemical factors of embryonic tendon development to guide stem/progenitor cell differentiation toward new tendon formation, and discuss the role this work may have in the future of tendon tissue engineering.

  8. Induction of embryonic stem cells to hematopoietic cells in vitro

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright's staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34+ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34-CD38+. Wright's staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.

  9. Toxicidade da combinação de dióxido de carbono e fosfina sob diferentes temperaturas para Tribolium castaneum Toxicity of the carbon dioxide and phosphine combination to Tribolium castaneum under different temperatures

    Directory of Open Access Journals (Sweden)

    Raimundo W. S. Aguiar

    2010-01-01

    Full Text Available O objetivo deste trabalho foi avaliar o efeito da temperatura sobre a toxicidade da combinação de dióxido de carbono e fosfina, para os estágios de desenvolvimento de Tribolium castaneum (Herbst (Coleoptera: Tenebrionidae. A toxicidade da combinação de 5% de dióxido de carbono e 1 g m-3 de fosfina para os estágios de ovo, larvas de 5, 10 e 15 dias, pupa e adulto de T. castaneum, foi estudada nas temperaturas de 25, 30, 35, 40 e 45 °C, por meio de estimativas dos tempos de exposição letais para 50 e 95% dos insetos (TL50 e TL95. Curvas tempo-resposta foram estabelecidas mediante bioensaios com períodos crescentes de exposição à combinação do dióxido de carbono com a fosfina. Observou-se que os TL50 e TL95 reduziram com a elevação da temperatura em todos os estágios de T. castaneum avaliados. O estágio de larva de cinco dias foi a mais susceptível à combinação de dióxido de carbono e fosfina. De acordo com os resultados, a combinação do dióxido de carbono com a fosfina é alternativa potencial para diminuir a quantidade de fosfina aplicada em produtos armazenados, por apresentar alta toxicidade para todos os estágios de T. castaneum sob diferentes temperaturas.The objective of this work was to assess the effect of temperature on the toxicity of the carbon dioxide-phosphine combination for the developmental stages of Tribolium castaneum (Herbst (Coleoptera: Tenebrionidae. The toxicity of combination of 5% carbon dioxide and 1 g m-3 phosphine in the developmental stages of egg, larvae of 5, 10 and 15 days, pupae and adult of T. castaneum was studied under the temperatures of 25, 30, 35, 40 and 45 °C, through the estimation of lethal insect exposure times of 50 and 95% (LT50 and LT95. For that, time-response curves were established through bioassays with increasing periods of exposure to the combination of carbon dioxide and phosphine. A reduction of LT50 and LT95 was observed with temperature increase in all

  10. Gamma radiation effects of Cobalt-60 on eggs of Tribolium castaneum (Herbst., 1797) (Coleoptera: tenebrionidae); Efeitos da radiacao gama do cobalto-60 em ovos de Tribolium castaneum (Herbst., 1797) (Coleoptera: Tenebrionidae)

    Energy Technology Data Exchange (ETDEWEB)

    Fontes, L.S.; Arthur, V. [Centro de Energia Nuclear na Agricultura (CENA), Piracicaba, SP (Brazil)

    1994-09-01

    The objective of this research was to verify the effects of gamma radiation of a cobalt-60 source on eggs of the red flour beetle Tribolium castaneum (Herbst., 1797). The used dose rate was 1.28 kGy per hour, and the irradiated insects were kept under controlled environmental conditions: 25 {+-} 2{sup 0} C and 70 {+-} 5% relative humidity. (author). 11 refs., 2 figs., 1 tab.

  11. FUMIGANT EFFICACY OF 1,8-CINEOLE AND EUGENOL ON THE PUPAL STAGE OF TRIBOLIUM CASTANEUM (HERBST (INSECTA: COLEOPTERA: TENEBRIONIDAE

    Directory of Open Access Journals (Sweden)

    Anita Liška

    2015-12-01

    Full Text Available The fumigant efficacy of 1,8-cineole and eugenol compounds was tested on the pupal stage of the red flour beetle Tribolium castaneum (Herbst. Effects of those compounds were determined as mortality as well as impact on development of treated male and female pupae. Also differences in sensitivity to the tested compounds between sexes of T. castaneum pupae were assessed. Compounds were tested at 3 doses rate 0.34, 0.86 and 1.71 ml L-1 vol. at 30±1°C and 70±10% r.h. during 48 h. Compounds toxicity was expressed in two action modes: lethal, and as an impediment to normal metamorphosis of pupae into adult stage, forming "adultoids" and deformed adults (at both sexes. Male pupae were generally more sensitive to both tested compounds. Overall, better efficiency was performed with 1,8-cineole, while eugenol had not accomplished promising effect on the tested pupae.

  12. PDP1 regulates energy metabolism through the IIS-TOR pathway in the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Li, Zhiqian; Jiang, Jianhao; Chen, Yazhou; You, Lang; Huang, Yongping; Tan, Anjiang; Li, Zhiqian; Jiang, Jianhao; Niu, Baolong; Meng, Zhiqi

    2014-03-01

    The PAR-domain protein 1 (PDP1) is essential for locomotor activity of insects. However, its functions in insect growth and development have not been studied extensively, which prompted our hypothesis that PDP1 acts in energy metabolism. Here we report identification of TcPDP1 in the red flour beetle, Tribolium castaneum, and its functional analysis by RNAi. Treating larvae with dsTcPDP1 induced pupae developmental arrestment, accompanied by accelerated fat body degradation. dsTcPDP1 treatments in adults resulted in reduced female fecundity. Disruption of TcPDP1 expression affected the transcription of genes involved in insulin signaling transduction and mechanistic target of rapamycin (mTOR) pathway. These results support our hypothesis that TcPDP1 acts in energy metabolism in T. castaneum.

  13. Evolutionary variations in the expression of dorso-ventral patterning genes and the conservation of pioneer neurons in Tribolium castaneum.

    Science.gov (United States)

    Biffar, Lucia; Stollewerk, Angelika

    2015-04-01

    Insects are ideally suited for gaining insight into the evolutionary developmental mechanisms that have led to adaptive changes of the nervous system since the specific structure of the nervous system can be directly linked to the neural stem cell (neuroblast) lineages, which in turn can be traced back to the last common ancestor of insects. The recent comparative analysis of the Drosophila melanogaster and Tribolium castaneum neuroblast maps revealed substantial differences in the expression profiles of neuroblasts. Here we show that despite the overall conservation of the dorso-ventral expression domains of muscle segment homeobox, intermediate neuroblasts defective and ventral nervous system defective, the expression of these genes relative to the neuroblasts in the respective domains has changed considerably during insect evolution. Furthermore, functional studies show evolutionary changes in the requirement of ventral nervous system defective in the formation of neuroblast 1-1 and the correct differentiation of its presumptive progeny, the pioneer neurons aCC and pCC. The inclusion of the expression data of the dorso-ventral genes into the recently established T. castaneum neuroblast map further increases the differences in the neuroblast expression profiles between D. melanogaster and T. castaneum. Despite these molecular variations, the Even-skipped positive pioneer neurons show an invariant arrangement, except for an additional Even-skipped positive cluster that we discovered in T. castaneum. Given the importance of these pioneer neurons in establishing the intersegmental nerves and the longitudinal tracts, which are part of the conserved axonal scaffold of arthropods, we discuss internal buffering mechanisms that might ensure that neuroblast lineages invariantly generate pioneer neurons over a wide range of molecular variations.

  14. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy...... are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  15. Effect of ozone on respiration of adult Sitophilus oryzae (L.), Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.).

    Science.gov (United States)

    Lu, Baoqian; Ren, Yonglin; Du, Yu-zhou; Fu, Yueguan; Gu, Jie

    2009-10-01

    The effect of ozone on the respiration of three species of adult stored-product Coleoptera was tested in an air-tight flask. Sitophilus oryzae (L.), Rhyzopertha dominica (F.) and Tribolium castaneum (Herbst) adults were exposed to atmosphere containing 0.1, 0.2 or 0.4microg/ml initial ozone at 23-25 degrees C and 50% r.h. Carbon dioxide (CO(2)) production reflected the respiration rates of insects and was determined with a gas chromatograph (GC). The experiments showed that the effects of ozone on respiration had two distinct phases. Phase 1 involved a lower respiration rate of the adult stored-product Coleoptera under ozone atmosphere and reflected the need for insects to reduce ozone toxicity. After 1h, CO(2) production of S. oryzae was 3.19, 2.63, 2.27 and 1.99microl/mg for the ozone concentration of 0, 0.1, 0.2 and 0.4microg/ml, respectively. The results also showed that there were decreases in the rate of respiration in R. dominica and T. castaneum with an increase in ozone concentration. During phase 2, respiration of S. oryzae, R. dominica, and T. castaneum adults treated with ozone increased as the ozone degraded to oxygen. After 7h, the effect of ozone on CO(2) production, relative to the control, changed from a decrease to an increase. The findings in relation to control strategies were discussed.

  16. Investigation of Natural Plant Aegle marmelos Essential Oil Bioactivity on Development and Toxicity of Tribolium castaneum (Coleoptera: Tenebrionidae

    Directory of Open Access Journals (Sweden)

    Bhuwan Bhaskar Mishra

    2016-01-01

    Full Text Available Background: The uncontrolled use of synthetic chemicals is a great hazard for the environment and consumers. Methodology: In the present study, essential oil from leaves of Aegle marmelos was isolated by hydro-distillation and tested their insecticidal activity against Tribolium castaneum (Herbst. Results: The results showed that the essential oil of A. marmelos have fumigant toxicity, oviposition and developmental inhibitory activity against T. castaneum. The percentage mortality increased with increasing exposure time and concentration. The median lethal concentration (LC50 of A. marmelos essential oil at 48 h was 14.172 and 17.752 FL against larvae and adults of T. castaneum, respectively. The essential oil significantly reduced oviposition (F3,20 = 304.734 in adults and also reduced pupation (F3,20 = 137.442 and adult emergence (F3,20 = 225.619 in larvae when fumigated with sub-lethal concentration. The percent grains infection was reduced 83.66% at 60% of sub-lethal concentration of 24 h LC50. Fumigation of insect with sub-lethal concentration of A. marmelos essential oil inhibited AChE activity. Reduction in AChE activity was 81.48 and 54.32% of the control, after 24 h of fumigation with sub-lethal concentration. Conclusion: In conclusion, this essential oil probably induces toxicity in insect by inhibiting AChE activity.

  17. Penetration through the peritrophic matrix is a key to lectin toxicity against Tribolium castaneum.

    Science.gov (United States)

    Walski, Tomasz; Van Damme, Els J M; Smagghe, Guy

    2014-11-01

    In the last decades lectins have received a lot of attention as potential tools in pest control. Despite substantial progress in the field not all the factors determining insecticidal potency and selectivity of these proteins have been described. Recently, three lectins, RSA (Rhizoctonia solani agglutinin), SNA-I and SNA-II (Sambucus nigra agglutinin I and II) have been shown to be toxic to aphids and caterpillars. In this project we investigated if these lectins are also toxic against larvae and a cell line of the red flour beetle, Tribolium castaneum, a model organism and important pest of stored products. Furthermore, we analyzed the stability of the lectins in the larval gut and used confocal microscopy to compare their efficiency in passing through the peritrophic matrix (PM). We observed that all three lectins were toxic against the T. castaneum cell line and their effectiveness in vitro was in decreasing order SNA-II>SNA-I>RSA with the respective EC50 being 0.1, 0.5 and 3.6 μg/ml. Larvae feeding for 16 day on diets containing 2% RSA, 2% SNA-II and 2% SNA-I weighed 0.14 ± 0.07 mg, 0.67 ± 0.44 mg and 1.89 ± 0.38 mg, corresponding to approximately 7%, 36% and 80% of control larvae, respectively. As a consequence, RSA increased the time to adult emergence by over 3-fold, SNA-II by 1.9-fold and SNA-I by 1.2-fold. RSA and SNA-II were stable in the larval gut, while SNA-I was digested and excreted with the feces. Finally, confocal microscopy confirmed that RSA passed through the PM more efficiently than SNA-II. In conclusion, our data suggest that the lectin ability to pass through the PM, governed by molecule dimensions, charge and size of PM pores, is one of the features that determine the toxicity of these insecticidal proteins.

  18. Embryonic death and the creation of human embryonic stem cells

    OpenAIRE

    Landry, Donald W.; Zucker, Howard A.

    2004-01-01

    The creation of human embryonic stem cells through the destruction of a human embryo pits the value of a potential therapeutic tool against that of an early human life. This contest of values has resulted in a polarized debate that neglects areas of common interest and perspective. We suggest that a common ground for pursuing research on human embryonic stem cells can be found by reconsidering the death of the human embryo and by applying to this research the ethical norms of essential organ ...

  19. Embryonic death and the creation of human embryonic stem cells.

    Science.gov (United States)

    Landry, Donald W; Zucker, Howard A

    2004-11-01

    The creation of human embryonic stem cells through the destruction of a human embryo pits the value of a potential therapeutic tool against that of an early human life. This contest of values has resulted in a polarized debate that neglects areas of common interest and perspective. We suggest that a common ground for pursuing research on human embryonic stem cells can be found by reconsidering the death of the human embryo and by applying to this research the ethical norms of essential organ donation.

  20. Mapping the stem cell state: eight novel human embryonic stem and embryonal carcinoma cell antibodies

    DEFF Research Database (Denmark)

    Wright, A; Andrews, N; Bardsley, K

    2011-01-01

    The antigenic profile of human embryonic stem (ES) and embryonal carcinoma (EC) cells has served as a key element of their characterization, with a common panel of surface and intracellular markers now widely used. Such markers have been used to identify cells within the 'undifferentiated state...... of reactivity for all antibodies against both ES and EC cells, suggesting that these markers will afford recognition of unique sub-states within the undifferentiated stem cell compartment....... and EC cells, and herein describe their characterization. The reactivity of these antibodies against a range of cell lines is reported, as well as their developmental regulation, basic biochemistry and reactivity in immunohistochemistry of testicular germ cell tumours. Our data reveal a range...

  1. Efficacies of spinosad and a combination of chlorpyrifos-methyl and deltamethrin against phosphine-resistant Rhyzopertha dominica (Coleoptera: Bostrichidae) and Tribolium castaneum (Coleoptera: Tenebrionidae) on wheat.

    Science.gov (United States)

    Bajracharya, N S; Opit, George P; Talley, J; Jones, C L

    2013-10-01

    Highly phosphine-resistant populations of Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) and Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) have recently been found in Oklahoma grain storage facilities. These findings necessitate development of a phosphine resistance management strategy to ensure continued effective use of phosphine. Therefore, we investigated the efficacies of two grain insecticides, namely, spinosad applied at label rate of 1 ppm and a mixture of chlorpyrifos-methyl and deltamethrin applied at label rates of 3 and 0.5 ppm, respectively, against highly phosphine-resistant R. dominica and T. castaneum. Adult mortality and progeny production suppression of spinosad- or chlorpyrifos-methyl + deltamethrin mixture-treated wheat that had been stored for 2, 84, 168, 252, and 336 d posttreatment were assessed. We found that both spinosad and chlorpyrifos-methyl + deltamethrin were effective against phosphine-resistant R. dominica and caused 83-100% mortality and also caused total progeny production suppression for all storage periods. Spinosad was not effective against phosphine-resistant T. castaneum; the highest mortality observed was only 3% for all the storage periods. Chlorpyrifos-methyl + deltamethrin was effective against phosphine-resistant T. castaneum only in treated wheat stored for 2 and 84 d, where it caused 93-99% mortality. However, chlorpyrifos-methyl + deltamethrin was effective and achieved total suppression of progeny production in T. castaneum for all the storage periods. Spinosad was not as effective as chlorpyrifos-methyl + deltamethrin mixture at suppressing progeny production of phosphine-resistant T. castaneum. These two insecticides can be used in a phosphine resistance management strategy for R. dominica and T. castaneum in the United States.

  2. The Tribolium castaneum cell line TcA: a new tool kit for cell biology.

    Science.gov (United States)

    Silver, Kristopher; Jiang, Hongbo; Fu, Jinping; Phillips, Thomas W; Beeman, Richard W; Park, Yoonseong

    2014-10-30

    The red flour beetle, Tribolium castaneum, is an agriculturally important insect pest that has been widely used as a model organism. Recently, an adherent cell line (BCIRL-TcA-CLG1 or TcA) was developed from late pupae of the red flour beetle. Next generation transcriptome sequencing of TcA cells demonstrated expression of a wide variety of genes associated with specialized functions in chitin metabolism, immune responses and cellular and systemic RNAi pathways. Accordingly, we evaluated the sensitivity of TcA cells to dsRNA to initiate an RNAi response. TcA cells were highly sensitive to minute amounts of dsRNA, with a minimum effective dose of 100 pg/mL resulting in significant suppression of gene expression. We have also developed a plasmid containing two TcA-specific promoters, the promoter from the 40S ribosomal protein subunit (TC006550) and a bi-directional heat shock promoter (TcHS70) from the intergenic space between heat shock proteins 68a and b. These promoters have been employed to provide high levels of either constitutive (TC006550) or inducible (TcHS70) gene expression of the reporter proteins. Our results show that the TcA cell line, with its sensitivity to RNAi and functional TcA-specific promoters, is an invaluable resource for studying basic molecular and physiological questions.

  3. Effect of selection and gamma-irradiation on fecundity in Tribolium castaneum

    Energy Technology Data Exchange (ETDEWEB)

    Bhat, P.P.; Raheja, K.L. (Indian Veterinary Research Inst., Izatnagar, Uttar Pradesh); Bhat, P.N. (Central Inst. for Research on Goats, Mathura (India)); Verma, S.B. (Rajendra Agricultural Univ., Patna (India))

    1981-10-01

    In order to investigate the joint effect of selection and gamma irradiation on a polygenic trait like egg production in Tribolium castaneum, a 3 x 3 x 2 fractorial experiment with 3 levels of selection, viz. O, high (S/sub 1/), and low (S/sub 2/); 3 levels of irradiation, viz. 0,1 1500R (R/sub 1/) 2300R(R/sub 2/); and 2 replicates with 24 females in each cell, was set up. The data were collected on 721 females of parental generation subjected to selection and radiation and the subsequent progeny generation. A significant divergence of 11.6 eggs were observed due to high and low selection methods. There was no difference between control and R/sub 1/ radiation dose. The R/sub 2/ radiation dose depressed the egg production of the progeny significantly. There was a linear decrease in egg number in the progeny with an increase in dose level of radiation in parents; this is indicative of significant number of gene mutations effecting a quantitative trait like egg production. It is suggested that reduction in fecundity in the progeny of parents subjected to gamma-irradiation can possibly arise due to either (i) molecular rearrangement at a point on the chromosome leading to gene mutation or (ii) chromosome breakage with subsequent rearrangement of gene position or both.

  4. Functional value of elytra under various stresses in the red flour beetle, Tribolium castaneum

    Science.gov (United States)

    Linz, David M.; Hu, Alan W.; Sitvarin, Michael I.; Tomoyasu, Yoshinori

    2016-01-01

    Coleoptera (beetles) is a massively successful order of insects, distinguished by their evolutionarily modified forewings called elytra. These structures are often presumed to have been a major driving force for the successful radiation of this taxon, by providing beetles with protection against a variety of harsh environmental factors. However, few studies have directly demonstrated the functional significance of the elytra against diverse environmental challenges. Here, we sought to empirically test the function of the elytra using Tribolium castaneum (the red flour beetle) as a model. We tested four categories of stress on the beetles: physical damage to hindwings, predation, desiccation, and cold shock. We found that, in all categories, the presence of elytra conferred a significant advantage compared to those beetles with their elytra experimentally removed. This work provides compelling quantitative evidence supporting the importance of beetle forewings in tolerating a variety of environmental stresses, and gives insight into how the evolution of elytra have facilitated the remarkable success of beetle radiation. PMID:27708390

  5. Evaluation of synergized pyrethrin aerosol for control of Tribolium castaneum and Tribolium confusum (Coleoptera: Tenebrionidae).

    Science.gov (United States)

    Kharel, Kabita; Arthur, Frank H; Zhu, Kun Yan; Campbell, James F; Subramanyam, Bhadriraju

    2014-02-01

    Aerosol insecticides are being used in flour mill pest management programs, but there is limited information on their efficacy on different insect life stages. In this study, we evaluated the efficacy of synergized pyrethrin applied as an aerosol against eggs, larvae, pupae, and adults of the red flour beetle, Tribolium castaneum (Herbst), and the confused flour beetle, Tribolium confusum Jacquelin du Val. Effects of direct and indirect exposure were evaluated by exposing each life stage to the aerosol and then transferring to untreated flour, transferring untreated insects to treated flour, or exposing both the insects and the flour to the aerosol. The aerosol produced >88% mortality of both species and all life stages when insects were directly treated and transferred to either treated or untreated flour. Mortality was significantly reduced when insects were either treated together with flour or untreated insects were transferred to treated flour (indirect exposure to the aerosol). Larvae and adults of both species were more tolerant compared with eggs and pupae. Recovery of moribund adults in the indirect exposure treatments was greater compared with recovery of moribund insects in the direct exposure treatments. Good sanitation before aerosol application could facilitate direct exposure of insects and thus increase aerosol efficacy inside flour mills.

  6. Are there genetic trade-offs between immune and reproductive investments in Tribolium castaneum?

    Science.gov (United States)

    Hangartner, Sandra; Sbilordo, Sonja H; Michalczyk, Łukasz; Gage, Matthew J G; Martin, Oliver Y

    2013-10-01

    Parasites impose strong selection on hosts to defend themselves, which is expected to result in trade-offs with other fitness traits such as reproduction. Here we test for genetic trade-offs between reproductive traits and immunity using Tribolium castaneum lines that were subject to experimental evolution. The lines have been exposed to contrasting sexual selection intensities via different sex ratios (female-biased, equal and male-biased). After 56 generations, the lines have significantly diverged and those experiencing high sexual selection have evolved males who are superior competitors for reproductive success, and females who are more resistant to multiple mating. All selected lines were assessed for both an immune measurement (phenoloxidase (PO) activity) and host resistance to the microsporidian Nosema whitei after two generations of relaxed selection. In contrast to our expectations we did not find any evidence for a genetic trade-off between investment in reproduction and immunity. Both PO and Nosema resistance did not differ between lines, despite their divergences in reproductive investment due to variation in sexual selection and conflict. Nevertheless, overall we found that females had higher PO activities and in the Nosema free control treatment survived longer than males, suggesting that females generally invest more in PO and survival under control conditions than males. This result fits the Bateman's principle, which states that females gain fitness through increased immunity and longevity, while males gain fitness through increased mating success.

  7. Direct and indirect genetic effects in life-history traits of flour beetles (Tribolium castaneum).

    Science.gov (United States)

    Ellen, Esther D; Peeters, Katrijn; Verhoeven, Merel; Gols, Rieta; Harvey, Jeffrey A; Wade, Michael J; Dicke, Marcel; Bijma, Piter

    2016-01-01

    Indirect genetic effects (IGEs) are the basis of social interactions among conspecifics, and can affect genetic variation of nonsocial and social traits. We used flour beetles (Tribolium castaneum) of two phenotypically distinguishable populations to estimate genetic (co)variances and the effect of IGEs on three life-history traits: development time (DT), growth rate (GR), and pupal body mass (BM). We found that GR was strongly affected by social environment with IGEs accounting for 18% of the heritable variation. We also discovered a sex-specific social effect: male ratio in a group significantly affected both GR and BM; that is, beetles grew larger and faster in male-biased social environments. Such sex-specific IGEs have not previously been demonstrated in a nonsocial insect. Our results show that beetles that achieve a higher BM do so via a slower GR in response to social environment. Existing models of evolution in age-structured or stage-structured populations do not account for IGEs of social cohorts. It is likely that such IGEs have played a key role in the evolution of developmental plasticity shown by Tenebrionid larvae in response to density. Our results document an important source of genetic variation for GR, often overlooked in life-history theory.

  8. Constraints on the evolution of function-valued traits: a study of growth in Tribolium castaneum.

    Science.gov (United States)

    Irwin, K K; Carter, P A

    2013-12-01

    Growth trajectories often impact individual fitness. They are continuous by nature and so are amenable to analysis using a function-valued (FV) trait framework to reveal their underlying genetic architecture. Previous studies have found high levels of standing additive genetic (co)variance for growth trajectories despite the expectation that growth should be responding to frequent strong directional selection. In this study, the FV framework is used to estimate the additive genetic covariance function for growth trajectories in larval Tribolium castaneum to address questions about standing additive genetic (co)variance and possible evolutionary constraints on growth and to predict responses to four plausible selection regimes. Results show that additive genetic (co)variance is high at the early ages, but decreases towards later ages in the larval period. A selection gradient function of the same size and in the same direction of the first eigenfunction of the G-function should give the maximal response. However, evolutionary constraints may be acting to keep this maximal response from being realized, through either conflicting effects on survivability and fecundity of larger body size, few evolutionary directions having sufficient additive variance for a response, genetic trade-offs with other traits or physiological regulatory mechanisms. More light may be shed on these constraints through the development of more sophisticated statistical approaches and implementation of additional empirical studies to explicitly test for specific types of constraints.

  9. Gender- and stressor-specific microRNA expression in Tribolium castaneum.

    Science.gov (United States)

    Freitak, Dalial; Knorr, Eileen; Vogel, Heiko; Vilcinskas, Andreas

    2012-10-23

    MicroRNAs (miRNAs) are small non-coding RNAs mediating post-transcriptional regulation of gene expression in eukaryotes. Addressing their role in regulation of physiological adaptations to environmental stress in insects, we selected the red flour beetle Tribolium castaneum as a model. Beetles were fed with the bacterial entomopathogen Pseudomonas entomophila (to mimic natural infection), injected with peptidoglycan (experimental setting of strong immune responses) or subjected to either mild heat shock or starvation. Differential expression of selected immunity- and stress-related genes was quantified using real-time PCR, and expression and induction of 455 mature arthropod miRNAs were determined using proprietary microarrays. We found that Tribolium exhibits both gender- and stressor-specific adjustment of immune gene and miRNA expression. Strikingly, we discovered that the number of stressor-induced miRNAs in females is remarkably higher than in males. This observation could support the hypothesis called Bateman's principle in immunity that predicts gender-specific immune responses because females gain fitness through increased longevity, whereas males gain fitness by increasing mating rates. Our results suggest that Tribolium males and females display differential regulatory elements, both pre- and post-transcriptional, likely resulting from different investment strategies in life-history traits.

  10. Insect gravitational biology: ground-based and shuttle flight experiments using the beetle Tribolium castaneum

    Science.gov (United States)

    Bennett, R. L.; Abbott, M. K.; Denell, R. E.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Many of the traditional experimental advantages of insects recommend their use in studies of gravitational and space biology. The fruit fly, Drosophila melanogaster, is an obvious choice for studies of the developmental significance of gravity vectors because of the unparalleled description of regulatory mechanisms controlling oogenesis and embryogenesis. However, we demonstrate that Drosophila could not survive the conditions mandated for particular flight opportunities on the Space Shuttle. With the exception of Drosophila, the red flour beetle, Tribolium castaneum, is the insect best characterized with respect to molecular embryology and most frequently utilized for past space flights. We show that Tribolium is dramatically more resistant to confinement in small sealed volumes. In preparation for flight experiments we characterize the course and timing of the onset of oogenesis in newly eclosed adult females. Finally, we present results from two shuttle flights which indicate that a number of aspects of the development and function of the female reproductive system are not demonstrably sensitive to microgravity. Available information supports the utility of this insect for future studies of gravitational biology.

  11. Copulatory courtship and cryptic female choice in red flour beetles Tribolium castaneum.

    Science.gov (United States)

    Edvardsson, M; Arnqvist, G

    2000-03-22

    Males of many animal species engage in courtship behaviours during and after copulation that appear to be solely aimed at stimulating the female. It has been suggested that these behaviours have evolved by cryptic female choice, whereby females are thought to impose biases on male postmating paternity success. Males of the red flour beetle Tribolium castaneum rub the lateral edges of the females' elytra with their tarsi during copulation. We manipulated female perception of this behaviour by tarsal ablation in males, thus preventing males from reaching the edge of the female elytra with their manipulated legs, and by subsequently performing a series of double-mating experiments where the copulatory behaviour was quantified. We found a positive relationship between the intensity of the copulatory courtship behaviour and relative fertilization success among unmanipulated males. This pattern, however, was absent in manipulated males, where female perception of male behaviour differed from that actually performed. Thus, female perception of male copulatory courtship behaviour, rather than male behaviour per se, apparently governs the fate of sperm competing over fertilizations within the female, showing that copulatory courtship is under selection by cryptic female choice.

  12. Hormonal enhancement of insecticide efficacy in Tribolium castaneum: oxidative stress and metabolic aspects.

    Science.gov (United States)

    Plavšin, Ivana; Stašková, Tereza; Šerý, Michal; Smýkal, Vlastimil; Hackenberger, Branimir K; Kodrík, Dalibor

    2015-04-01

    Insect anti-stress responses, including those induced by insecticides, are controlled by adipokinetic hormones (AKHs). We examined the physiological consequences of Pyrap-AKH application on Tribolium castaneum adults (AKH-normal and AKH-deficient prepared by the RNAi technique) treated by two insecticides, pirimiphos-methyl and deltamethrin. Co-application of pirimiphos-methyl and/or deltamethrin with AKH significantly increased beetle mortality compared with application of the insecticides alone. This co-treatment was accompanied by substantial stimulation of general metabolism, as monitored by carbon dioxide production. Further, the insecticide treatment alone affected some basic markers of oxidative stress: it lowered total antioxidative capacity as well as the activity of superoxide dismutase in the beetle body; in addition, it enhanced the activity of catalase and glutathione-S-transferase. However, these discrepancies in oxidative stress markers were eliminated/reduced by co-application with Pyrap-AKH. We suggest that the elevation of metabolism, which is probably accompanied with faster turnover of toxins, might be responsible for the higher mortality that results after AKH and insecticide co-application. Changes in oxidative stress markers are probably not included in the mechanisms responsible for increased mortality.

  13. Satellite DNA as a driver of population divergence in the red flour beetle Tribolium castaneum.

    Science.gov (United States)

    Feliciello, Isidoro; Akrap, Ivana; Brajković, Josip; Zlatar, Ivo; Ugarković, Đurđica

    2014-12-19

    Tandemly repeated satellite DNAs are among most rapidly evolving sequences in eukaryotic genome, usually differing significantly among closely related species. By inducing changes in heterochromatin and/or centromere, satellite DNAs are expected to drive population and species divergence. However, despite high evolutionary dynamics, divergence of satellite DNA profiles at the level of natural population which precedes and possibly triggers speciation process is not readily detected. Here, we characterize minor TCAST2 satellite DNA of the red flour beetle Tribolium castaneum and follow its dynamics among wild-type strains originating from diverse geographic locations. The investigation revealed presence of three distinct subfamilies of TCAST2 satellite DNA which differ in monomer size, genome organization, and subfamily specific mutations. Subfamilies Tcast2a and Tcast2b are tandemly arranged within pericentromeric heterochromatin whereas Tcast2c is preferentially dispersed within euchromatin of all chromosomes. Among strains, TCAST2 subfamilies are conserved in sequence but exhibit a significant content variability. This results in overrepresentation or almost complete absence of particular subfamily in some strains and enables discrimination between strains. It is proposed that homologous recombination, probably stimulated by environmental stress, is responsible for the emergence of TCAST2 satellite subfamilies, their copy number variation and dispersion within genome. The results represent the first evidence for the existence of population-specific satellite DNA profiles. Partial organization of TCAST2 satellite DNA in the form of single repeats dispersed within euchromatin additionally contributes to the genome divergence at the population level.

  14. The lethal giant larvae Gene in Tribolium castaneum: Molecular Properties and Roles in Larval and Pupal Development as Revealed by RNA Interference

    Directory of Open Access Journals (Sweden)

    Da Xiao

    2014-04-01

    Full Text Available We identified and characterized the TcLgl gene putatively encoding lethal giant larvae (Lgl protein from the red flour beetle (Tribolium castaneum. Analyses of developmental stage and tissue-specific expression patterns revealed that TcLgl was constitutively expressed. To examine the role of TcLgl in insect development, RNA interference was performed in early (1-day larvae, late (20-day larvae, and early (1-day pupae. The early larvae injected with double-stranded RNA of TcLgl (dsTcLgl at 100, 200, and 400 ng/larva failed to pupate, and 100% mortality was achieved within 20 days after the injection or before the pupation. The late larvae injected with dsTcLgl at these doses reduced the pupation rates to only 50.3%, 36.0%, and 18.2%, respectively. The un-pupated larvae gradually died after one week, and visually unaffected pupae failed to emerge into adults and died during the pupal stage. Similarly, when early pupae were injected with dsTcLgl at these doses, the normal eclosion rates were reduced to only 22.5%, 18.0%, and 11.2%, respectively, on day 7 after the injection, and all the adults with abnormal eclosion died in two days after the eclosion. These results indicate that TcLgl plays an essential role in insect development, especially during their metamorphosis.

  15. Childhood Central Nervous System Embryonal Tumors Treatment

    Science.gov (United States)

    ... Cord Tumors Treatment Childhood Astrocytomas Treatment Childhood Brain Stem Glioma ... Central nervous system (CNS) embryonal tumors may begin in embryonic (fetal) cells that remain in the brain after birth. ...

  16. Development of neural precursor cells from mouse embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    WU Xuan; LI Hai-di; Li Shu-nong; XU Hai-wei; XU Ling

    2001-01-01

    Objective: To explore the serum-free culture conditions for differentiating mouse embryonic stem cells (ES cells)into neural precursor cells (NPC) and compare the effects of human embryonic fibroblasts (HEF) as the feeder layer of ES with that of mouse embryonic fibroblasts (MEF)in vitro. Methods: Mouse ES cells were cultured in or not in feeder layer cells medium containing or not leukemia inhibitory factor to suppress their differentiation. Immunocytochemical method was used to identify NPC by detecting nestin antigen and alkaline phosphatase. Results: The ES cells cultured in HEF were positive to alkaline phosphatase. Serum-free medium allowed the differentiation of ES cells into NPC. Conclusion:HEF could replace MEF and keep the undifferentiated condition of ES cells with more benefits. NPC of high purity could be cultured from ES cells by serum-free culture method.

  17. Embryonal rhabdomyosarcoma of the cervix

    Directory of Open Access Journals (Sweden)

    Ocheke A

    2011-01-01

    Full Text Available Embryonal rhabdomyosarcoma (sarcoma botyroides of the cervix, which is rare, is described in a 16-year-old. 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 and loss to follow-up.

  18. Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum.

    Science.gov (United States)

    Gilles, Anna F; Schinko, Johannes B; Averof, Michalis

    2015-08-15

    Gene-editing techniques are revolutionizing the way we conduct genetics in many organisms. The CRISPR/Cas nuclease has emerged as a highly versatile, efficient and affordable tool for targeting chosen sites in the genome. Beyond its applications in established model organisms, CRISPR technology provides a platform for genetic intervention in a wide range of species, limited only by our ability to deliver it to cells and to select mutations efficiently. Here, we test the CRISPR technology in an emerging insect model and pest, the beetle Tribolium castaneum. We use simple assays to test CRISPR/Cas activity, we demonstrate efficient expression of guide RNAs and Cas9 from Tribolium U6 and hsp68 promoters and we test the efficiency of knockout and knock-in approaches in Tribolium. We find that 55-80% of injected individuals carry mutations (indels) generated by non-homologous end joining, including mosaic bi-allelic knockouts; 71-100% carry such mutations in their germ line and transmit them to the next generation. We show that CRISPR-mediated gene knockout of the Tribolium E-cadherin gene causes defects in dorsal closure, which is consistent with RNAi-induced phenotypes. Homology-directed knock-in of marker transgenes was observed in 14% of injected individuals and transmitted to the next generation by 6% of injected individuals. Previous work in Tribolium mapped a large number of transgene insertions associated with developmental phenotypes and enhancer traps. We present an efficient method for re-purposing these insertions, via CRISPR-mediated replacement of these transgenes by new constructs.

  19. Anatomical and morphological spine variation in Gymnocalycium kieslingii subsp. castaneum (Cactaceae)

    Science.gov (United States)

    Gebauer, Roman; Řepka, Radomír; Šmudla, Radek; Mamoňová, Miroslava; Ďurkovič, Jaroslav

    2016-01-01

    Abstract Although spine variation within cacti species or populations is assumed to be large, the minimum sample size of different spine anatomical and morphological traits required for species description is less studied. There are studies where only 2 spines were used for taxonomical comparison amnog species. Therefore, the spine structure variation within areoles and individuals of one population of Gymnocalycium kieslingii subsp. castaneum (Ferrari) Slaba was analyzed. Fifteen plants were selected and from each plant one areole from the basal, middle and upper part of the plant body was sampled. A scanning electron microscopy was used for spine surface description and a light microscopy for measurements of spine width, thickness, cross-section area, fiber diameter and fiber cell wall thickness. The spine surface was more visible and damaged less in the upper part of the plant body than in the basal part. Large spine and fiber differences were found between upper and lower parts of the plant body, but also within single areoles. In general, the examined traits in the upper part had by 8–17% higher values than in the lower parts. The variation of spine and fiber traits within areoles was lower than the differences between individuals. The minimum sample size was largely influenced by the studied spine and fiber traits, ranging from 1 to 70 spines. The results provide pioneer information useful in spine sample collection in the field for taxonomical, biomechanical and structural studies. Nevertheless, similar studies should be carried out for other cacti species to make generalizations. The large spine and fiber variation within areoles observed in our study indicates a very complex spine morphogenesis. PMID:27698579

  20. Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae.

    Science.gov (United States)

    Shirley, Derek; Oppert, Cris; Reynolds, Todd B; Miracle, Bethany; Oppert, Brenda; Klingeman, William E; Jurat-Fuentes, Juan Luis

    2014-10-01

    Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full-length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde-3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β-1,4-endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments.

  1. TrOn: an anatomical ontology for the beetle Tribolium castaneum.

    Science.gov (United States)

    Dönitz, Jürgen; Grossmann, Daniela; Schild, Inga; Schmitt-Engel, Christian; Bradler, Sven; Prpic, Nikola-Michael; Bucher, Gregor

    2013-01-01

    In a morphological ontology the expert's knowledge is represented in terms, which describe morphological structures and how these structures relate to each other. With the assistance of ontologies this expert knowledge is made processable by machines, through a formal and standardized representation of terms and their relations to each other. The red flour beetle Tribolium castaneum, a representative of the most species rich animal taxon on earth (the Coleoptera), is an emerging model organism for development, evolution, physiology, and pest control. In order to foster Tribolium research, we have initiated the Tribolium Ontology (TrOn), which describes the morphology of the red flour beetle. The content of this ontology comprises so far most external morphological structures as well as some internal ones. All modeled structures are consistently annotated for the developmental stages larva, pupa and adult. In TrOn all terms are grouped into three categories: Generic terms represent morphological structures, which are independent of a developmental stage. In contrast, downstream of such terms are concrete terms which stand for a dissectible structure of a beetle at a specific life stage. Finally, there are mixed terms describing structures that are only found at one developmental stage. These terms combine the characteristics of generic and concrete terms with features of both. These annotation principles take into account the changing morphology of the beetle during development and provide generic terms to be used in applications or for cross linking with other ontologies and data resources. We use the ontology for implementing an intuitive search function at the electronic iBeetle-Base, which stores morphological defects found in a genome wide RNA interference (RNAi) screen. The ontology is available for download at http://ibeetle-base.uni-goettingen.de.

  2. Signalling properties and pharmacology of a 5-HT7 -type serotonin receptor from Tribolium castaneum.

    Science.gov (United States)

    Vleugels, R; Lenaerts, C; Vanden Broeck, J; Verlinden, H

    2014-04-01

    In the last decade, genome sequence data and gene structure information on invertebrate receptors has been greatly expanded by large sequencing projects and cloning studies. This information is of great value for the identification of receptors; however, functional and pharmacological data are necessary for an accurate receptor classification and for practical applications. In insects, an important group of neurotransmitter and neurohormone receptors, for which ample sequence information is available but pharmacological information is missing, are the biogenic amine G protein-coupled receptors (GPCRs). In the present study, we investigated the sequence information, pharmacology and signalling properties of a 5-HT7 -type serotonin receptor from the red flour beetle, Tribolium castaneum (Trica5-HT7 ). The receptor encoding cDNA shows considerable sequence similarity with cognate 5-HT7 receptors and phylogenetic analysis also clusters the receptor within this 5-HT receptor group. Real-time reverse transcription PCR demonstrated high expression levels in the brain, indicating the possible importance of this receptor in neural processes. Trica5-HT7 was dose-dependently activated by 5-HT, which induced elevated intracellular cyclic AMP levels but had no effect on calcium signalling. The synthetic agonists, α-methyl 5-HT, 5-methoxytryptamine, 5-carboxamidotryptamine and 8-hydroxy-2-(dipropylamino)tetralin hydrobromide, showed a response, although with a much lower potency and efficacy than 5-HT. Ketanserin and methiothepin were the most potent antagonists. Both showed characteristics of competitive inhibition on Trica5-HT7 . The signalling pathway and pharmacological profile offer important information that will facilitate functional and comparative studies of 5-HT receptors in insects and other invertebrates. The pharmacology of invertebrate 5-HT receptors differs considerably from that of vertebrates. The present study may therefore contribute to establishing a more

  3. Efficacy of Spinosad and Abamectin against Different Populations of Red Flour Beetle (Tribolium castaneum Herbst in Treated Wheat Grain

    Directory of Open Access Journals (Sweden)

    Goran Andrić

    2013-01-01

    Full Text Available The efficacy of spinosad and abamectin against T. castaneum adults from a laboratorypopulation with normal susceptibility to contact insecticides and against malathion-resistantpopulations from Nikinci and Jakovo was tested in the laboratory (25±1ºC and 60±5%r.h.. The insecticides were applied to 500 g of untreated wheat grain for each of the followingapplication doses: 0.25, 0.5, 1.0, 2.5 and 5.0 mg a.i./kg. After treatment, wheat was dividedinto three equal subsamples and 50 T. castaneum adults from each of the three test populationswere released the next day into jars for each dose. Mortality was evaluated after 7,14 and 21 days of exposure to treated wheat grain.Generally, higher concentrations and longer exposure periods resulted in higher efficacyof both insecticides, but abamectin was significantly more effective than spinosadagainst all three tested populations. After 7 days of exposure, mortality did not exceed30% in any test variant. Fourteen days after treatment with the highest dose (5 mg/kgof spinosad, mortality was highest (75% in the laboratory population, while treatmentwith the same dose of abamectin achieved the highest mortality (58% in the laboratoryand Jakovo populations. After 21 days, spinosad applied at the rate of 5 mg/kg was mosteffective (97% mortality in the laboratory population, while 88% efficacy was recorded inJakovo population and 87% in Nikinci population. Abamectin doses of 2.5 and 5 mg/kgcaused high adult mortality of 94-100% in the laboratory and Jakovo populations, and a significantlylower mortality in Nikinci population (75 and 86%, respectively.Statistically significant differences in the efficacy of spinosad, and particularly ofabamectin, were detected among the three tested populations, the greatest differencebeing between the laboratory and Nikinci populations, which clearly indicates that resistanceof T. castaneum adults to malathion had a significant influence.

  4. Comparative characterization of two intracellular Ca²⁺-release channels from the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Liu, Yaping; Li, Chengjun; Gao, Jingkun; Wang, Wenlong; Huang, Li; Guo, Xuezhu; Li, Bin; Wang, Jianjun

    2014-10-21

    Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) are members of a family of tetrameric intracellular Ca(2+)-release channels (CRCs). While it is well known in mammals that RyRs and IP3Rs modulate multiple physiological processes, the roles of these two CRCs in the development and physiology of insects remain poorly understood. In this study, we cloned and functionally characterized RyR and IP3R cDNAs (named TcRyR and TcIP3R) from the red flour beetle, Tribolium castaneum. The composite TcRyR gene contains an ORF of 15,285 bp encoding a protein of 5,094 amino acid residues. The TcIP3R contains an 8,175 bp ORF encoding a protein of 2,724 amino acids. Expression analysis of TcRyR and TcIP3R revealed significant differences in mRNA expression levels among T. castaneum during different developmental stages. When the transcript levels of TcRyR were suppressed by RNA interference (RNAi), an abnormal folding of the adult hind wings was observed, while the RNAi-mediated knockdown of TcIP3R resulted in defective larval-pupal and pupal-adult metamorphosis. These results suggested that TcRyR is required for muscle excitation-contraction (E-C) coupling in T. castaneum, and that calcium release via IP3R might play an important role in regulating ecdysone synthesis and release during molting and metamorphosis in insects.

  5. Comparative toxicity and micronuclei formation in Tribolium castaneum, Callosobruchus maculatus and Sitophilus oryzae exposed to high doses of gamma radiation.

    Science.gov (United States)

    Ahmadi, Mehrdad; Mozdarani, Hossein; Abd-Alla, Adly M M

    2015-07-01

    The effects of gamma radiation on mortality and micronucleus formation in Tribolium castaneum Herbst, Callosobruchus maculatus (F.) and Sitophilus oryzae (L.) genital cells were evaluated. Two groups of healthy and active adult insects 1-3 and 8-10 days old were irradiated with various doses (50-200 Gy) gamma ray. Seven days post-irradiation; mortality rates and micronucleus formation were assessed in genital cells of the irradiated insects. The results show that with increasing gamma doses, the mortality rate of each species increased and T. castaneum and S. oryzae showed the low and high sensitivity respectively. It was shown that the micronucleus appearance in the tested insects had correlation with amount and intensity of radiation doses. Moreover our results indicate different levels in the genotoxicity of gamma radiation among the insects' genital cells under study. The frequency of micronuclei in genital cells of 1-3 days old insects exposed to 50 and 200 Gy were 12.6 and 38.8 Mn/1000 cells in T. castaneum, 20.8 and 46.8 Mn/1000 cells in C. maculatus and 16.8 and 57.2 Mn/1000 cells in S. oryzae respectively. A high sensitivity of the genital cells to irradiation exposure was seen in S. oryzae correlated with its high mortality rate compared with the other two species. These results might be indicative of inflicting chromosomal damage expressed as micronucleus in high mortality rates observed in the pest population; an indication of genotoxic effects of radiation on the studied species.

  6. Determination of mortality of different life stages of Tribolium castaneum (Coleoptera: Tenebrionidae) in stored barley using microwaves.

    Science.gov (United States)

    Vadivambal, R; Jayas, D S; White, N D G

    2008-06-01

    Barley, Hordeum vulgare L., one of the important crops in Canada, is used in malting, feed, and food industries. Disinfestation of barley using microwaves can be an alternative to chemical methods used to kill insects. A pilot-scale industrial microwave system operating at 2.45 MHz was used in this study to determine the mortality of life stages (egg, larva, pupa, and adult) of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Barley samples of 50 g each at 14, 16, and 18% moisture content (MC; wet basis) were infested with various life stages of T. castaneum and exposed to microwave energy at different power levels and exposure times, and the mortality of the insects was determined. The average temperature of the 14% MC sample exposed to 28 s at 0, 200, 300, 400, and 500 W were 27.4, 42.6, 53.7, 66.9, and 73.0 degrees C, respectively, and those exposed to 56 s at 0, 200, 300, and 400 W were 27.4, 57.3, 75.5, and 91.2 degrees C, respectively. A similar range of temperature was observed for 16 and 18% MC barley. Complete mortality of all life stages of T. castaneum can be achieved at a power level of 400 W and an exposure time of 56 s or at 500 W for 28 s. Among the life stages of T. castaneum, eggs were the most susceptible to microwave energy and adults were the least susceptible. There was no significant difference in the mortality of larvae and adults at 14, 16, and 18% MC, but the mortality was significantly different for eggs and pupae at different MCs. There was a significant increase in the mortality with an increase in power level or exposure time or both. Germination capacity of the seeds was decreased with an increase in power level or exposure time or both. Quality characteristics such as alpha-amylase, diastatic power, soluble protein, viscosity, and density of the barley malt treated at 500 W for 28 s were same as the control sample, whereas the samples treated at 400 W for 56 s were significantly lower.

  7. Chemical composition of four essential oils from Eupatorium spp: Biological activities toward Tribolium castaneum (Coleoptera: Tenebrionidae Composición química de cuatro aceites esenciales provenientes de Eupatorium spp. y su toxicidad para Tribolium castaneum (Coleoptera: Tenebrionidae

    Directory of Open Access Journals (Sweden)

    Hugo G. Lancelle

    2009-12-01

    Full Text Available Toxic and repellent properties of whole essential oils from four Eupatorium (Asteraceae species (E. buniifolium Hook. et Arn, E. inulaefolium Kunth, E. arnottii Baker, and E. viscidum Hook. & Arn were investigated in different concentrations toward Tribolium castaneum Herbst adults. The essential oils were isolated by hydrodistillation techniques from the aerial parts. The analysis was performed by GC-FID and GC-MS methods. Contact toxicity assays showed that all the evaluated essential oils were toxic. Furthermore, in all the cases mortality was dose dependent. The main repellency was observed for the essential oil recovered from E. buniifolium.Se evaluaron las propiedades tóxicas y repelentes de los aceites esenciales de cuatro especies del género Eupatorium (Asteraceae: E. buniifolium Hook. et Arn, E. inulaefolium Kunth, E. arnottii Baker y E. viscidum Hook. & Arn, en diferentes concentraciones frente a adultos de Tribolium castaneum Herbst. Los aceites esenciales se aislaron de las partes aéreas de las plantas, mediante técnicas de hidrodestilación y se analizaron por los métodos GC-FID y GC-MS. Los ensayos de toxicidad por contacto demostraron que todos los aceites fueron tóxicos y la mortalidad fue, en todos los casos, dependiente de la dosis. El aceite esencial de E. buniifolium presentó la mayor actividad repelente.

  8. The birth of embryonic pluripotency

    OpenAIRE

    Boroviak, Thorsten; Nichols, Jennifer

    2014-01-01

    This is the final published version. It first appeared at http://rstb.royalsocietypublishing.org/content/369/1657/20130541. Formation of a eutherian mammal requires concurrent establishment of embryonic and extraembryonic lineages. The functions of the trophectoderm and primitive endoderm are to enable implantation in the maternal uterus, axis specification and delivery of nutrients. The pluripotent epiblast represents the founding cell population of the embryo proper, which is...

  9. 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 also...... be induced rapidly to differentiate. Maintaining this balance of stability versus plasticity is a challenge, and extensive studies in recent years have focused on understanding the contributions of transcription factors and epigenetic enzymes to the "stemness" properties of these cells. Identifying...... the molecular switches that regulate ES cell self-renewal versus differentiation can provide insights into the nature of the pluripotent state and enhance the potential use of these cells in therapeutic applications. Here, we review the latest models for how changes in chromatin methylation can modulate ES cell...

  10. Lethal dose determination of Cobalt-60 for adult Tribolium castaneum (Coleoptera: Tenebrionidae) and Cryptolestes ferrugineus (Coleoptera: Laemophloeidae)

    Energy Technology Data Exchange (ETDEWEB)

    Farias, Anderson Aparecido, E-mail: potenza@biologico.sp.gov.br [Instituto Biologico, Centro de Pesquisa e Desenvolvimento de Protecao Ambiental, Sao Paulo, SP (Brazil); Potenza, Marcos Roberto, E-mail: fcreis@usp.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Reis, Fabricio Caldeira; Sato, Mario Eidi, E-mail: mesato@biologico.sp.gov.br [Instituto Biologico, Centro Experimental Central, Laboratorio de Acarologia, Campinas, SP (Brazil)

    2015-07-01

    The insect infestation is a major problem in the grain storage. The pesticides are most widely used method for disinfestation and prevention. Treatment with gamma irradiation may increase the product shelf life without encountering formation of waste can be used in packaged foods and ready for commercialization, representing an important alternative to the use of pesticides. This study aimed to determine the immediate lethal dose of gamma radiation for adults of Tribolium castaneum and Cryptolestes ferrugineus. The study was conducted in the Instituto Biologico and the radiations held at the Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN, Sao Paulo city, using a multipurpose irradiator Cobalt-60 with 3.31 dose rate and 3.23 kGy in months of November/2014 and January/2015. Each experimental unit consisted of 20 adult insects, confined in a 10 mL polyethylene container. The experimental plots in number 10 per dose were subjected to increasing doses of gamma radiation: 0; 0.5; 0.75; 1.0; 1.25; 1.50; 1.75; 2.00; 2:50 and 3.00 kGy. Mortality was assessed within two to four hours after irradiation. The data were submitted to Probit analysis, using the POLO PLUS program. The LD90 to control 90% (LD90) to control adult Cryptolestes ferrugineus and Tribolium castaneum were 2.73 and 2.91 kGy, respectively. (author)

  11. Evaluation of Reference Genes for RT-qPCR in Tribolium castaneum (Coleoptera: Tenebrionidae) Under UVB Stress.

    Science.gov (United States)

    Sang, Wen; He, Li; Wang, Xiao-Ping; Zhu-Salzman, Keyan; Lei, Chao-Liang

    2015-04-01

    Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) has become a widely used technique to quantify gene expression. It is necessary to select appropriate reference genes for normalization. In the present study, we assessed the expression stability of seven candidate genes in Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) irradiated by ultraviolet B (UVB) at different developmental stages for various irradiation time periods. The algorithms of geNorm, NormFinder, and BestKeeper were applied to determine the stability of these candidate genes. Ribosomal protein genes RpS3, RpL13A, and β-actin gene (ActB) showed the highest stability across all UVB irradiation time points, whereas expression of other normally used reference genes, such as those encoding the β-tubulin gene TUBB and the E-cadherin gene CAD, varied at different developmental stages. This study will potentially provide more suitable reference gene candidates for RT-qPCR analysis in T. castaneum subjected to environmental stresses, particularly UV irradiation.

  12. Proteome response of Tribolium castaneum larvae to Bacillus thuringiensis toxin producing strains.

    Directory of Open Access Journals (Sweden)

    Estefanía Contreras

    Full Text Available Susceptibility of Tribolium castaneum (Tc larvae was determined against spore-crystal mixtures of five coleopteran specific and one lepidopteran specific Bacillus thuringiensis Cry toxin producing strains and those containing the structurally unrelated Cry3Ba and Cry23Aa/Cry37Aa proteins were found toxic (LC(50 values 13.53 and 6.30 µg spore-crystal mixture/µL flour disc, respectively. Using iTRAQ combined with LC-MS/MS allowed the discovery of seven novel differentially expressed proteins in early response of Tc larvae to the two active spore-crystal mixtures. Proteins showing a statistically significant change in treated larvae compared to non-intoxicated larvae fell into two major categories; up-regulated proteins were involved in host defense (odorant binding protein C12, apolipophorin-III and chemosensory protein 18 and down-regulated proteins were linked to metabolic pathways affecting larval metabolism and development (pyruvate dehydrogenase Eα subunit, cuticular protein, ribosomal protein L13a and apolipoprotein LI-II. Among increased proteins, Odorant binding protein C12 showed the highest change, 4-fold increase in both toxin treatments. The protein displayed amino acid sequence and structural homology to Tenebrio molitor 12 kDa hemolymph protein b precursor, a non-olfactory odorant binding protein. Analysis of mRNA expression and mortality assays in Odorant binding protein C12 silenced larvae were consistent with a general immune defense function of non-olfactory odorant binding proteins. Regarding down-regulated proteins, at the transcriptional level, pyruvate dehydrogenase and cuticular genes were decreased in Tc larvae exposed to the Cry3Ba producing strain compared to the Cry23Aa/Cry37Aa producing strain, which may contribute to the developmental arrest that we observed with larvae fed the Cry3Ba producing strain. Results demonstrated a distinct host transcriptional regulation depending upon the Cry toxin treatment. Knowledge

  13. Evaluation of structural treatment efficacy against Tribolium castaneum and Tribolium confusum (Coleoptera: Tenebrionidae) using meta-analysis of multiple studies conducted in food facilities

    Science.gov (United States)

    The phase out of methyl bromide for the treatment of structures where grain is milled and processed has triggered a need to evaluate the effectiveness of alternative structural treatments such as sulfuryl fluoride and heat. The red flour beetle (Tribolium castaneum (Herbst)) (RFB) and confused flour...

  14. Impact of varying levels of sanitation on mortality of Tribolium castaneum eggs and adults during heat treatment of a pilot flour mill

    Science.gov (United States)

    The influence of sanitation on responses of life stages of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), an economically important pest in flour mills, was investigated in a pilot flour mill subjected to two, 24-h heat treatments. One hundred eggs or 100 adults of T...

  15. Evaluation of Standard Loose Plastic Packaging for the Management of Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) and Tribolium castaneum (Herbst) (Coleoptera: Tenebriondiae).

    Science.gov (United States)

    Hassan, Muhammad Waqar; Gulraize; Ali, Usman; Ur Rehman, Fazal; Najeeb, Hafsa; Sohail, Maryam; Irsa, Bakhtawar; Muzaffar, Zubaria; Chaudhry, Muhammad Shafiq

    2016-01-01

    Three standard foodstuff plastic packaging namely polyethylene (PE), polypropylene (PP), and polyvinylchloride (PVC) were evaluated for management of lesser grain borer Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) and red flour beetle Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Resistance parameters in packaging were recorded as punctures, holes, penetrations, sealing defects, and invasions with two thicknesses and tested for two lengths of time. Damages like punctures, holes and penetrations by both insects were more in PE packaging however R. dominica made more penetrations in PP than in PE. For both insects sealing defects and invasions were predominant in PVC than in others. Thickness did not affect significantly damage types but significantly more holes and penetrations by R. dominica were in less thickness. Punctures and holes by R. dominica were more after less time period but other damages in packaging were more after more time period. However for T. castaneum all sorts of damages were seen more after more time period. Overall categorization between two insects showed R. dominica made more penetrations and T. castaneum made more invasions compared with their counterparts. Pictures were taken under camera fitted microscope to magnify punctures and holes in different packaging and thicknesses. Insect mortality due to phosphine was more in PP and PE packaging and least in PVC packaging and thickness effect was marginal. T. castaneum mortality was significantly more after 48 h than after 24 h. Damages extent in packaging and fumigation results showed PP to be the best of three packaging materials to manage these insects.

  16. Evaluation of Standard Loose Plastic Packaging for the Management of Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) and Tribolium castaneum (Herbst) (Coleoptera: Tenebriondiae)

    Science.gov (United States)

    Hassan, Muhammad Waqar; Gulraize; Ali, Usman; Ur Rehman, Fazal; Najeeb, Hafsa; Sohail, Maryam; Irsa, Bakhtawar; Muzaffar, Zubaria; Chaudhry, Muhammad Shafiq

    2016-01-01

    Three standard foodstuff plastic packaging namely polyethylene (PE), polypropylene (PP), and polyvinylchloride (PVC) were evaluated for management of lesser grain borer Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) and red flour beetle Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Resistance parameters in packaging were recorded as punctures, holes, penetrations, sealing defects, and invasions with two thicknesses and tested for two lengths of time. Damages like punctures, holes and penetrations by both insects were more in PE packaging however R. dominica made more penetrations in PP than in PE. For both insects sealing defects and invasions were predominant in PVC than in others. Thickness did not affect significantly damage types but significantly more holes and penetrations by R. dominica were in less thickness. Punctures and holes by R. dominica were more after less time period but other damages in packaging were more after more time period. However for T. castaneum all sorts of damages were seen more after more time period. Overall categorization between two insects showed R. dominica made more penetrations and T. castaneum made more invasions compared with their counterparts. Pictures were taken under camera fitted microscope to magnify punctures and holes in different packaging and thicknesses. Insect mortality due to phosphine was more in PP and PE packaging and least in PVC packaging and thickness effect was marginal. T. castaneum mortality was significantly more after 48 h than after 24 h. Damages extent in packaging and fumigation results showed PP to be the best of three packaging materials to manage these insects. PMID:27638958

  17. Residual efficacy of methoprene for control of Tribolium castaneum (Coleoptera: Tenebrionidae) larvae at different temperatures on varnished wood, concrete, and wheat

    Science.gov (United States)

    The residual efficacy of the juvenile hormone analogue, methoprene (Diacon II), was evaluated in bioassays using larvae of Tribolium castaneum (Herbst) exposed on varnished wood or unsealed concrete treated with a liquid formulation and held at different temperatures. When these surfaces were stored...

  18. Transcriptional profiling of ectoderm specification to keratinocyte fate in human embryonic stem cells.

    Science.gov (United States)

    Tadeu, Ana Mafalda Baptista; Lin, Samantha; Hou, Lin; Chung, Lisa; Zhong, Mei; Zhao, Hongyu; Horsley, Valerie

    2015-01-01

    In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ-secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions.

  19. Epigenetic influence on embryonic development

    DEFF Research Database (Denmark)

    Donkin, Ida; Barrès, Romain; Pinborg, Anja

    2016-01-01

    The epigenome is sensitive to environmental changes and can sustainably alter gene expression, notably during embryonic development. New research indicates that epigenetic factors are heritable, which is why paternal lifestyle may affect fetal development and risk of disease. Children conceived...... by assisted reproduction technology (ART) have an increased risk of peri- and postnatal complications, and as specific ART protocols associate with specific risk profiles, the procedures themselves may cause epigenetic changes contributing to the altered outcomes of the 5,000 Danish children annually...

  20. Inhibition of β-catenin/p300 interaction proximalizes mouse embryonic lung epithelium

    OpenAIRE

    Sasaki, Tomoyo; Kahn, Michael

    2014-01-01

    Background Wnt/β-catenin signaling has been suggested to regulate proximal-distal determination of embryonic lung epithelium based upon genetically modified mouse models. The previously identified and characterized small molecule inhibitor IQ1 can pharmacologically decrease the interaction between β-catenin and its transcriptional coactivator p300, thereby enhancing the β-catenin/CBP interaction. Inhibition of the β-catenin/p300 interaction by IQ1 blocks the differentiation of embryonic stem ...

  1. Progresses in research on the functional genomics on Tribolium castaneum%赤拟谷盗功能基因组学研究进展

    Institute of Scientific and Technical Information of China (English)

    李承军; 王艳允; 刘幸; 桑明; 李斌

    2011-01-01

    赤拟谷盗Tribolium castaneum是一种重要的模式生物,在遗传、发育、生化与免疫等研究领域均取得了重要的研究进展.同时它也是一种危害极大的鞘翅目类储粮害虫,在世界各地都有分布,每年给储藏物造成了数十亿美元的经济损失.其全基因组测序的完成、遗传操作体系的构建及系统RNAi方法的应用都极大地促进了其功能基因组学的研究.本文综述了近年来赤拟谷盗基因组计划及功能基因组学的研究进展,拟为赤拟谷盗的生物学研究和防治奠定基础.%Tribolium castaneum is a powerful model organism for research in such fields as insect genetics, biology development, biochemistry and immunity, and rapid and useful progress has been made in these fields in recent years. T. Castaneum is also an important coleopteran pest of stored agricultural products. This globally distributed pest causes billions of dollars of damage to such stored products each year. Recently, the sequencing of the entire genome of T. Castaneum, the construction of their genetics operation systems, and the utilization of the systemic RNA interference, have greatly facilitated and accelerated research on their functional genomics and the related fields. In order to facilitate research on both the fundamental biology and control of T. Castaneum, we here review progresses and achievements in research on T. Castaneum genome projects and functional genomics over the past few years.

  2. Isolation, identification and differentiation of human embryonic cartilage stem cells.

    Science.gov (United States)

    Fu, Changhao; Yan, Zi; Xu, Hao; Zhang, Chen; Zhang, Qi; Wei, Anhui; Yang, Xi; Wang, Yi

    2015-07-01

    We isolated human embryonic cartilage stem cells (hECSCs), a novel stem cell population, from the articular cartilage of eight-week-old human embryos. These stem cells demonstrated a marker expression pattern and differentiation potential intermediate to those of human embryonic stem cells (hESCs) and human adult stem cells (hASCs). hECSCs expressed markers associated with both hESCs (OCT4, NANOG, SOX2, SSEA-3 and SSEA-4) and human adult stem cells (hASCs) (CD29, CD44, CD90, CD73 and CD10). These cells also differentiated into adipocytes, osteoblasts, chondrocytes, neurons and islet-like cells under specific inducing conditions. We identified N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) as an inducer of chondrogenic differentiation in hECSCs. Similar results using N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) were obtained for two other types of human embryonic tissue-derived stem cells, human embryonic hepatic stem cells (hEHSCs) and human embryonic amniotic fluid stem cells (hEASCs), both of which exhibited a marker expression pattern similar to that of hECSCs. The isolation of hECSCs and the discovery that N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) induces chondrogenic differentiation in different stem cell populations might aid the development of strategies in tissue engineering and cartilage repair.

  3. The cys-loop ligand-gated ion channel gene superfamily of the red flour beetle, Tribolium castaneum

    Directory of Open Access Journals (Sweden)

    Sattelle David B

    2007-09-01

    Full Text Available Abstract Background Members of the cys-loop ligand-gated ion channel (cys-loop LGIC superfamily mediate chemical neurotransmission and are studied extensively as potential targets of drugs used to treat neurological disorders such as Alzheimer's disease. Insect cys-loop LGICs are also of interest as they are targets of highly successful insecticides. The red flour beetle, Tribolium castaneum, is a major pest of stored agricultural products and is also an important model organism for studying development. Results As part of the T. castaneum genome sequencing effort, we have characterized the beetle cys-loop LGIC superfamily which is the third insect superfamily to be described after those of Drosophila melanogaster and Apis mellifera, and also the largest consisting of 24 genes. As with Drosophila and Apis, Tribolium possesses ion channels gated by acetylcholine, γ-amino butyric acid (GABA, glutamate and histamine as well as orthologs of the Drosophila pH-sensitive chloride channel subunit (pHCl, CG8916 and CG12344. Similar to Drosophila and Apis, Tribolium cys-loop LGIC diversity is broadened by alternative splicing although the beetle orthologs of RDL and GluCl possess more variants of exon 3. Also, RNA A-to-I editing was observed in two Tribolium nicotinic acetylcholine receptor subunits, Tcasα6 and Tcasβ1. Editing in Tcasα6 is evolutionarily conserved with D. melanogaster, A. mellifera and Heliothis virescens, whereas Tcasβ1 is edited at a site so far only observed in the beetle. Conclusion Our findings reveal that in diverse insect species the cys-loop LGIC superfamily has remained compact with only minor changes in gene numbers. However, alternative splicing, RNA editing and the presence of divergent subunits broadens the cys-loop LGIC proteome and generates species-specific receptor isoforms. These findings on Tribolium castaneum enhance our understanding of cys-loop LGIC functional genomics and provide a useful basis for the

  4. Embryonic anti-aging niche.

    Science.gov (United States)

    Conboy, Irina M; Yousef, Hanadie; Conboy, Michael J

    2011-05-01

    Although functional organ stem cells persist in the old, tissue damage invariably overwhelms tissue repair, ultimately causing the demise of an organism. The poor performance of stem cells in an aged organ, such as skeletal muscle, is caused by the changes in regulatory pathways such as Notch, MAPK and TGF-β, where old differentiated tissue actually inhibits its own regeneration. This perspective analyzes the current literature on regulation of organ stem cells by their young versus old niches and suggests that determinants of healthy and prolonged life might be under a combinatorial control of cell cycle check point proteins and mitogens, which need to be tightly balanced in order to promote tissue regeneration without tumor formation. While responses of adult stem cells are regulated extrinsically and age-specifically, we put forward experimental evidence suggesting that embryonic cells have an intrinsic youthful barrier to aging and produce soluble pro-regenerative proteins that signal the MAPK pathway for rejuvenating myogenesis. Future identification of this activity will improve our understanding of embryonic versus adult regulation of tissue regeneration suggesting novel strategies for organ rejuvenation. Comprehensively, the current intersection of aging and stem cell science indicates that if the age-imposed decline in the regenerative capacity of stem cells was understood, the debilitating lack of organ maintenance in the old could be ameliorated and perhaps, even reversed.

  5. Embryonic Development: Chicken and Zebrafish

    Directory of Open Access Journals (Sweden)

    Veerle M. Darras

    2011-01-01

    Full Text Available Chicken and zebrafish are two model species regularly used to study the role of thyroid hormones in vertebrate development. Similar to mammals, chickens have one thyroid hormone receptor α (TRα and one TRβ gene, giving rise to three TR isoforms: TRα, TRβ2, and TRβ0, the latter with a very short amino-terminal domain. Zebrafish also have one TRβ gene, providing two TRβ1 variants. The zebrafish TRα gene has been duplicated, and at least three TRα isoforms are expressed: TRαA1-2 and TRαB are very similar, while TRαA1 has a longer carboxy-terminal ligand-binding domain. All these TR isoforms appear to be functional, ligand-binding receptors. As in other vertebrates, the different chicken and zebrafish TR isoforms have a divergent spatiotemporal expression pattern, suggesting that they also have distinct functions. Several isoforms are expressed from the very first stages of embryonic development and early chicken and zebrafish embryos respond to thyroid hormone treatment with changes in gene expression. Future studies in knockdown and mutant animals should allow us to link the different TR isoforms to specific processes in embryonic development.

  6. ETS transcription factors in embryonic vascular development.

    Science.gov (United States)

    Craig, Michael P; Sumanas, Saulius

    2016-07-01

    At least thirteen ETS-domain transcription factors are expressed during embryonic hematopoietic or vascular development and potentially function in the formation and maintenance of the embryonic vasculature or blood lineages. This review summarizes our current understanding of the specific roles played by ETS factors in vasculogenesis and angiogenesis and the implications of functional redundancies between them.

  7. LONG-TERM EFFICACY OF PROTEIN-ENRICHED PEA FLOUR AGAINST TRIBOLIUM CASTANEUM (COLEOPTERA: TENEBRIONIDAE IN WHEAT FLOUR

    Directory of Open Access Journals (Sweden)

    P Pretheep-Kumar

    2007-07-01

    Full Text Available Long-term efficacy of the protein-enriched flour of pea (Pisum sativum L. var. Bonneville in its toxicity, progeny reduction and organoleptic properties was evaluated by combining it with wheat flour and testing the admixture against the red flour beetle, Tribolium castaneum (Herbst. The toxicity and progeny-reducing effects of the wheat flour treated with protein-enriched pea flour were stable for a period of 5 months when stored at 28°C with 75% r.h. Heat treatment destroyed the biological activity of the protein-enriched pea flour containing the active ingredient due to the denaturation of proteins. The organoleptic properties of stored wheat flour were not affected by the treatment with protein-enriched pea flour.

  8. Estagiamento de embriões de Macrobrachium olfersi (Wiegman (Crustacea, Palaemonidae através de critérios morfológicos nos dias embrionários Macrobrachium olfersi (Wiegman (Crustacea, Palaemonidae embryo staging through morphological landmarks identified in each embryonic day

    Directory of Open Access Journals (Sweden)

    Marcos S. Simões-Costa

    2005-06-01

    Full Text Available Em embriões de Macrobrachium olfersi (Wiegman, 1836 foram analisadas as características morfológicas bem como o dia do desenvolvimento em que estas características surgiram. Machos e fêmeas de M. olfersi foram coletados na Ilha de Santa Catarina e colocados em aquários de água doce, na temperatura de 26°C e ciclo escuro e claro de 10:14 horas. Fêmeas ovígeras foram monitoradas diariamente para retirada de uma amostra de 20 ovos da câmara incubadora. O desenvolvimento embrionário foi caracterizado através do sistema de estagiamento diário. Embriões vivos e fixados foram analisados (48x em intervalos de 24 horas (dia embrionário. O índice do olho foi calculado em cada dia embrionário, a partir do aparecimento da pigmentação no olho. O desenvolvimento de M. olfersi foi caracterizado em 14 dias embrionários (E, onde entre E1 a E4 ocorreu a clivagem, gastrulação, disco germinativo e organização do nauplius embrionizado. Nos dias subseqüentes foi caracterizado o crescimento do nauplius embrionizado bem como a formação e encurvamento do pós-nauplius. Em E7 observou-se a pigmentação no olho, seguida do início dos batimentos cardíacos em E8. Entre E9 e E14, ocorreu de forma mais intensa o processo de organogênese, principalmente dos sistemas nervoso, cardiovascular e digestivo. O estagiamento diário do desenvolvimento de M. olfersi permitiu o reconhecimento de diferentes formas embrionárias, bem como de ritmos de crescimento e diferenciação do embrião, os quais são essenciais à formação gradual do plano do corpo.Morphological landmarks of Macrobrachium olfersi embryos were examined and their appearance times were related to each embryonic day. Males and females of M. olfersi (Wiegman, 1836 were captured in Santa Catarina Island and kept in freshwater small tanks at 26ºC and 10:14 dark: light cycle. Ovigerous females were monitored daily to remove samples of 20 eggs from brood pouch. The embryonic development

  9. A Defensin from the Model Beetle Tribolium castaneum Acts Synergistically with Telavancin and Daptomycin against Multidrug Resistant Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Rajmohan Rajamuthiah

    Full Text Available The red flour beetle Tribolium castaneum is a common insect pest and has been established as a model beetle to study insect development and immunity. This study demonstrates that defensin 1 from T. castaneum displays in vitro and in vivo antimicrobial activity against drug resistant Staphylococcus aureus strains. The minimum inhibitory concentration (MIC of defensin 1 against 11 reference and clinical staphylococcal isolates was between 16-64 μg/ml. The putative mode of action of the defensin peptide is disruption of the bacterial cell membrane. The antibacterial activity of defensin 1 was attenuated by salt concentrations of 1.56 mM and 25 mM for NaCl and CaCl2 respectively. Treatment of defensin 1 with the reducing agent dithiothreitol (DTT at concentrations 1.56 to 3.13 mM abolished the antimicrobial activity of the peptide. In the presence of subinhibitory concentrations of antibiotics that also target the bacterial cell envelope such as telavancin and daptomycin, the MIC of the peptide was as low as 1 μg/ml. Moreover, when tested against an S. aureus strain that was defective in D-alanylation of the cell wall, the MIC of the peptide was 0.5 μg/ml. Defensin 1 exhibited no toxicity against human erythrocytes even at 400 μg/ml. The in vivo activity of the peptide was validated in a Caenorhabditis elegans-MRSA liquid infection assay. These results suggest that defensin 1 behaves similarly to other cationic AMPs in its mode of action against S. aureus and that the activity of the peptide can be enhanced in combination with other antibiotics with similar modes of action or with compounds that have the ability to decrease D-alanylation of the bacterial cell wall.

  10. Phosphine resistance in the rust red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae): inheritance, gene interactions and fitness costs.

    Science.gov (United States)

    Jagadeesan, Rajeswaran; Collins, Patrick J; Daglish, Gregory J; Ebert, Paul R; Schlipalius, David I

    2012-01-01

    The recent emergence of heritable high level resistance to phosphine in stored grain pests is a serious concern among major grain growing countries around the world. Here we describe the genetics of phosphine resistance in the rust red flour beetle Tribolium castaneum (Herbst), a pest of stored grain as well as a genetic model organism. We investigated three field collected strains of T. castaneum viz., susceptible (QTC4), weakly resistant (QTC1012) and strongly resistant (QTC931) to phosphine. The dose-mortality responses of their test- and inter-cross progeny revealed that most resistance was conferred by a single major resistance gene in the weakly (3.2×) resistant strain. This gene was also found in the strongly resistant (431×) strain, together with a second major resistance gene and additional minor factors. The second major gene by itself confers only 12-20× resistance, suggesting that a strong synergistic epistatic interaction between the genes is responsible for the high level of resistance (431×) observed in the strongly resistant strain. Phosphine resistance is not sex linked and is inherited as an incompletely recessive, autosomal trait. The analysis of the phenotypic fitness response of a population derived from a single pair inter-strain cross between the susceptible and strongly resistant strains indicated the changes in the level of response in the strong resistance phenotype; however this effect was not consistent and apparently masked by the genetic background of the weakly resistant strain. The results from this work will inform phosphine resistance management strategies and provide a basis for the identification of the resistance genes.

  11. A Defensin from the Model Beetle Tribolium castaneum Acts Synergistically with Telavancin and Daptomycin against Multidrug Resistant Staphylococcus aureus.

    Science.gov (United States)

    Rajamuthiah, Rajmohan; Jayamani, Elamparithi; Conery, Annie L; Fuchs, Beth Burgwyn; Kim, Wooseong; Johnston, Tatiana; Vilcinskas, Andreas; Ausubel, Frederick M; Mylonakis, Eleftherios

    2015-01-01

    The red flour beetle Tribolium castaneum is a common insect pest and has been established as a model beetle to study insect development and immunity. This study demonstrates that defensin 1 from T. castaneum displays in vitro and in vivo antimicrobial activity against drug resistant Staphylococcus aureus strains. The minimum inhibitory concentration (MIC) of defensin 1 against 11 reference and clinical staphylococcal isolates was between 16-64 μg/ml. The putative mode of action of the defensin peptide is disruption of the bacterial cell membrane. The antibacterial activity of defensin 1 was attenuated by salt concentrations of 1.56 mM and 25 mM for NaCl and CaCl2 respectively. Treatment of defensin 1 with the reducing agent dithiothreitol (DTT) at concentrations 1.56 to 3.13 mM abolished the antimicrobial activity of the peptide. In the presence of subinhibitory concentrations of antibiotics that also target the bacterial cell envelope such as telavancin and daptomycin, the MIC of the peptide was as low as 1 μg/ml. Moreover, when tested against an S. aureus strain that was defective in D-alanylation of the cell wall, the MIC of the peptide was 0.5 μg/ml. Defensin 1 exhibited no toxicity against human erythrocytes even at 400 μg/ml. The in vivo activity of the peptide was validated in a Caenorhabditis elegans-MRSA liquid infection assay. These results suggest that defensin 1 behaves similarly to other cationic AMPs in its mode of action against S. aureus and that the activity of the peptide can be enhanced in combination with other antibiotics with similar modes of action or with compounds that have the ability to decrease D-alanylation of the bacterial cell wall.

  12. Tribolium castaneum RR-1 cuticular protein TcCPR4 is required for formation of pore canals in rigid cuticle.

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    Mi Young Noh

    2015-02-01

    Full Text Available Insect cuticle is composed mainly of structural proteins and the polysaccharide chitin. The CPR family is the largest family of cuticle proteins (CPs, which can be further divided into three subgroups based on the presence of one of the three presumptive chitin-binding sequence motifs denoted as Rebers-Riddiford (R&R consensus sequence motifs RR-1, RR-2 and RR-3. The TcCPR27 protein containing the RR-2 motif is one of the most abundant CPs present both in the horizontal laminae and in vertical pore canals in the procuticle of rigid cuticle found in the elytron of the red flour beetle, Tribolium castaneum. Depletion of TcCPR27 by RNA interference (RNAi causes both unorganized laminae and pore canals, resulting in malformation and weakening of the elytron. In this study, we investigated the function(s of another CP, TcCPR4, which contains the RR-1 motif and is easily extractable from elytra after RNAi to deplete the level of TcCPR27. Transcript levels of the TcCPR4 gene are dramatically increased in 3 d-old pupae when adult cuticle synthesis begins. Immunohistochemical studies revealed that TcCPR4 protein is present in the rigid cuticles of the dorsal elytron, ventral abdomen and leg but not in the flexible cuticles of the hindwing and dorsal abdomen of adult T. castaneum. Immunogold labeling and transmission electron microscopic analyses revealed that TcCPR4 is predominantly localized in pore canals and regions around the apical plasma membrane protrusions into the procuticle of rigid adult cuticles. RNAi for TcCPR4 resulted in an abnormal shape of the pore canals with amorphous pore canal fibers (PCFs in their lumen. These results support the hypothesis that TcCPR4 is required for achieving proper morphology of the vertical pore canals and PCFs that contribute to the assembly of a cuticle that is both lightweight and rigid.

  13. Human embryonic stem cells derived by somatic cell nuclear transfer.

    Science.gov (United States)

    Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L; Wolf, Don; Mitalipov, Shoukhrat

    2013-06-06

    Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

  14. 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....

  15. Role of microglia in embryonic neurogenesis

    Science.gov (United States)

    Tong, Chih Kong

    2016-01-01

    Microglia begin colonizing the developing brain as early as embryonic day 9, prior to the emergence of neurons and other glia. Their ontogeny is also distinct from other central nervous system cells, as they derive from yolk sac hematopoietic progenitors and not neural progenitors. In this review, we feature these unique characteristics of microglia and assess the spatiotemporal similarities between microglia colonization of the central nervous system and embryonic neurogenesis. We also infer to existing evidence for microglia function from embryonic through to postnatal neurodevelopment to postulate roles for microglia in neurogenesis. PMID:27555616

  16. Embryonic stem cell research: an ethical problem

    OpenAIRE

    Рамазанова, А.

    2014-01-01

    Embryonic stem cells offer hope for new therapies, but their use and research entail an ethical problem, which does not have a certain solution. Therefore, we can ask: What exactly are the ethical arguments? Why are they so tricky to resolve?Embryonic stem cell research poses a moral dilemma. It forces us to choose between two moral principles: The duty to prevent or alleviate suffering The duty to respect the value of human life To obtain embryonic stem cells, the early embryo has to be dest...

  17. Establishment of human embryonic stem cell line from gamete donors

    Institute of Scientific and Technical Information of China (English)

    LI Tao; ZHOU Can-quan; MAI Qing-yun; ZHUANG Guang-lun

    2005-01-01

    Background Human embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study. Methods Three oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed.Results Four ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line (cHES-1) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types.Conclusions HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.

  18. MicroRNAs in human embryonic and cancer stem cells.

    Science.gov (United States)

    Navarro, Alfons; Monzo, Mariano

    2010-09-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate messenger RNAs at the post-transcriptional level. They play an important role in the control of cell physiological functions, and their alterations have been related to cancer, where they can function as oncogenes or tumor suppressor genes. Recently, they have emerged as key regulators of "stemness", collaborating in the maintenance of pluripotency, control of self-renewal, and differentiation of stem cells. The miRNA pathway has been shown to be crucial in embryonic development and in embryonic stem (ES) cells, as shown by Dicer knockout analysis. Specific patterns of miRNAs have been reported to be expressed only in ES cells and in early phases of embryonic development. Moreover, many cancers present small populations of cells with stem cell characteristics, called cancer stem cells (CSCs). CSCs are responsible for relapse and treatment failure in many cancer patients, and the comparative analysis of expression patterns between ES cells and tumors can lead to the identification of a miRNA signature to define CSCs. Most of the key miRNAs identified to date in ES cells have been shown to play a role in tumor diagnosis or prognosis, and may well prove to be essential in cancer therapy in the foreseeable future.

  19. RNAi screening of developmental toolkit genes: a search for novel wing genes in the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Linz, David M; Tomoyasu, Yoshinori

    2015-01-01

    The amazing array of diversity among insect wings offers a powerful opportunity to study the mechanisms guiding morphological evolution. Studies in Drosophila (the fruit fly) have identified dozens of genes important for wing development. These genes are often called candidate genes, serving as an ideal starting point to study wing development in other insects. However, we also need to explore beyond the candidate genes to gain a more comprehensive view of insect wing evolution. As a first step away from the traditional candidate genes, we utilized Tribolium (the red flour beetle) as a model and assessed the potential involvement of a group of developmental toolkit genes (embryonic patterning genes) in beetle wing development. We hypothesized that the highly pleiotropic nature of these developmental genes would increase the likelihood of finding novel wing genes in Tribolium. Through the RNA interference screening, we found that Tc-cactus has a less characterized (but potentially evolutionarily conserved) role in wing development. We also found that the odd-skipped family genes are essential for the formation of the thoracic pleural plates, including the recently discovered wing serial homologs in Tribolium. In addition, we obtained several novel insights into the function of these developmental genes, such as the involvement of mille-pattes and Tc-odd-paired in metamorphosis. Despite these findings, no gene we examined was found to have novel wing-related roles unique in Tribolium. These results suggest a relatively conserved nature of developmental toolkit genes and highlight the limited degree to which these genes are co-opted during insect wing evolution.

  20. Biological effects of gamma radiation on stored product insects. 4 - radiation effects on sex pheromone production and perception by the rust-red flour beetle. Tribolium castaneum (herbst)

    OpenAIRE

    Abdu, R. M.; Abdel-Kader, Maissa M.; M. A. Hussein; Abdel-Rahman, H. A.

    1985-01-01

    Irradiation of the rust-red flour beetle, T. castaneum at different doses of gamma radiation considerably affected sex pheromone production by females and perception by males. The production of sex pheromone by virgin females decreased with the increase of radiation doses from 4 to 10 krad., and a dose of 12 krad could almost inhibit pheromone production. Males were more radiosensitive in their response to sex pheromone; and a radiation dose of 8 krad could brought inhibition of male respo...

  1. Impact of varying levels of sanitation on mortality of Tribolium castaneum eggs and adults during heat treatment of a pilot flour mill.

    Science.gov (United States)

    Brijwani, Monika; Subramanyam, Bhadriraju; Flinn, Paul W

    2012-04-01

    The influence of sanitation on responses of life stages of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), an economically important pest in flour mills, was investigated in a pilot flour mill subjected to two, 24-h heat treatments. One hundred eggs or 100 adults of T. castaneum were exposed inside each 20-cm diameter by 15-cm high PVC rings holding 0.1-, 0.2-, 1.0-, 3.0-, 6.0-, and 10.0-cm-deep wheat flour to simulate different sanitation levels that may exist in a flour mill. These rings were placed on the first and third floors of a pilot flour mill. On the first floor, temperatures inside rings with eggs reached 50 degrees C in 7-11 h only in 0.1- and 0.2-cm-deep flour treatments. In all other treatments the maximum temperatures attained generally were below 50 degrees C and inversely related to flour depth. Adults of T. castaneum on this floor were less susceptible than eggs. The egg mortality decreased linearly with an increase in flour depth, whereas that of adults decreased exponentially. All eggs and adults in rings on the third floor were killed irrespective of flour depth, because temperatures inside rings reached 50 degrees C in 15-17 h and were held above 50 degrees C for 6-8 h with the maximum temperatures ranging between 55.0 and 57.0 degrees C. Although the protective effects of flour on survival of T. castaneum eggs and adults were evident only if temperatures did not reach 50 degrees C, removal of flour accumulations is essential to improve heat treatment effectiveness.

  2. Effect of five diatomaceous earth formulations against Tribolium castaneum (Coleoptera: Tenebrionidae),Oryzaephilus surinamensis (Coleoptera: Silvanidae) and Rhyzopertha dominica (Coleoptera: Bostrychidae)

    Institute of Scientific and Technical Information of China (English)

    MASUMEH ZIAEE; ADEL KHASHAVEH

    2007-01-01

    Laboratory bioassays were conducted to determine the effect of food source on the survival of Tribolium castaneum Herbst, Oryzaephilus surinamensis L. and Rhyzopertha dominica F., after exposure to five diatomaceous earth (DE) formulations: Protect-It(R),Insecto(R), Perma-GuardTM, Dryacide(R) and SilicoSec(R). Adults of these species were exposed to DEs at the rate of 0.5 mg/cm2 for 1 day on filter paper inside plastic Petri dishes. After exposure,the initial mortality was counted and live individuals of the three species were held for a week in glass vials containing 50 mg wheat flour, rice and whole wheat, respectively. In the second experiment, after 1 day exposure to DEs, beetles were transferred to Petri dishes without food and held for a week to determine if the presence of food source would decrease the mortality of beetles. Experiments were carried out at 27℃ and 55% RH in the dark. The initial morality in both of the experiments reached 100% for the three species exposed to Protect-It(R) and in the case orR. dominica and O. surinamensis exposed to Dryacide(R). In contrast, low level of mortality (< 10%) was observed for T. castaneum exposed to Perma-GuardTM and Insecto(R). The mortality after the post-treatment period on food was decreased for the three species exposed to Perma-GuardTM and in the case of T. castaneum and R. dominica exposed to Insecto(R) and SilicoSec(R). Adults of O. surinamensis were the most susceptible followed by R. dominica and 100% adult mortality was obtained, whereas T. castaneum were the least susceptible beetles to DEs. Protect-It(R) and Dryacide(R) were the most efficient DE formulations and can be used effectively in a stored grain integrated pest management program.

  3. Composition, Repellent, and Insecticidal Activities of Two South American Plants against the Stored Grain Pests Tribolium castaneum and Tribolium confusum (Coleoptera: Tenebrionidae)

    OpenAIRE

    Benzi, Verónica; Stefanazzi, Natalia; Murray, Ana Paula; Werdin González, Jorge O.; Ferrero, Adriana

    2014-01-01

    As part of a screening program to evaluate the biological activity of indigenous plants, we report the composition and the bioactivity of essential oils (EOs) extracted from Té de Burro Aloysia polystachya [(Griseb.) Moldenke] and Lemon Verbena Aloysia citriodora [Palau] against two of the most widespread secondary pests of stored products, the red flour beetle Tribolium castaneum [Herbst] and the confused flour beetle Tribolium confusum [Jacqueline du Val]. Analysis by gas chromatography-mas...

  4. carboxypeptidase E-ΔN, a neuroprotein transiently expressed during development protects embryonic neurons against glutamate neurotoxicity.

    Directory of Open Access Journals (Sweden)

    Xiao-Yan Qin

    Full Text Available Neuroprotective proteins expressed in the fetus play a critical role during early embryonic neurodevelopment, especially during maternal exposure to alcohol and drugs that cause stress, glutamate neuroexcitotoxicity, and damage to the fetal brain, if prolonged. We have identified a novel protein, carboxypeptidase E-ΔN (CPE-ΔN, which is a splice variant of CPE that has neuroprotective effects on embryonic neurons. CPE-ΔN is transiently expressed in mouse embryos from embryonic day 5.5 to postnatal day 1. It is expressed in embryonic neurons, but not in 3 week or older mouse brains, suggesting a function primarily in utero. CPE-ΔN expression was up-regulated in embryonic hippocampal neurons in response to dexamethasone treatment. CPE-ΔN transduced into rat embryonic cortical and hippocampal neurons protected them from glutamate- and H2O2-induced cell death. When transduced into embryonic cortical neurons, CPE-ΔN was found in the nucleus and enhanced the transcription of FGF2 mRNA. Embryonic cortical neurons challenged with glutamate resulted in attenuated FGF2 levels and cell death, but CPE-ΔN transduced neurons treated in the same manner showed increased FGF2 expression and normal viability. This neuroprotective effect of CPE-ΔN was mediated by secreted FGF2. Through receptor signaling, FGF2 activated the AKT and ERK signaling pathways, which in turn increased BCL-2 expression. This led to inhibition of caspase-3 activity and cell survival.

  5. 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.

  6. Actin cytoskeleton contributes to the elastic modulus of embryonic tendon during early development.

    Science.gov (United States)

    Schiele, Nathan R; von Flotow, Friedrich; Tochka, Zachary L; Hockaday, Laura A; Marturano, Joseph E; Thibodeau, Jeffrey J; Kuo, Catherine K

    2015-06-01

    Tendon injuries are common and heal poorly. Strategies to regenerate or replace injured tendons are challenged by an incomplete understanding of normal tendon development. Our previous study showed that embryonic tendon elastic modulus increases as a function of developmental stage. Inhibition of enzymatic collagen crosslink formation abrogated increases in tendon elastic modulus at late developmental stages, but did not affect increases in elastic modulus of early stage embryonic tendons. Here, we aimed to identify potential contributors to the mechanical properties of these early stage embryonic tendons. We characterized tendon progenitor cells in early stage embryonic tendons, and the influence of actin cytoskeleton disruption on tissue elastic modulus. Cells were closely packed in embryonic tendons, and did not change in density during early development. We observed an organized network of actin filaments that seemed contiguous between adjacent cells. The actin filaments exhibited a crimp pattern with a period and amplitude that matched the crimp of collagen fibers at each developmental stage. Chemical disruption of the actin cytoskeleton decreased tendon tissue elastic modulus, measured by atomic force microscopy. Our results demonstrate that early developmental stage embryonic tendons possess a well organized actin cytoskeleton network that contributes significantly to tendon tissue mechanical properties.

  7. Cortical network from human embryonic stem cells

    OpenAIRE

    2010-01-01

    Abstract The connection of embryonic stem cell technology and developmental biology provides valuable tools to decipher the mechanisms underlying human brain development and diseases, especially among neuronal populations, that are not readily available in primary cultures. It is obviously the case of neurons forming the human cerebral cortex. In the images that are presented, the neurons were generated in vitro from human embryonic stem cells via forebrain-like progenitors. Maintained in cul...

  8. Very small embryonic like(VSEL) stem cells

    Institute of Scientific and Technical Information of China (English)

    Ruinan Lu; Dengshun Miao

    2008-01-01

    It is generally accepted that adult bone marrow(BM) contains both hematopoietic stem cells(HSCs) and mesenchymal stem cells(MSCs). Recently, a rare population of stem cells different from HSCs and MSCs were identified in routine BM and human cord blood(CB), named as very small embryonic like(VSEL) stem cells. These cells are tiny round and CXCR4+ Sca-l+ Lin- CD45-, expressing SSEA-1/4, Oct-4 and Nanog, which have potent of differentiation into all three germ-layer lineages, such as cardiomnyocytes, neural and pancreatic cells.

  9. 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 chromosom

  10. Experimental removal of sexual selection leads to decreased investment in an immune component in female Tribolium castaneum.

    Science.gov (United States)

    Hangartner, Sandra; Michalczyk, Łukasz; Gage, Matthew J G; Martin, Oliver Y

    2015-07-01

    Because of divergent selection acting on males and females arising from different life-history strategies, polyandry can be expected to promote sexual dimorphism of investment into immune function. In previous work we have established the existence of such divergence within populations where males and females are exposed to varying degrees of polyandry. We here test whether the removal of sexual selection via enforced monogamy generates males and females that have similar levels of investment in immune function. To test this prediction experimentally, we measured differences between the sexes in a key immune measurement (phenoloxidase (PO) activity) and resistance to the microsporidian Paranosema whitei in Tribolium castaneum lines that evolved under monogamous (sexual selection absent) vs polyandrous (sexual selection present) mating systems. At generation 49, all selected lines were simultaneously assessed for PO activity and resistance to their natural parasite P. whitei after two generations of relaxed selection. We found that the polyandrous regime was associated with a clear dimorphism in immune function: females had significantly higher PO activities than males in these lines. In contrast, there was no such difference between the sexes in the lines evolving under the monogamous regime. Survival in the infection experiment did not differ between mating systems or sexes. Removing sexual selection via enforced monogamy thus seems to erase intersexual differences in immunity investment. We suggest that higher PO activities in females that have evolved under sexual selection might be driven by the increased risk of infections and/or injuries associated with exposure to multiple males.

  11. Differences in Attack Avoidance and Mating Success between Strains Artificially Selected for Dispersal Distance in Tribolium castaneum.

    Directory of Open Access Journals (Sweden)

    Kentarou Matsumura

    Full Text Available Individuals of both dispersal and non-dispersal types (disperser and non-disperser are found in a population, suggesting that each type has both costs and benefits for fitness. However, few studies have examined the trade-off between the costs and benefits for the types. Here, we artificially selected for walking distance, i.e., an indicator of dispersal ability, in the red flour beetle Tribolium castaneum and established strains with longer (L-strains or shorter (S-strains walking distances. We then compared the frequency of predation by the assassin bug Amphibolus venator and the mating frequency of the selected strains. L-strain beetles suffered higher predation risk, than did S-strain beetles. L-strain males had significantly increased mating success compared to S-strain males, but females did not show a significant difference between the strains. The current results showed the existence of a trade-off between predation avoidance and mating success associated with dispersal types at a genetic level only in males. This finding can help to explain the maintenance of variation in dispersal ability within a population.

  12. Effects of Flour and Milling Debris on Efficacy of Beta-Cyfluthrin for Control of Tribolium castaneum (Coleoptera: Tenebrionidae).

    Science.gov (United States)

    Arthur, Frank H; Starkus, Laura A; Mckay, Tanja

    2015-04-01

    Laboratory and field studies were conducted to determine if accumulation of a flour food source or milling debris affected residual efficacy of beta-cyfluthrin for control of Tribolium castaneum (Herbst), the red flour beetle. In the laboratory study, the high label rate of 20 mg active ingredient (AI)/m2 gave effective control for 8 wks, regardless of whether or not the concrete was sealed prior to application or the presence of flour contamination. However, with the low label rate of 10 mg AI/m2, the flour apparently absorbed the insecticide residues from the treated surface, and sealing the concrete did not have a beneficial effect on efficacy. Two field studies with the low label rate were conducted during autumn of 2012 and 2013 and summer of 2013, using only unsealed concrete. Accumulated milling debris caused a reduction in efficacy in the autumn studies, as shown by increased time to 100% knockdown, decreased mortality, and decreased residual efficacy. There was no such corresponding decrease in residual efficacy in the summer study. Overall, results of both studies show that accumulated food and milling debris can absorb residue of beta-cyfluthrin from a treated surface and have a negative impact on residual efficacy, particularly with the low label rate of 10 mg AI/m2.

  13. RNAi-mediated knockdown of the voltage gated sodium ion channel TcNav causes mortality in Tribolium castaneum

    Science.gov (United States)

    Abd El Halim, Hesham M.; Alshukri, Baida M. H.; Ahmad, Munawar S.; Nakasu, Erich Y. T.; Awwad, Mohammed H.; Salama, Elham M.; Gatehouse, Angharad M. R.; Edwards, Martin G.

    2016-01-01

    The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30–60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control. PMID:27411529

  14. 3D standard brain of the red flour beetle Tribolium castaneum: a tool to study metamorphic development and adult plasticity

    Directory of Open Access Journals (Sweden)

    David Dreyer

    2010-03-01

    Full Text Available The red flour beetle Tribolium castaneum is emerging as a further standard insect model beside Drosophila. Its genome is fully sequenced and it is susceptible for genetic manipulations including RNA-interference. We use this beetle to study adult brain development and plasticity primarily with respect to the olfactory system. In the current study, we provide 3D standard brain atlases of freshly eclosed adult female and male beetles (A0. The atlases include eight paired and three unpaired neuropils including antennal lobes, optic lobe neuropils, mushroom body calyces and pedunculi, and central complex. For each of the two standard brains, we averaged brain areas of 20 individual brains. Additionally, we characterized eight selected olfactory glomeruli from 10 A0 female and male beetles respectively, which we could unequivocally recognize from individual to individual owing to their size and typical position in the antennal lobes. In summary, comparison of the averaged neuropil volumes revealed no sexual dimorphism in any of the reconstructed neuropils in A0 Tribolium brains. Both, the female and male 3D standard brain are also used for interspecies comparisons, and, very importantly, will serve as future volumetric references after genetical manipulation especially regarding metamorphic development and adult plasticity.

  15. Computational analysis of expression of human embryonic stem cell-associated signatures in tumors

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    Wang Xiaosheng

    2011-10-01

    Full Text Available Abstract Background The cancer stem cell model has been proposed based on the linkage between human embryonic stem cells and human cancer cells. However, the evidences supporting the cancer stem cell model remain to be collected. In this study, we extensively examined the expression of human embryonic stem cell-associated signatures including core genes, transcription factors, pathways and microRNAs in various cancers using the computational biology approach. Results We used the class comparison analysis and survival analysis algorithms to identify differentially expressed genes and their associated transcription factors, pathways and microRNAs among normal vs. tumor or good prognosis vs. poor prognosis phenotypes classes based on numerous human cancer gene expression data. We found that most of the human embryonic stem cell- associated signatures were frequently identified in the analysis, suggesting a strong linkage between human embryonic stem cells and cancer cells. Conclusions The present study revealed the close linkage between the human embryonic stem cell associated gene expression profiles and cancer-associated gene expression profiles, and therefore offered an indirect support for the cancer stem cell theory. However, many interest issues remain to be addressed further.

  16. Periods of cardiovascular susceptibility to hypoxia in embryonic american alligators (Alligator mississippiensis).

    Science.gov (United States)

    Tate, Kevin B; Rhen, Turk; Eme, John; Kohl, Zachary F; Crossley, Janna; Elsey, Ruth M; Crossley, Dane A

    2016-06-01

    During embryonic development, environmental perturbations can affect organisms' developing phenotype, a process known as developmental plasticity. Resulting phenotypic changes can occur during discrete, critical windows of development. Critical windows are periods when developing embryos are most susceptible to these perturbations. We have previously documented that hypoxia reduces embryo size and increases relative heart mass in American alligator, and this study identified critical windows when hypoxia altered morphological, cardiovascular function and cardiac gene expression of alligator embryos. We hypothesized that incubation in hypoxia (10% O2) would increase relative cardiac size due to cardiac enlargement rather than suppression of somatic growth. We exposed alligator embryos to hypoxia during discrete incubation periods to target windows where the embryonic phenotype is altered. Hypoxia affected heart growth between 20 and 40% of embryonic incubation, whereas somatic growth was affected between 70 and 90% of incubation. Arterial pressure was depressed by hypoxic exposure during 50-70% of incubation, whereas heart rate was depressed in embryos exposed to hypoxia during a period spanning 70-90% of incubation. Expression of Vegf and PdgfB was increased in certain hypoxia-exposed embryo treatment groups, and hypoxia toward the end of incubation altered β-adrenergic tone for arterial pressure and heart rate. It is well known that hypoxia exposure can alter embryonic development, and in the present study, we have identified brief, discrete windows that alter the morphology, cardiovascular physiology, and gene expression in embryonic American alligator.

  17. 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

  18. Establishment of a molecular embryonic stem cell developmental toxicity assay.

    Science.gov (United States)

    Panzica-Kelly, Julieta M; Brannen, Kimberly C; Ma, Yan; Zhang, Cindy X; Flint, Oliver P; Lehman-McKeeman, Lois D; Augustine-Rauch, Karen A

    2013-02-01

    The mouse embryonic stem cell test (EST) is a 10-day screen for teratogenic potential developed to reduce animal use for embryotoxicity testing of chemicals (Spielmann, 2005; Spielmann et al., 1997). In this study, we used the cytotoxicity IC(50) values and transcriptional expression changes as primary endpoints in a shorter 4-day version of the EST, the molecular embryonic stem cell assay. Mouse D3 embryonic stem cells were used for cytotoxicity assessment (monolayers) or grown as embryoid bodies in low attachment plates for transcriptional profiling. Sixty-five compounds with known in vivo teratogenicity (33 teratogens and 32 nonteratogens) were evaluated to develop a model for classifying compounds with teratogenic potential. The expression of 12 developmentally regulated gene targets (nanog, fgf5, gsc, cd34, axin2, apln, chst7, lhx1, fgf8, sox17, foxa2, and cxcr4) was measured following exposure of embryoid bodies to a single compound concentration (0.1 × the cytotoxicity IC(20)) for 4 days. In the decision-tree model, compounds with IC(50) values teratogens, whereas compounds in the two groups with IC(50) values between 22-200 µM and > 200 µM were categorized as teratogens if ≥ 8 and 12 genes, respectively, were deregulated by at least 10%. Forty-seven of 65 compounds of the training set were correctly identified (72% total concordance). In a test set of 12 additional compounds (5 teratogens, 7 nonteratogens), 10 were correctly classified by this approach (83% concordance). The false positive rate in the training and test sets was 24 and 0%, respectively, indicating that this assay has potential to identify teratogens.

  19. Embryonic Stem Cells: Isolation, Characterization and Culture

    Science.gov (United States)

    Amit, Michal; Itskovitz-Eldor, Joseph

    Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

  20. REDOX DISRUPTING POTENTIAL OF TOXCAST CHEMICALS RANKED BY ACTIVITY IN MOUSE EMBRYONIC STEM CELLS

    Science.gov (United States)

    To gain insight regarding the adverse outcome pathways leading to developmental toxicity following exposure to chemicals, we evaluated ToxCast™ Phase I chemicals in an adherent mouse embryonic stem cell (mESC) assay and identified a redox sensitive pathway that correlated with al...

  1. Embryonic stem cells: testing the germ-cell theory.

    Science.gov (United States)

    Hochedlinger, Konrad

    2011-10-25

    The exact cellular origin of embryonic stem cells remains elusive. Now a new study provides compelling evidence that embryonic stem cells, established under conventional culture conditions, originate from a transient germ-cell state.

  2. Insecticide-Mediated Up-Regulation of Cytochrome P450 Genes in the Red Flour Beetle (Tribolium castaneum

    Directory of Open Access Journals (Sweden)

    Xiao Liang

    2015-01-01

    Full Text Available Some cytochrome P450 (CYP genes are known for their rapid up-regulation in response to insecticide exposures in insects. To date, however, limited information is available with respect to the relationships among the insecticide type, insecticide concentration, exposure duration and the up-regulated CYP genes. In this study, we examined the transcriptional response of eight selected CYP genes, including CYP4G7, CYP4Q4, CYP4BR3, CYP12H1, CYP6BK11, CYP9D4, CYP9Z5 and CYP345A1, to each of four insecticides in the red flour beetle, Tribolium castaneum. Reverse transcription quantitative PCR (RT-qPCR revealed that CYP4G7 and CYP345A1 can be significantly up-regulated by cypermethrin (1.97- and 2.06-fold, respectively, permethrin (2.00- and 2.03-fold and lambda-cyhalothrin (1.73- and 1.81-fold, whereas CYP4BR3 and CYP345A1 can be significantly up-regulated by imidacloprid (1.99- and 1.83-fold when 20-day larvae were exposed to each of these insecticides at the concentration of LC20 for 24 h. Our studies also showed that similar levels of up-regulation can be achieved for CYP4G7, CYP4BR3 and CYP345A1 by cypermethrin, permethrin, lambda-cyhalothrin or imidacloprid with approximately one fourth of LC20 in 6 h. Our study demonstrated that up-regulation of these CYP genes was rapid and only required low concentrations of insecticides, and the up-regulation not only depended on the CYP genes but also the type of insecticides. Our results along with those from previous studies also indicated that there were no specific patterns for predicting the up-regulation of specific CYP gene families based on the insecticide classification.

  3. Dissecting systemic RNA interference in the red flour beetle Tribolium castaneum: parameters affecting the efficiency of RNAi.

    Directory of Open Access Journals (Sweden)

    Sherry C Miller

    Full Text Available The phenomenon of RNAi, in which the introduction of dsRNA into a cell triggers the destruction of the corresponding mRNA resulting in a gene silencing effect, is conserved across a wide array of plant and animal phyla. However, the mechanism by which the dsRNA enters a cell, allowing the RNAi effect to occur throughout a multicellular organism (systemic RNAi, has only been studied extensively in certain plants and the nematode Caenorhabditis elegans. In recent years, RNAi has become a popular reverse genetic technique for gene silencing in many organisms. Although many RNAi techniques in non-traditional model organisms rely on the systemic nature of RNAi, little has been done to analyze the parameters required to obtain a robust systemic RNAi response. The data provided here show that the concentration and length of dsRNA have profound effects on the efficacy of the RNAi response both in regard to initial efficiency and duration of the effect in Tribolium castaneum. In addition, our analyses using a series of short dsRNAs and chimeric dsRNA provide evidence that dsRNA cellular uptake (and not the RNAi response itself is the major step affected by dsRNA size in Tribolium. We also demonstrate that competitive inhibition of dsRNA can occur when multiple dsRNAs are injected together, influencing the effectiveness of RNAi. These data provide specific information essential to the design and implementation of RNAi based studies, and may provide insight into the molecular basis of the systemic RNAi response in insects.

  4. Insecticide-mediated up-regulation of cytochrome P450 genes in the red flour beetle (Tribolium castaneum).

    Science.gov (United States)

    Liang, Xiao; Xiao, Da; He, Yanping; Yao, Jianxiu; Zhu, Guonian; Zhu, Kun Yan

    2015-01-19

    Some cytochrome P450 (CYP) genes are known for their rapid up-regulation in response to insecticide exposures in insects. To date, however, limited information is available with respect to the relationships among the insecticide type, insecticide concentration, exposure duration and the up-regulated CYP genes. In this study, we examined the transcriptional response of eight selected CYP genes, including CYP4G7, CYP4Q4, CYP4BR3, CYP12H1, CYP6BK11, CYP9D4, CYP9Z5 and CYP345A1, to each of four insecticides in the red flour beetle, Tribolium castaneum. Reverse transcription quantitative PCR (RT-qPCR) revealed that CYP4G7 and CYP345A1 can be significantly up-regulated by cypermethrin (1.97- and 2.06-fold, respectively), permethrin (2.00- and 2.03-fold) and lambda-cyhalothrin (1.73- and 1.81-fold), whereas CYP4BR3 and CYP345A1 can be significantly up-regulated by imidacloprid (1.99- and 1.83-fold) when 20-day larvae were exposed to each of these insecticides at the concentration of LC20 for 24 h. Our studies also showed that similar levels of up-regulation can be achieved for CYP4G7, CYP4BR3 and CYP345A1 by cypermethrin, permethrin, lambda-cyhalothrin or imidacloprid with approximately one fourth of LC20 in 6 h. Our study demonstrated that up-regulation of these CYP genes was rapid and only required low concentrations of insecticides, and the up-regulation not only depended on the CYP genes but also the type of insecticides. Our results along with those from previous studies also indicated that there were no specific patterns for predicting the up-regulation of specific CYP gene families based on the insecticide classification.

  5. Molecular Cloning and Functional Analysis of ESGP, an Embryonic Stem Cell and Germ Cell Specific Protein

    Institute of Scientific and Technical Information of China (English)

    Yan-Mei CHEN; Zhong-Wei DU; Zhen YAO

    2005-01-01

    Several putative Oct-4 downstream genes from mouse embryonic stem (ES) cells have been identified using the suppression-subtractive hybridization method. In this study, one of the novel genes encoding an ES cell and germ cell specific protein (ESGP) was cloned by rapid amplification of cDNA ends.ESGP contains 801 bp encoding an 84 amino acid small protein and has no significant homology to any known genes. There is a signal peptide at the N-terminal of ESGP protein as predicted by SeqWeb (GCG)(SeqWeb version 2.0.2, http://gcg.biosino.org:8080/). The result of immunofluorescence assay suggested that ESGP might encode a secretory protein. The expression pattern of ESGP is consistent with the expression of Oct-4 during embryonic development. ESGP protein was detected in fertilized oocyte, from 3.5 day postcoital (dpc) blastocyst to 17.5 dpc embryo, and was only detected in testis and ovary tissues in adult. In vitro, ESGP was only expressed in pluripotent cell lines, such as embryonic stem cells, embryonic carcinoma cells and embryonic germ cells, but not in their differentiated progenies. Despite its specific expression,forced expression of ESGP is not indispensable for the effect of Oct-4 on ES cell self-renewal, and does not affect the differentiation to three germ layers.

  6. UCP2 Regulates Embryonic Neurogenesis via ROS-mediated Yap Alternation in the Developing Neocortex.

    Science.gov (United States)

    Ji, Fen; Shen, Tianjin; Zou, Wenzheng; Jiao, Jianwei

    2017-03-09

    Mitochondrial metabolism is a fundamental process in tissue development. How this process play functions in embryonic neurogenesis remains largely unknown. Here, we show that mitochondrial uncoupling protein 2 (UCP2) regulates the embryonic neurogenesis by inhibiting the production of reactive oxygen species (ROS), which affect the proliferation of progenitors. In the embryonic brains of UCP2 knockdown or condition knockout mice, the proliferation of progenitors is significantly increased, while the differentiation of progenitors is reduced. Furthermore, we identify that Yap is the response protein of UCP2 mediated ROS production. When UCP2 is inactive, the production of ROS are increased. The amount of Yap protein is increased as Yap degradation through ubiquitin-proteasome proteolytic pathway is decreased. The defect caused by UCP2 depression can be rescued by Yap down-regulation. Collectively, our results demonstrate that UCP2 regulates embryonic neurogenesis through ROS-mediated Yap alternation, thus shedding new sight on mitochondrial metabolism involved in embryonic neurogenesis. This article is protected by copyright. All rights reserved.

  7. A data integration approach to mapping OCT4 gene regulatory networks operative in embryonic stem cells and embryonal carcinoma cells.

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    Marc Jung

    Full Text Available It is essential to understand the network of transcription factors controlling self-renewal of human embryonic stem cells (ESCs and human embryonal carcinoma cells (ECs if we are to exploit these cells in regenerative medicine regimes. Correlating gene expression levels after RNAi-based ablation of OCT4 function with its downstream targets enables a better prediction of motif-specific driven expression modules pertinent for self-renewal and differentiation of embryonic stem cells and induced pluripotent stem cells.We initially identified putative direct downstream targets of OCT4 by employing CHIP-on-chip analysis. A comparison of three peak analysis programs revealed a refined list of OCT4 targets in the human EC cell line NCCIT, this list was then compared to previously published OCT4 CHIP-on-chip datasets derived from both ES and EC cells. We have verified an enriched POU-motif, discovered by a de novo approach, thus enabling us to define six distinct modules of OCT4 binding and regulation of its target genes.A selection of these targets has been validated, like NANOG, which harbours the evolutionarily conserved OCT4-SOX2 binding motif within its proximal promoter. Other validated targets, which do not harbour the classical HMG motif are USP44 and GADD45G, a key regulator of the cell cycle. Over-expression of GADD45G in NCCIT cells resulted in an enrichment and up-regulation of genes associated with the cell cycle (CDKN1B, CDKN1C, CDK6 and MAPK4 and developmental processes (BMP4, HAND1, EOMES, ID2, GATA4, GATA5, ISL1 and MSX1. A comparison of positively regulated OCT4 targets common to EC and ES cells identified genes such as NANOG, PHC1, USP44, SOX2, PHF17 and OCT4, thus further confirming their universal role in maintaining self-renewal in both cell types. Finally we have created a user-friendly database (http://biit.cs.ut.ee/escd/, integrating all OCT4 and stem cell related datasets in both human and mouse ES and EC cells.In the current

  8. Physiopathology of human embryonic implantation: clinical incidences.

    Directory of Open Access Journals (Sweden)

    Pauline Demailly

    2010-01-01

    Full Text Available Embryo implantation consists of a series of events promoting the invasion of the endometrium and then the uterine arterial system by the extra-embryonic trophoblast. In order for this semi-heterologous implantation to succeed, the endometrium has to first undergo a number of structural and biochemical changes (decidualization. The decidua's various constituents subsequently play a role in the embryonic implantation. The third step is the transformation of the uterine vascular system and the growth of the placenta, which will provide the foetoplacental unit with nutrients. Several physiopathological aspects will be discussed: 1 the implantation window, regulated by maternal and embryonic hormonal secretions and thus influenced by any defects in the latter: dysharmonic luteal phase, 21-hydroxylase block, abnormal integrin expression, 2 the successive trophoblast invasions of uterine vessels which, when defective, lead to early embryo loss or late-onset vascular pathologies, as preeclampsia, 3 the pregnancy's immunological equilibrium, with a spontaneously tolerated semi-allogeneic implant, 4 the impact of pro-coagulant factors (thrombophilia on the pregnancy's progression, 5 the environment of the uterus, ranging from hydrosalpinx to uterine contractions. In summary, the least anatomical or physiological perturbation can interfere with human embryonic implantation - a very particular phenomenon and a true biological paradox.

  9. Skeletal tissue engineering using embryonic stem cells

    NARCIS (Netherlands)

    Jukes, Jojanneke Maria

    2009-01-01

    Tissue engineering aims at repairing or replacing damaged or diseased tissue. In this thesis, we investigated the potential of embryonic stem cells (ESCs) for cartilage tissue engineering. After differentiation of mouse and human ESCs into the chondrogenic and osteogenic lineage had been established

  10. Actividad insecticida del aceite esencial de Lippia alba (Mill.) N. E. Brown sobre Tribolium castaneum Herbst. en granos de trigo (Triticum aestivum L.)

    OpenAIRE

    Ringuelet, Jorge Abel; Ocampo, Rafael; Henning, Cynthia; Padín, Susana; Urrutia, María Inés; Dal Bello, Gustavo

    2014-01-01

    Tribolium castaneum (Coleoptera: Tenebrionidae) es un insecto plaga de granos, muy destructivo en la etapa de almacenamiento y altamente resistente a los fitosanitarios sintéticos. Por esta razón su control químico se realiza con altas concentraciones de insecticidas que aumentan la contaminación ambiental y los peligros para la salud humana. Las tendências actuales en el manejo integrado de plagas se orientan hacia el uso de plaguicidas biológicos o biopesticidas como los extractos vegetales...

  11. Identifying Activity

    CERN Document Server

    Lewis, Adrian S

    2009-01-01

    Identification of active constraints in constrained optimization is of interest from both practical and theoretical viewpoints, as it holds the promise of reducing an inequality-constrained problem to an equality-constrained problem, in a neighborhood of a solution. We study this issue in the more general setting of composite nonsmooth minimization, in which the objective is a composition of a smooth vector function c with a lower semicontinuous function h, typically nonsmooth but structured. In this setting, the graph of the generalized gradient of h can often be decomposed into a union (nondisjoint) of simpler subsets. "Identification" amounts to deciding which subsets of the graph are "active" in the criticality conditions at a given solution. We give conditions under which any convergent sequence of approximate critical points finitely identifies the activity. Prominent among these properties is a condition akin to the Mangasarian-Fromovitz constraint qualification, which ensures boundedness of the set of...

  12. Amniotic fluid may act as a transporting pathway for signaling molecules and stem cells during the embryonic development of amniotes.

    Science.gov (United States)

    Tong, Xinglong

    2013-11-01

    Amniotic fluid (AF) is formed at the very early stages of pregnancy, and is present throughout embryonic development of amniotes. It is well-known that AF provides a protective sac around the fetus that allows fetal movement and growth, and prevents mechanical and thermal shock. However, a growing body of evidence has shown that AF contains a number of proteins and peptides, including growth factors and cytokines, which potently affect cellular growth and proliferation. In addition, pluripotent stem cells have recently been identified in AF. Herein, this article reviews the biological properties of AF during embryonic development and speculates that AF may act as a transporting pathway for signaling molecules and stem cells during amniote embryonic development. Defining this novel function of AF is potentially significant for further understanding embryonic development and regenerative medicine, preventing genetic diseases, and developing therapeutic options for human malignancies.

  13. Deciphering the Mechanisms of Developmental Disorders (DMDD: a new programme for phenotyping embryonic lethal mice

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    Timothy Mohun

    2013-05-01

    International efforts to test gene function in the mouse by the systematic knockout of each gene are creating many lines in which embryonic development is compromised. These homozygous lethal mutants represent a potential treasure trove for the biomedical community. Developmental biologists could exploit them in their studies of tissue differentiation and organogenesis; for clinical researchers they offer a powerful resource for investigating the origins of developmental diseases that affect newborns. Here, we outline a new programme of research in the UK aiming to kick-start research with embryonic lethal mouse lines. The ‘Deciphering the Mechanisms of Developmental Disorders’ (DMDD programme has the ambitious goal of identifying all embryonic lethal knockout lines made in the UK over the next 5 years, and will use a combination of comprehensive imaging and transcriptomics to identify abnormalities in embryo structure and development. All data will be made freely available, enabling individual researchers to identify lines relevant to their research. The DMDD programme will coordinate its work with similar international efforts through the umbrella of the International Mouse Phenotyping Consortium [see accompanying Special Article (Adams et al., 2013] and, together, these programmes will provide a novel database for embryonic development, linking gene identity with molecular profiles and morphology phenotypes.

  14. 敲减Wingless/Wnt1基因表达对赤拟谷盗发育中的影响%Knocking-down of Wingless/Wnt1 Influences the Development of Tribolium castaneum

    Institute of Scientific and Technical Information of China (English)

    彭亚男; 李承军; 李斌; 李卓玉

    2012-01-01

    Known as highly conserved during evolution, the Wnt signaling pathway plays a vital role in regulating animal embryonic axis, embryonic differentiation, and deciding cell polarity and maintaining adult dynamic equilibrium. Mutations or deregulations of its components might cause the occurrence of carcinoma. We studied the role of Wingless/Wnt1 during larva-adult development of the red flour beetle, Tribolium castaneum, with dsRNA-mediated Wingless(Wg)/Wntl gene knocked down. The treated late larvae metamorphosed into pupae with drastically increased wing interval and decreased wing width ( P < 0. 01 ). The pursuant pupa-adult eclosion was also severely affected and most of pupae died during this period. The qPCR result showed that the mRNA level of Cadherin-like and Smoothened (Smo) genes were up-regulated greatly, and that of armadillo-2 was slightly higher, after Wingless/Wntl gene was knocked down. We drew the conclusion that Wnt-1 signaling pathway is closely related to the proper wing development and adult metamorphosis of Tribolium. In addition, the elevated expression of Cadherin-like and Armadillo-2 may be accountable for the reduced wing width and enlarged wing interval caused by wg gene silencing, because those alterations can either enhance cell adhesion or change cell morphology. Importantly, the up-regulation of smo gene indicates that Hedgehog signaling pathway may be affected by the RNAi of Wg and involved in the abnormal or lethal phenotypes observed in our experiment.%Wnt信号通路是进化中高度保守的一条信号转导途径,在调控动物的胚胎轴向正常发育、胚胎分化、决定细胞极性、维持成体动态平衡等方面发挥重要作用.该信号通路的异常激活还与肿瘤的发生密切相关.本实验将体外人工合成的Wingless( Wg)/Wnt1基因dsRNA显微注射入赤拟谷盗晚期幼虫体内,研究Wingless/Wnt1蛋白在赤拟谷盗发育过程中发挥的作用.实验结果显示,注射Wingless(wg)/Wnt1基

  15. Ojo blanco (obl : un gene marcador del grupo de ligamiento IV en Tribolium castaneum Herbst Ojo blanco (obl: un gene marcador del grupo de ligamiento IV en Tribolium castaneum Herbst

    Directory of Open Access Journals (Sweden)

    Nuñez Fernando

    1988-06-01

    Full Text Available

    Some genetic aspects of a spontaneous mutation in T. castaneum, discovered in the Department of Biology of National University of Colombia (Bogota, were studied. The mutant gene christened "ojo blanco" (obl, is recessive autosomic, of excellent viability, complete penetrance and variable expression. The gene is phenotipically characterized by a depigmentation of the compound eyes and can be observed from the larval stage. Linkage tests with marker mutants for different groups were carried out. The marker "sooty", of the linkage group IV, gave positive results. The distance was estimated from 2843 descendants obtained from backcrosses in repulsion plase and 1630 in coupling phase. This distance was worked out as 15.64 ± 0.92 units between obl ands. It was not possible to determine if obl is on the left or the right hand side of s. Nor discrepancies between sexes, neither between phases were found in the recombination frequencies for the region between the two loci of chromosome IV.

    Se estudiaron algunos aspectos genéticos de una mutación espontanea en T. casteneum, descubierta en el Departamento de Biología de la Universidad Nacional de Colombia (Bogotá. EI gene mutante llamado "ojo blanco" (obl es autosómico recesivo, de excelente viabilidad, penetrancia completa y expresividad variable. Fenotípicamente se caracteriza por una despigmentación de los ojos compuestos; puede observarse desde el estado de larva. Se le hicieron pruebas de ligamiento con marcadores para diferentes grupos. Se obtuvieron resultados positivos con el marcador "sooty" del grupo de ligamiento IV. La  distancia se calcu1ó a partir de 2.843 descendientes de retrocruzas en posición de repulsión y 1.630 descendientes de retrocruzas en posición de acoplamiento. Esta distancia fue de 15.ó4 ± 0.92 unidades entre obl ys. No se pudo determinar si obl esta a la izquierda 0 a la

  16. Zebrafish Caudal Haematopoietic Embryonic Stromal Tissue (CHEST) Cells Support Haematopoiesis

    Science.gov (United States)

    Wolf, Anja; Aggio, Julian; Campbell, Clyde; Wright, Francis; Marquez, Gabriel; Traver, David; Stachura, David L.

    2017-01-01

    Haematopoiesis is an essential process in early vertebrate development that occurs in different distinct spatial locations in the embryo that shift over time. These different sites have distinct functions: in some anatomical locations specific hematopoietic stem and progenitor cells (HSPCs) are generated de novo. In others, HSPCs expand. HSPCs differentiate and renew in other locations, ensuring homeostatic maintenance. These niches primarily control haematopoiesis through a combination of cell-to-cell signalling and cytokine secretion that elicit unique biological effects in progenitors. To understand the molecular signals generated by these niches, we report the generation of caudal hematopoietic embryonic stromal tissue (CHEST) cells from 72-hours post fertilization (hpf) caudal hematopoietic tissue (CHT), the site of embryonic HSPC expansion in fish. CHEST cells are a primary cell line with perivascular endothelial properties that expand hematopoietic cells in vitro. Morphological and transcript analysis of these cultures indicates lymphoid, myeloid, and erythroid differentiation, indicating that CHEST cells are a useful tool for identifying molecular signals critical for HSPC proliferation and differentiation in the zebrafish. These findings permit comparison with other temporally and spatially distinct haematopoietic-supportive zebrafish niches, as well as with mammalian haematopoietic-supportive cells to further the understanding of the evolution of the vertebrate hematopoietic system. PMID:28300168

  17. CK2α is essential for embryonic morphogenesis.

    Science.gov (United States)

    Dominguez, Isabel; Degano, Irene R; Chea, Kathleen; Cha, Julie; Toselli, Paul; Seldin, David C

    2011-10-01

    CK2 is a highly conserved serine-threonine kinase involved in biological processes such as embryonic development, circadian rhythms, inflammation, and cancer. Biochemical experiments have implicated CK2 in the control of several cellular processes and in the regulation of signal transduction pathways. Our laboratory is interested in characterizing the cellular, signaling, and molecular mechanisms regulated by CK2 during early embryonic development. For this purpose, animal models, including mice deficient in CK2 genes, are indispensable tools. Using CK2α gene-deficient mice, we have recently shown that CK2α is a critical regulator of mid-gestational morphogenetic processes, as CK2α deficiency results in defects in heart, brain, pharyngeal arch, tail bud, limb bud, and somite formation. Morphogenetic processes depend upon the precise coordination of essential cellular processes in which CK2 has been implicated, such as proliferation and survival. Here, we summarize the overall phenotype found in CK2α (-/- ) mice and describe our initial analysis aimed to identify the cellular processes affected in CK2α mutants.

  18. Optical coherence tomography for embryonic imaging: a review

    Science.gov (United States)

    Raghunathan, Raksha; Singh, Manmohan; Dickinson, Mary E.; Larin, Kirill V.

    2016-05-01

    Embryogenesis is a highly complex and dynamic process, and its visualization is crucial for understanding basic physiological processes during development and for identifying and assessing possible defects, malformations, and diseases. While traditional imaging modalities, such as ultrasound biomicroscopy, micro-magnetic resonance imaging, and micro-computed tomography, have long been adapted for embryonic imaging, these techniques generally have limitations in their speed, spatial resolution, and contrast to capture processes such as cardiodynamics during embryogenesis. Optical coherence tomography (OCT) is a noninvasive imaging modality with micrometer-scale spatial resolution and imaging depth up to a few millimeters in tissue. OCT has bridged the gap between ultrahigh resolution imaging techniques with limited imaging depth like confocal microscopy and modalities, such as ultrasound sonography, which have deeper penetration but poorer spatial resolution. Moreover, the noninvasive nature of OCT has enabled live imaging of embryos without any external contrast agents. We review how OCT has been utilized to study developing embryos and also discuss advances in techniques used in conjunction with OCT to understand embryonic development.

  19. Embryonic and neonatal mortality from salmonellosis in captive bred raptors.

    Science.gov (United States)

    Battisti, A; Di Guardo, G; Agrimi, U; Bozzano, A I

    1998-01-01

    In a captive breeding center near Rome (Italy), cases of embryonic and neonatal death were recorded during the breeding seasons in the European eagle owl (Bubo bubo), peregrine falcon (Falco peregrinus), buzzard (Buteo buteo), and lanner falcon. (Falco biarmicus). Salmonella havana and S. virchow were isolated. Three pulli, clinically infected with S. havana, were successfully treated with enrofloxacin. From two groups of healthy 3- to 4-wk-old eagle owls, Salmonella sp. group 61 (61:r:-) and S. havana were collected. A strain of S. paratyphi B was detected in a pharyngeal swab and a fecal sample from an adult female goshawk (Accipiter gentilis), affected with pharyngeal trichomoniasis. A S. hadar strain was collected from a healthy 1-yr-old female eagle owl and S. livingstone was isolated from a 1-mo-old female peregrine, dead of an acute respiratory syndrome. Lesions of fibrinous polyserositis and multivisceral congestion were observed. From frozen 1-day-old chicks, on which adult and young raptors were fed, S. havana and S. livingstone isolates with similar biochemical and drug susceptibility patterns to those isolated from raptors were identified. A surveillance program on infectious diseases reduced embryonic and neonatal death rates in the following breeding seasons.

  20. SETD7 Regulates the Differentiation of Human Embryonic Stem Cells.

    Science.gov (United States)

    Castaño, Julio; Morera, Cristina; Sesé, Borja; Boue, Stephanie; Bonet-Costa, Carles; Martí, Merce; Roque, Alicia; Jordan, Albert; Barrero, Maria J

    2016-01-01

    The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation.

  1. Tribolium castaneum immune defense genes are differentially expressed in response to Bacillus thuringiensis toxins sharing common receptor molecules and exhibiting disparate toxicity.

    Science.gov (United States)

    Contreras, Estefanía; Benito-Jardón, María; López-Galiano, M José; Real, M Dolores; Rausell, Carolina

    2015-06-01

    In Tribolium castaneum larvae we have demonstrated by RNA interference knockdown that the Bacillus thuringiensis Cry3Ba toxin receptors Cadherin-like and Sodium solute symporter proteins are also functional receptors of the less active Cry3Aa toxin. Differences in susceptibility to B. thuringiensis infection might not only rely on toxin-receptor interaction but also on host defense mechanisms. We compared the expression of the immune related genes encoding Apolipophorin-III and two antimicrobial peptides, Defensin3 and Defensin2 after B. thuringiensis challenge. All three genes were up-regulated following Cry3Ba spore-crystal intoxication whereas only Defensins gene expression was induced upon Cry3Aa spore-crystal treatment, evidencing a possible association between host immune response and larval susceptibility to B. thuringiensis. We assessed the antimicrobial activity spectra of T. castaneum defensins peptide fragments and found that a peptide fragment of Defensin3 was effective against the human microbial pathogens, Escherichia coli, Staphylococcus aureus and Candida albicans, being S. aureus the most susceptible one.

  2. The life cycle of Gregarina cuneata in the midgut of Tribolium castaneum and the effects of parasitism on the development of insects.

    Science.gov (United States)

    Gigliolli, A A S; Julio, A H F; Conte, H

    2016-04-01

    Tribolium castaneum Herbst 1797 (Coleoptera: Tenebrionidae), an important pest of stored grains and byproducts, is naturally infected by Gregarina cuneata Stein 1848 (Apicomplexa: Gregarinidae). Changes in the life cycle of insects caused by the parasite development in the midgut were studied. Trophozoites, gamonts (solitary and associated), and gametocysts were present in the midgut of the insects. In young trophozoites, the apical region differentiated into an epimerite that firmly attached the parasite to the host epithelial cells. With maturation, trophozoites developed in gamonts that were associated with the initiation of sexual reproduction in the cell cycle, culminating in the formation of the spherical gametocyst. Morpho-functional analyses indicated that gregarines absorb nutrients from infected cells and can occlude the midgut as they develop. Consequently, nutritional depletion may interfere with the host's physiology, causing decreased growth, delayed development, and high mortality rates of the parasitized insects. These results suggest G. cuneata could be an important biological agent for controlling T. castaneum in integrated pest management programs.

  3. Toxicity and repellency of Hoslundia opposita Vahl (Lamiaceae) leaves' essential oil against rust-red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae).

    Science.gov (United States)

    Babarinde, Samuel Adelani; Akinyemi, Adeyemi Oluseye; Usman, Lamidi Ajao; Odewole, Adeola Foluke; Sangodele, Abraham Opeola; Iyiola, Oluwaseun Olasupo; Olalere, Oluwatoyin Deborah

    2014-01-01

    The insecticidal properties of Hoslundia opposita Vahl (Lamiaceae) leaves' essential oil (EO) against Tribolium castaneum were investigated using contact toxicity and repellency bioassays. Mortality progressed with exposure period and ranged from 61.13% observed at 24 h after treatment (HAT) to 88.86% at 168 HAT. The LT50 (lethal time for 50% of treated adults) of H. opposita EO against T. castaneum was 10.42 h. Application of EO at 20-30 μL/30 cm(2) caused significantly (P < 0.05) higher percentage repellency than what was observed in control, 10 and 15 μL/30 cm(2) at 1 and 3 HAT, with significant repellency at 24 HAT regardless of dosage. Repellency class increased with EO dosage, with class V observed at 30 μL/30 cm(2), regardless of exposure duration. The RD50 (repellency dose for 50% of treated adults) of 15.88 and 13.37 μL/30 cm(2) for 1 and 2 HAT, respectively, was significantly higher than 0.09 μL/30 cm(2) at 24 HAT.

  4. Gene function in early mouse embryonic stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Campbell Pearl A

    2007-03-01

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

  5. Isolation and differentiation of embryonic stem cells from BALB/c mouse

    Institute of Scientific and Technical Information of China (English)

    Wei GONG; Zhuo-Jing LUO; Hua HAN; Hong-Yan QIN; You-Biao CHU; Xue-Yu HU; Li-Feng LAN

    2006-01-01

    Objective To invest the efficient method which can culture and induce embryonic stem cells to neurocyte in vitro. Methods Isolate the blastula o f 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass (inner cell mass, ICM) which were isolated by mechanical method on the mouse embryonic fibroblaste cell (MEF) feeder layer or 0.1% gelatin coated dishes. The stem cells were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunochemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 (stage specific embryonic antigen 1 ). Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated cells presented the characters of ESCs. Then the isolated cells were able to differentiate into neurocytes in vitro. Conclusion Mouse embryonic stem cells isolation, culture and differentiation system has been established.

  6. Complex adaptive responses during antagonistic coevolution between Tribolium castaneum and its natural parasite Nosema whitei revealed by multiple fitness components

    Directory of Open Access Journals (Sweden)

    Bérénos Camillo

    2012-01-01

    Full Text Available Abstract Background Host-parasite coevolution can lead to local adaptation of either parasite or host if there is specificity (GxG interactions and asymmetric evolutionary potential between host and parasite. This has been demonstrated both experimentally and in field studies, but a substantial proportion of studies fail to detect such clear-cut patterns. One explanation for this is that adaptation can be masked by counter-adaptation by the antagonist. Additionally, genetic architecture underlying the interaction is often highly complex thus preventing specific adaptive responses. Here, we have employed a reciprocal cross-infection experiment to unravel the adaptive responses of two components of fitness affecting both parties with different complexities of the underlying genetic architecture (i.e. mortality and spore load. Furthermore, our experimental coevolution of hosts (Tribolium castaneum and parasites (Nosema whitei included paired replicates of naive hosts from identical genetic backgrounds to allow separation between host- and parasite-specific responses. Results In hosts, coevolution led to higher resistance and altered resistance profiles compared to paired control lines. Host genotype × parasite genotype interactions (GH × GP were observed for spore load (the trait of lower genetic complexity, but not for mortality. Overall parasite performance correlated with resistance of its matching host coevolution background reflecting a directional and unspecific response to strength of selection during coevolution. Despite high selective pressures exerted by the obligatory killing parasite, and host- and parasite-specific mortality profiles, no general pattern of local adaptation was observed, but one case of parasite maladaptation was consistently observed on both coevolved and control host populations. In addition, the use of replicate control host populations in the assay revealed one case of host maladaptation and one case of parasite

  7. Sequence characterization of a human embryonic craniofacial cDNA library

    Energy Technology Data Exchange (ETDEWEB)

    Padanilam, B.J.; Barsel, S.; Solursh, M. [and others

    1994-09-01

    Broad-based sequencing approaches for the characterization of human cDNA libraries have proven successful in identifying large numbers of novel genes of specific tissue or developmental stages. To pursue our interests in human craniofacial development, stages. To pursue our interests in human craniofacial development, we have made use of both subtracted and unsubtracted cDNA libraries constructed from embryonic craniofacial tissue obtained from pooled samples at 42-54 days gestation. Single-pass sequencing was carried out using an ABI automated sequencer and T3 or T7 primers. Sequences were characterized using BLAST and GRAIL, and the identified homologous sequences grouped according to gene class and family. Four genes have been mapped using repeat sequence elements identified in the clones. Using primers developed from sequence data, other genes are being mapped using a panel of somatic cell hybrids. To date, a total of 786 sequences have been returned with 35% identifying no homologies, and 35% with strong homologies to previously identified genes. A number of genes previously identified to play a role in human embryonic development have been returned from the sequence comparisons providing evidence that the library is representative of this tissue and stage of development. Previous characterization of the library has also identified a number of novel embryonically expressed human homeobox genes. Genes felt to be of special relevance based on their homology to characterized genes known to play a role in development or that are members of novel classes but with high scores on GRAIL searches are being characterized using whole mount in situ hybridization with mouse embryos. Characterization of the library with respect to chromosomal mapping, gene types and make-up, and embryonic expression patterns will be presented.

  8. Mechanically patterning the embryonic airway epithelium

    Science.gov (United States)

    Varner, Victor D.; Gleghorn, Jason P.; Miller, Erin; Radisky, Derek C.; Nelson, Celeste M.

    2015-01-01

    Collections of cells must be patterned spatially during embryonic development to generate the intricate architectures of mature tissues. In several cases, including the formation of the branched airways of the lung, reciprocal signaling between an epithelium and its surrounding mesenchyme helps generate these spatial patterns. Several molecular signals are thought to interact via reaction-diffusion kinetics to create distinct biochemical patterns, which act as molecular precursors to actual, physical patterns of biological structure and function. Here, however, we show that purely physical mechanisms can drive spatial patterning within embryonic epithelia. Specifically, we find that a growth-induced physical instability defines the relative locations of branches within the developing murine airway epithelium in the absence of mesenchyme. The dominant wavelength of this instability determines the branching pattern and is controlled by epithelial growth rates. These data suggest that physical mechanisms can create the biological patterns that underlie tissue morphogenesis in the embryo. PMID:26170292

  9. Progress with nonhuman primate embryonic stem cells.

    Science.gov (United States)

    Wolf, Don P; Kuo, Hung-Chih; Pau, K-Y Francis; Lester, Linda

    2004-12-01

    Embryonic stem cells hold potential in the fields of regenerative medicine, developmental biology, tissue regeneration, disease pathogenicity, and drug discovery. Embryonic stem (ES) cell lines are now available in primates, including man, rhesus, and cynomologous monkeys. Monkey ES cells serve as invaluable clinically relevant models for studies that can't be conducted in humans because of practical or ethical limitations, or in rodents because of differences in physiology and anatomy. Here, we review the current status of nonhuman primate research with ES cells, beginning with a description of their isolation, characterization, and availability. Substantial limitations still plague the use of primate ES cells, such as their required growth on feeder layers, poor cloning efficiency, and restricted availability. The ability to produce homogenous populations of both undifferentiated as well as differentiated phenotypes is an important challenge, and genetic approaches to achieving these objectives are discussed. Finally, safety, efficiency, and feasibility issues relating to the transplantation of ES-derived cells are considered.

  10. Cytokine signalling in embryonic stem cells

    DEFF Research Database (Denmark)

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

    2006-01-01

    Cytokines play a central role in maintaining self-renewal in mouse embryonic stem (ES) cells through a member of the interleukin-6 type cytokine family termed leukemia inhibitory factor (LIF). LIF activates the JAK-STAT3 pathway through the class I cytokine receptor gp130, which forms a trimeric...... pathways seem to converge on c-myc as a common target to promote self-renewal. Whereas LIF does not seem to stimulate self-renewal in human embryonic stem cells it cannot be excluded that other cytokines are involved. The pleiotropic actions of the increasing number of cytokines and receptors signalling...... via JAKs, STATs and SOCS exhibit considerable redundancy, compensation and plasticity in stem cells in accordance with the view that stem cells are governed by quantitative variations in strength and duration of signalling events known from other cell types rather than qualitatively different stem...

  11. 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.

  12. Embryonic stem cell factors and pancreatic cancer.

    Science.gov (United States)

    Herreros-Villanueva, Marta; Bujanda, Luis; Billadeau, Daniel D; Zhang, Jin-San

    2014-03-07

    Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic tumor, is a highly aggressive human cancer with the lowest five-year survival rate of any human maligancy primarily due to its early- metastasis and lack of response to chemotherapy and radiation. Recent research suggests that PDAC cells comprise a hierarchy of tumor cells that develop around a population of cancer stem cells (CSCs), a small and distinct population of cancer cells that mediates tumoregenesis, metastasis and resistance to standard treatments. Thus, CSCs could be a target for more effective treatment options. Interestingly, pancreatic CSCs are subject to regulation by some of key embryonic stem cell (ESC) transctiption factors abberently expressed in PDAC, such as SOX2, OCT4 and NANOG. ESC transcription factors are important DNA-binding proteins present in both embryonic and adult somatic cells. The critical role of these factors in reprogramming processes makes them essential not only for embryonic development but also tumorigenesis. Here we provide an overview of stem cell transcription factors, particularly SOX2, OCT4, and NANOG, on their expression and function in pancreatic cancer. In contrast to embryonic stem cells, in which OCT4 and SOX2 are tightly regulated and physically interact to regulate a wide spectrum of target genes, de novo SOX2 expression alone in pancreatic cancer cells is sufficient to promote self-renewal, de-differentiation and imparting stemness characteristics via impacting specific cell cycle regulatory genes and epithelial-mesnechymal transtion driver genes. Thus, targeting ESC factors, particularly SOX2, could be a worthy strategy for pancreatic cancer therapy.

  13. Directed hepatic differentiation from embryonic stem cells

    OpenAIRE

    Chen, Xuesong; Zeng, Fanyi

    2011-01-01

    The liver is the largest internal organ in mammals, and is important for the maintenance of normal physiological functions of other tissues and organs. Hepatitis, cirrhosis, liver cancer and other chronic liver diseases are serious threats to human health, and these problems are compounded by a scarcity of liver donors for transplantation therapies. Directed differentiation of embryonic stem cells to liver cells is a promising strategy for obtaining hepatocytes that can be used for cell trans...

  14. Embryonic stem cell differentiation: A chromatin perspective

    OpenAIRE

    Rasmussen Theodore P

    2003-01-01

    Abstract Embryonic stem (ES) cells hold immense promise for the treatment of human degenerative disease. Because ES cells are pluripotent, they can be directed to differentiate into a number of alternative cell-types with potential therapeutic value. Such attempts at "rationally-directed ES cell differentiation" constitute attempts to recapitulate aspects of normal development in vitro. All differentiated cells retain identical DNA content, yet gene expression varies widely from cell-type to ...

  15. Hedgehog Signalling in the Embryonic Mouse Thymus

    OpenAIRE

    Barbarulo, Alessandro; Lau, Ching-In; Mengrelis, Konstantinos; Ross, Susan; Solanki, Anisha; Saldaña, José Ignacio; Crompton, Tessa

    2016-01-01

    T cells develop in the thymus, which provides an essential environment for T cell fate\\ud specification, and for the differentiation of multipotent progenitor cells into major histocompatibility\\ud complex (MHC)-restricted, non-autoreactive T cells. Here we review the role of the Hedgehog\\ud signalling pathway in T cell development, thymic epithelial cell (TEC) development, and\\ud thymocyte–TEC cross-talk in the embryonic mouse thymus during the last week of gestation.\\ud

  16. Current Progress with Primate Embryonic Stem Cells

    OpenAIRE

    Byrne, James A.; Mitalipov, Shoukhrat M.; Wolf, Don P

    2006-01-01

    Embryonic stem cells (ESCs) can proliferate indefinitely, maintain an undifferentiated pluripotent state and differentiate into any cell type. Differentiation of ESCs into various specific cell-types may be able to cure or alleviate the symptoms of various degenerative diseases. Unresolved issues regarding maintaining function, possible apoptosis and tumor formation in vivo mean a prudent approach should be taken towards advancing ESCs into human clinical trials. Rhesus macaques provide the i...

  17. Embryonic growth and mobilization of energy and material during incubation in the checkered keelback snake, Xenochrophis piscator.

    Science.gov (United States)

    Lu, Hong-Liang; Hu, Rui-Bin; Ji, Xiang

    2009-02-01

    We collected 20 checkered keelback snakes (Xenochrophis piscator) to study embryonic growth and mobilization of energy and material during incubation. Females laid eggs between late May and late June. The eggs were incubated at 27 degrees C (+/-0.3). One egg from each clutch was dissected at five-day intervals starting at oviposition. The mean incubation length at 27 degrees C was 48.9 days. We identified three phases of embryonic growth or yolk depletion in X. piscator. Phase 1, between oviposition and Day 20, was one of minimal transfer of energy and material from yolk to embryo. Phase 2, between Day 20 and Day 39-40, was characterized by increasingly rapid embryonic growth or yolk depletion. Phase 3, between Day 39-40 and hatching, was characterized by reduced embryonic growth or yolk depletion. Approximately 71% of dry mass, 53% of non-polar lipids and 66% of energy were transferred from the egg contents to the hatchling during incubation. Our data confirm that oviposition is not timed to coincide with the onset of rapid embryonic growth in oviparous squamate reptiles. The greater conversion efficiencies of energy and material from egg to hatchling in snakes can be attributed to their lower energetic costs of embryonic development and greater residual yolk sizes.

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

    Institute of Scientific and Technical Information of China (English)

    WANG Weimin; Khalid ABBAS; YAN Ansheng

    2006-01-01

    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℃, 6.7±0.4 mg/L and 7.4±0.2, 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 ℃, the ontogenesis spent about33 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.

  19. Embryonic mortality in buffalo naturally mated

    Directory of Open Access Journals (Sweden)

    G. Campanile

    2010-02-01

    Full Text Available The aim of this work was to evaluate the incidence of embryonic mortality in three different period of year in buffaloes naturally mated. The trial was carried out in a buffalo farm located in Caserta province between 2000-2006. In this period were registered natural insemination on 200 buffaloes. Pregnancy diagnosis was carried out on Day 30, confirmed on Day 45 and every 15th days until 90 days after natural mating. Buffaloes that were pregnant on Day 30 but not on Day 45 or Day 90 were considered to have undergone embryonic (EM or fetal mortality (FM respectively. EM and FM were 8.8% and 13.4% respectively throughout the experimental period. A high incidence (P<0.01 of FM was found in the transitional period (December-March than in other months of the year. The incidence of embryonic mortality was significantly (P<0.01 higher between 28-60 days of gestation and lower after 71 day of gestation. The higher fetal mortality found in this study could be due the lower serum levels of progesterone normally found in transitional period in buffalo cows.

  20. [Heart tissue from embryonic stem cells].

    Science.gov (United States)

    Zimmermann, W-H

    2008-09-01

    Embryonic stem cells can give rise to all somatic cells, making them an attractive cell source for tissue engineering applications. The propensity of cells to form tissue-like structures in a culture dish has been well documented. We and others made use of this intrinsic property to generate bioartificial heart muscle. First proof-of-concept studies involved immature heart cells mainly from fetal chicken, neonatal rats and mice. They eventually provided evidence that force-generating heart muscle can be engineered in vitro. Recently, the focus shifted to the application of stem cells to eventually enable the generation of human heart muscle and reach following long-term goals: (1) development of a simplified in vitro model of heart muscle development; (2) generation of a human test-bed for drug screening and development; (3) allocation of surrogate heart tissue to myocardial repair applications. This overview will provide the background for cell-based myocardial repair, introduce the main myocardial tissue engineering concepts, discuss the use of embryonic and non-embryonic stem cells, and lays out the potential direct and indirect therapeutic use of human tissue engineered myocardium.

  1. Random mitotic activities across human embryonic stem cell colonies.

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Q.; Duggan, R.; Dasa, S.; Li, F.; Chen, L. (Biosciences Division)

    2010-08-01

    A systemic and quantitative study was performed to examine whether different levels of mitotic activities, assessed by the percentage of S-phase cells at any given time point, existed at different physical regions of human embryonic stem (hES) cell colonies at 2, 4, 6 days after cell passaging. Mitotically active cells were identified by the positive incorporation of 5-bromo-2-deoxyuridine (BrdU) within their newly synthesized DNA. Our data indicated that mitotically active cells were often distributed as clusters randomly across the colonies within the examined growth period, presumably resulting from local deposition of newly divided cells. This latter notion was further demonstrated by the confined growth of enhanced green florescence protein (EGFP) expressing cells amongst non-GFP expressing cells. Furthermore, the overall percentage of mitotically active cells remained constantly at about 50% throughout the 6-day culture period, indicating mitotic activities of hES cell cultures were time-independent under current growth conditions.

  2. Biobanking human embryonic stem cell lines: policy, ethics and efficiency.

    Science.gov (United States)

    Holm, Søren

    2015-12-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy discussions relating to their establishment or maintenance. It is argued (1) that 'ethical arguments' are often more important in the establishment phase and 'efficiency arguments' more important in the maintenance phase, and (2) that arguments relating to the interests of embryo and gamete donors are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being ignored in the policy making process.

  3. 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.

  4. Color photographic index of fall Chinook salmon embryonic development and accumulated thermal units.

    Directory of Open Access Journals (Sweden)

    James W Boyd

    Full Text Available BACKGROUND: Knowledge of the relationship between accumulated thermal units and developmental stages of Chinook salmon embryos can be used to determine the approximate date of egg fertilization in natural redds, thus providing insight into oviposition timing of wild salmonids. However, few studies have documented time to different developmental stages of embryonic Chinook salmon and no reference color photographs are available. The objectives of this study were to construct an index relating developmental stages of hatchery-reared fall Chinook salmon embryos to time and temperature (e.g., degree days and provide high-quality color photographs of each identified developmental stage. METHODOLOGY/PRINCIPAL FINDINGS: Fall Chinook salmon eggs were fertilized in a hatchery environment and sampled approximately every 72 h post-fertilization until 50% hatch. Known embryonic developmental features described for sockeye salmon were used to describe development of Chinook salmon embryos. A thermal sums model was used to describe the relationship between embryonic development rate and water temperature. Mean water temperature was 8.0 degrees C (range; 3.9-11.7 degrees C during the study period. Nineteen stages of embryonic development were identified for fall Chinook salmon; two stages in the cleavage phase, one stage in the gastrulation phase, and sixteen stages in the organogenesis phase. The thermal sums model used in this study provided similar estimates of fall Chinook salmon embryonic development rate in water temperatures varying from 3.9-11.7 degrees C (mean=8 degrees C to those from several other studies rearing embryos in constant 8 degrees C water temperature. CONCLUSIONS/SIGNIFICANCE: The developmental index provides a reasonable description of timing to known developmental stages of Chinook salmon embryos and was useful in determining developmental stages of wild fall Chinook salmon embryos excavated from redds in the Columbia River. This index

  5. 甲酸乙酯对面粉中赤拟谷盗的熏蒸效果%The fumigation effect of Ethyl formate against Triboloum castaneum in flour.

    Institute of Scientific and Technical Information of China (English)

    游源浅; 詹开瑞; 张晓燕; 陈艳

    2011-01-01

    The purpose of this research is to evaluate the fumigation activity of Ethyl formate against Tribolium castaneum. The Lct99 of T. castaneum was calculated by detecting concentration of Ethyl formate during exposure. The results indicate that Ethyl formate was effective in disinfecting all life stage of T. castaneum in flour, and the sensitivities of life stages of T. castaneum to Ethyl formate, which arranged from strongest to weakest, were egg, low instar larva, adult, high instar larva and pupa. The Lct99 for disinfecting all life stages of T. castaneum in 6 and 12 h at 23℃ were 448.42 and 599.34rag.h/L, respectively.%本文研究了甲酸乙酯对面粉中赤拟谷盗不同虫态的熏蒸作用。通过对熏蒸期间熏蒸剂浓度检测,计算浓度时间乘积(Cr)计算不同时间下甲酸乙酯对赤拟谷盗各虫态达到99%控制的处理指标(Lct99)。结果表明甲酸乙酯熏蒸能有效防治面粉中的赤拟谷盗各虫态,赤拟谷盗不同虫态对甲酸乙酯的敏感性由强到弱依次为:卵〉低龄幼虫〉成虫〉高龄幼虫〉蛹。在23℃条件下处理6h和12h对面粉中赤拟谷盗的厶‰分别为448.42和599.34mg·h/L

  6. Pigment pattern evolution by differential deployment of neural crest and post-embryonic melanophore lineages in Danio fishes.

    Science.gov (United States)

    Quigley, Ian K; Turner, Jessica M; Nuckels, Richard J; Manuel, Joan L; Budi, Erine H; MacDonald, Erin L; Parichy, David M

    2004-12-01

    Latent precursors or stem cells of neural crest origin are present in a variety of post-embryonic tissues. Although these cells are of biomedical interest for roles in human health and disease, their potential evolutionary significance has been underappreciated. As a first step towards elucidating the contributions of such cells to the evolution of vertebrate form, we investigated the relative roles of neural crest cells and post-embryonic latent precursors during the evolutionary diversification of adult pigment patterns in Danio fishes. These pigment patterns result from the numbers and arrangements of embryonic melanophores that are derived from embryonic neural crest cells, as well as from post-embryonic metamorphic melanophores that are derived from latent precursors of presumptive neural crest origin. In the zebrafish D. rerio, a pattern of melanophore stripes arises during the larval-to-adult transformation by the recruitment of metamorphic melanophores from latent precursors. Using a comparative approach in the context of new phylogenetic data, we show that adult pigment patterns in five additional species also arise from metamorphic melanophores, identifying this as an ancestral mode of adult pigment pattern development. By contrast, superficially similar adult stripes of D. nigrofasciatus (a sister species to D. rerio) arise by the reorganization of melanophores that differentiated at embryonic stages, with a diminished contribution from metamorphic melanophores. Genetic mosaic and molecular marker analyses reveal evolutionary changes that are extrinsic to D. nigrofasciatus melanophore lineages, including a dramatic reduction of metamorphic melanophore precursors. Finally, interspecific complementation tests identify a candidate genetic pathway for contributing to the evolutionary reduction in metamorphic melanophores and the increased contribution of early larval melanophores to D. nigrofasciatus adult pigment pattern development. These results

  7. Identification, Characterization, and Expression of P450 Gene Encoding CYP6BQ13v2 from the Red Flour Beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

    Institute of Scientific and Technical Information of China (English)

    XU Yong-qiang; WANG Jin-jun; JIANG Hong-bo; DOU Wei; TANG Pei-an; AN Feng-ming

    2009-01-01

    An allele of CYP6BQ13, named CYP6BQ13v2 (GenBank accession no. FJ209361), was isolated from the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) by RT-PCR. The cDNA sequence of CYP6BQ13v2, 1 563 bp in length, contains an open reading frame of 1 554 nucleotides encoding a putative protein of 518 amino acid residues with a predicted molecular weight of 59.92 kDa and a theoretical pI of 7.60. The putative protein contains the classic berne-binding sequence motif F××G×××C×G (residues 456-465) conserved among all P450 enzymes as well as other characteristic motifs of all cytochrome P450s. It shares 98% identity with the previously published sequence of CYP6BQ13 (GenBank accession no. XP_967146) from the T. castaneum genome project. Phylogenetic analysis of amino acid sequences from members of various P450 families indicated that there was closer phylogenetic relationship of CYP6BQ13v2 with CYP302A1 and CYP307A1 mediating synthesis of the insect molting hormone, distant relationship with CYP6B1 metabolizing plant allelochemicals, CYP6D1 linking to pyrethroid resistance and other members of CYP6 family. Expression test of the gene in the adults and immature stages of T. castaneum by quantitative real-time PCR revealed that CYP6BQ13v2 is expressed in all life stages investigated. The mRNA expression level in 1st instar larvae was 14.9-and 3.86-fold higher than those in pupae and adults, respectively. The CYP6BQ13v2 expression levels appeared in the order of 1st instar larvae, followed by 4th instar larvae, 7th instar larvae, adult, and pupae from high to low. The more bioinformation of CYP6BQ13v2 was also analyzed.

  8. [Microglial cells and development of the embryonic central nervous system].

    Science.gov (United States)

    Legendre, Pascal; Le Corronc, Hervé

    2014-02-01

    Microglia cells are the macrophages of the central nervous system with a crucial function in the homeostasis of the adult brain. However, recent studies showed that microglial cells may also have important functions during early embryonic central nervous system development. In this review we summarize recent works on the extra embryonic origin of microglia, their progenitor niche, the pattern of their invasion of the embryonic central nervous system and on interactions between embryonic microglia and their local environment during invasion. We describe microglial functions during development of embryonic neuronal networks, including their roles in neurogenesis, in angiogenesis and developmental cell death. These recent discoveries open a new field of research on the functions of neural-microglial interactions during the development of the embryonic central nervous system.

  9. Immunocytochemical analysis of intermediate filaments in embryonic heart cells with monoclonal antibodies to desmin.

    Science.gov (United States)

    Danto, S I; Fischman, D A

    1984-06-01

    Monoclonal antibodies ( McAbs ) have been generated against a preparation of intermediate filament proteins (IFP) from adult chicken gizzard. Two antibodies, D3 and D76 , have been characterized in detail. They bind specifically to desmin but recognize different epitopes. In the adult chicken, both McAbs produced equivalent immunofluorescent staining patterns, reacting in frozen sections with all forms of muscle tissue, including vascular smooth muscle, but with no other tissue types. In isolated skeletal myofibrils and in longitudinal frozen sections of cardiac and skeletal muscle, desmin was detected with both McAbs at the Z-band and in longitudinally-oriented filament bundles between myofibrils. In contrast to these results in the adult, the intermediate filaments (IF) of embryonic cardiac myocytes in primary cultures were decorated only with McAb D3, whereas McAb D76 was completely unreactive with these cells. Similarly, frozen sections through the heart at early stages of embryonic chick development (Hamburger-Hamilton stages 17-18) revealed regions of myocytes, identified by double immunofluorescence with myosin-specific McAbs , that were unstained with McAb D76 even though similar regions were stained by McAb D3. That McAb D76 reacted with desmin in all adult cardiac myocytes but not with all embryonic heart cells indicates that embryonic and adult cardiac IF are immunologically distinct and implies a conversion in IF immunoreactivity during cardiac development.

  10. Imaging features of undifferentiated embryonal sarcoma of the liver: a series of 15 children

    Energy Technology Data Exchange (ETDEWEB)

    Gabor, Flaviu; Franchi-Abella, Stephanie; Pariente, Daniele [Bicetre Hospital, Department of Pediatric Radiology, Le Kremlin-Bicetre (France); Merli, Laura [Bambino Gesu Children' s Hospital, Unit of Hepato-Biliary and Transplant Surgery, Department of Surgery and Transplantation Centre, Rome (Italy); Adamsbaum, Catherine [Bicetre Hospital, Department of Pediatric Radiology, Le Kremlin-Bicetre (France); Paris Sud University, Faculty of Medicine, Le Kremlin-Bicetre (France); Universite Paris-Saclay, LTCI, CNRS, Telecom Paris Tech, Paris (France)

    2016-11-15

    Undifferentiated embryonal sarcoma of the liver is a rare malignant mesenchymal tumour occurring mostly in children ages 6-10 years. The discrepancy between its solid appearance on US and cystic-like appearance on CT has been described. To study the imaging particularities and similarities among our cases of undifferentiated embryonal sarcoma and to report the errors in initial diagnoses. We conducted a retrospective study of 15 children with undifferentiated embryonal sarcoma diagnosed or referred to our hospital during 1997-2015 and analysed the clinical, biological and imaging data. We identified eight boys and seven girls ages 9 months to 14 years. Ten children presented with abdominal pain. Alpha-fetoprotein was slightly increased in one. Initial US and CT had been performed for all, while additional MRI had been done in two children. Initial CT demonstrated a hypoattenuated mass in all. Rupture was seen in five and intratumoural bleeding in seven children. Tumour volumes reduced during neoadjuvant chemotherapy in 10 children. Undifferentiated embryonal sarcoma might be suggested in a non-secreting unifocal tumour with well-defined borders, fluid-filled spaces on US, hypoattenuation and serpiginous vessels on CT, and if there are signs of internal bleeding or rupture on CT or MRI. (orig.)

  11. Expression and function on embryonic development of lissencephaly-1 genes in zebrafish

    Institute of Scientific and Technical Information of China (English)

    Chengfu Sun; Mafei Xu; Zhen Xing; Zhili Wu; Yiping Li; Tsaiping Li; Mujun Zhao

    2009-01-01

    Lissencephaly is a severe disease characterized by brain malformation. The main causative gene of lissencephaly is LIS1. Mutation or deletion of LIS1 leads to prolifer-ation and migration deficiency of neurons in brain devel-opment. However, little is known about its biological function in embryonic development. In this article, we identified the expression patterns of zebrafish LIS1 gene and investigated its function in embryonic development. We demonstrated that zebrafish consisted of two LIS1 genes, LIS1a and LIS1b. Bioinformatics analysis revealed that LIS1 genes were conserved in evolution both in protein sequences and genomic structures. The expression patterns of zebrafish LIS1a and LIS1b showed that both transcripts were ubiquitously expressed at all embryonic developmental stages and in adult tissues examined. At the protein level, the LIS1 products mainly exist in brain tissue and in embryos at early stages as shown by western blotting analysis. The whole-mount immunostaining data showed that LIS1 proteins were distributed all over the embryos from 1-cell stage to 5 day post-fertilization. Knockdown of LIS1 protein expression through morpholino antisense oligonucleotides resulted in many developmental deficiencies in zebrafish, including brain malformation, circulation abnormality, and body curl. Taken together, our study suggested that zebrafish LIS1 plays a very important role in embryonic development.

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

    Science.gov (United States)

    2016-01-01

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

  13. The embryonic development of the malacostracan crustacean Porcellio scaber (Isopoda, Oniscidea).

    Science.gov (United States)

    Wolff, Carsten

    2009-12-01

    To examine the evolution of development and put it into a phylogenetic context, it is important to have, in addition to a model organism like Drosophila, more insights into the huge diversity of arthropod morphologies. In recent years, the malacostracan crustacean Porcellio scaber Latreille, 1804 has become a popular animal for studies in evolutionary and developmental biology, but a detailed and complete description of its embryonic development is still lacking. Therefore, the embryonic development of the woodlouse P. scaber is described in a series of discrete stages easily identified by examination of living animals and the widely used technique of nuclei staining on fixed specimens. It starts with the first cleavage of the zygote and ends with a hatched manca that eventually leaves the mother's brood pouch. Classical methods like normal light microscopy, scanning electron microscopy and fluorescence microscopy are used, in addition to confocal LCM and computer-aided 3D reconstruction in order to visualise important processes during ontogeny. The purpose of these studies is to offer an easy way to define the different degrees of development for future comparative analyses of embryonic development amongst crustaceans in particular, as well as between different arthropod groups. In addition, several aspects of Porcellio embryonic development, such as the mouth formation, limb differentiations and modifications or the formation of the digestive tract, make this species particularly interesting for future studies in evolutionary and developmental biology.

  14. Cell polarity and spindle orientation in the distal epithelium of embryonic lung.

    Science.gov (United States)

    El-Hashash, Ahmed H; Warburton, David

    2011-02-01

    A proper balance between self-renewal and differentiation of lung-specific progenitors at the distal epithelial tips is absolutely required for normal lung morphogenesis. Cell polarity and mitotic spindle orientation play a critical role in the self-renewal/differentiation of epithelial cells and can impact normal physiological processes, including epithelial tissue branching and differentiation. Therefore, understanding the behavior of lung distal epithelial progenitors could identify innovative solutions to restoring normal lung morphogenesis. Yet little is known about cell polarity, spindle orientation, and segregation of cell fate determinant in the embryonic lung epithelium, which contains progenitor cells. Herein, we provide the first evidence that embryonic lung distal epithelium is polarized and highly mitotic with characteristic perpendicular cell divisions. Consistent with these findings, mInsc, LGN, and NuMA polarity proteins, which control spindle orientation, are asymmetrically localized in mitotic distal epithelial progenitors of embryonic lungs. Furthermore, the cell fate determinant Numb is asymmetrically distributed at the apical side of distal epithelial progenitors and segregated to one daughter cell in most mitotic cells. These findings provide evidence for polarity in distal epithelial progenitors of embryonic lungs and provide a framework for future translationally oriented studies in this area.

  15. Microsatellite repeat instability fuels evolution of embryonic enhancers in Hawaiian Drosophila.

    Science.gov (United States)

    Brittain, Andrew; Stroebele, Elizabeth; Erives, Albert

    2014-01-01

    For ∼30 million years, the eggs of Hawaiian Drosophila were laid in ever-changing environments caused by high rates of island formation. The associated diversification of the size and developmental rate of the syncytial fly embryo would have altered morphogenic gradients, thus necessitating frequent evolutionary compensation of transcriptional responses. We investigate the consequences these radiations had on transcriptional enhancers patterning the embryo to see whether their pattern of molecular evolution is different from non-Hawaiian species. We identify and functionally assay in transgenic D. melanogaster the Neurogenic Ectoderm Enhancers from two different Hawaiian Drosophila groups: (i) the picture wing group, and (ii) the modified mouthparts group. We find that the binding sites in this set of well-characterized enhancers are footprinted by diverse microsatellite repeat (MSR) sequences. We further show that Hawaiian embryonic enhancers in general are enriched in MSR relative to both Hawaiian non-embryonic enhancers and non-Hawaiian embryonic enhancers. We propose embryonic enhancers are sensitive to Activator spacing because they often serve as assembly scaffolds for the aggregation of transcription factor activator complexes. Furthermore, as most indels are produced by microsatellite repeat slippage, enhancers from Hawaiian Drosophila lineages, which experience dynamic evolutionary pressures, would become grossly enriched in MSR content.

  16. Expanded CAG repeats in the murine Huntington's disease gene increases neuronal differentiation of embryonic and neural stem cells.

    Science.gov (United States)

    Lorincz, Matthew T; Zawistowski, Virginia A

    2009-01-01

    Huntington's disease is an uncommon autosomal dominant neurodegenerative disorder caused by expanded polyglutamine repeats. Increased neurogenesis was demonstrated recently in Huntington's disease post-mortem samples. In this manuscript, neuronally differentiated embryonic stem cells with expanded CAG repeats in the murine Huntington's disease homologue and neural progenitors isolated from the subventricular zone of an accurate mouse Huntington's disease were examined for increased neurogenesis. Embryonic stem cells with expanded CAG repeats in the murine Huntington's disease homologue were demonstrated to undergo facilitated differentiation first into neural progenitors, then into more mature neurons. Neural progenitor cells isolated from the subventricular zone of a Huntington's disease knock-in animal displayed increased production of neural progenitors and increased neurogenesis. These findings suggested that neuronally differentiating embryonic stem cells with expanded CAG repeats is a reasonable system to identify factors responsible for increased neurogenesis in Huntington's disease. Expression profiling analysis comparing neuronally differentiating embryonic stem cells with expanded CAG repeats to neuronally differentiating embryonic stem cells without expanded CAG repeats identified transcripts involved in development and transcriptional regulation as factors possibly mediating increased neurogenesis in response to expanded CAG repeats.

  17. Undifferentiated embryonic cell transcription factor 1 regulates ESC chromatin organization and gene expression

    DEFF Research Database (Denmark)

    Kooistra, Susanne M; van den Boom, Vincent; Thummer, Rajkumar P;

    2010-01-01

    cell chromatin structure. Using chromatin immunoprecipitation-on-chip analysis, we identified >1,700 UTF1 target genes that significantly overlap with previously identified Nanog, Oct4, Klf-4, c-Myc, and Rex1 targets. Gene expression profiling showed that UTF1 knock down results in increased expression......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...... to dimethyl sulfoxide (DMSO) or after LIF withdrawal and display increased colony formation. UTF1 KD ES cells display extensive chromatin decondensation, reflected by a dramatic increase in nucleosome release on micrococcal nuclease (MNase) treatment and enhanced MNase sensitivity of UTF1 target genes in UTF1...

  18. Relationship between Intrauterine Bacterial Infection and Early Embryonic Developmental Arrest

    Institute of Scientific and Technical Information of China (English)

    Shao-Fei Yan; Xin-Yan Liu; Yun-Fei Cheng; Zhi-Yi Li; Jie Ou; Wei Wang; Feng-Qin Li

    2016-01-01

    Background:Early embryonic developmental arrest is the most commonly understudied adverse outcome of pregnancy.The relevance of intrauterine infection to spontaneous embryonic death is rarely studied and remains unclear.This study aimed to investigate the relationship between intrauterine bacterial infection and early embryonic developmental arrest.Methods:Embryonic chorion tissue and uterine swabs for bacterial detection were obtained from 33 patients who underwent artificial abortion (control group) and from 45 patients who displayed early embryonic developmental arrest (trial group).Results:Intrauterine bacterial infection was discovered in both groups.The infection rate was 24.44% (11/45) in the early embryonic developmental arrest group and 9.09% (3/33) in the artificial abortion group.Classification analysis revealed that the highest detection rate for Micrococcus luteus in the early embryonic developmental arrest group was 13.33% (6/45),and none was detected in the artificial abortion group.M.luteus infection was significantly different between the groups (P < 0.05 as shown by Fisher's exact test).In addition,no correlation was found between intrauterine bacterial infection and history of early embryonic developmental arrest.Conclusions:M.luteus infection is related to early embryonic developmental arrest and might be one of its causative factors.

  19. Primary “Botryoid” Embryonal Rhabdomyosarcoma in Mesentery

    Directory of Open Access Journals (Sweden)

    Kiran AGARWAL

    2011-01-01

    Full Text Available Rhabdomyosarcoma is a soft tissue neoplasm arising from primitive embryonal mesenchyma. Embryonal rhabdomyosarcoma mostly affects children younger than 10 years of age, but it also occurs in adolescents and young adults. Pleomorphic rhabdomyosarcoma is a rare variant that almost always arises in adults older than 45 years of age. Mesentery is a rare site for botyroid embryonal rhabdomyosarcoma and on extensive search we found only one case of a botryoid rhabdomyosarcoma in a child of 2 years. We report a rare case of botyroid embryonal rhabdomyosarcoma occurring in the mesentery of a 30 year old female.

  20. Simulated Microgravity Modulates Differentiation Processes of Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Vaibhav Shinde

    2016-04-01

    Full Text Available Background/Aims: Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of altered gravity on the embryonic development processes we established an in vitro methodology allowing differentiation of mouse embryonic stem cells (mESCs under simulated microgravity within a fast-rotating clinostat (clinorotation and capture of microarray-based gene signatures. Methods: The differentiating mESCs were cultured in a 2D pipette clinostat. The microarray and bioinformatics tools were used to capture genes that are deregulated by simulated microgravity and their impact on developmental biological processes. Results: The data analysis demonstrated that differentiation of mESCs in pipettes for 3 days resultet to early germ layer differentiation and then to the different somatic cell types after further 7 days of differentiation in the Petri dishes. Clinorotation influences differentiation as well as non-differentiation related biological processes like cytoskeleton related 19 genes were modulated. Notably, simulated microgravity deregulated genes Cyr61, Thbs1, Parva, Dhrs3, Jun, Tpm1, Fzd2 and Dll1 are involved in heart morphogenesis as an acute response on day 3. If the stem cells were further cultivated under normal gravity conditions (1 g after clinorotation, the expression of cardiomyocytes specific genes such as Tnnt2, Rbp4, Tnni1, Csrp3, Nppb and Mybpc3 on day 10 was inhibited. This correlated well with a decreasing beating activity of the 10-days old embryoid bodies (EBs. Finally, we captured Gadd45g, Jun, Thbs1, Cyr61and Dll1 genes whose expressions were modulated by simulated microgravity and by real microgravity in various reported studies. Simulated microgravity also deregulated genes belonging to the MAP kinase and focal dhesion signal transduction pathways. Conclusion: One of the most prominent biological processes affected by simulated microgravity was the process of cardiomyogenesis. The

  1. Embryonic senescence and laminopathies in a progeroid zebrafish model.

    Directory of Open Access Journals (Sweden)

    Eriko Koshimizu

    Full Text Available BACKGROUND: Mutations that disrupt the conversion of prelamin A to mature lamin A cause the rare genetic disorder Hutchinson-Gilford progeria syndrome and a group of laminopathies. Our understanding of how A-type lamins function in vivo during early vertebrate development through aging remains limited, and would benefit from a suitable experimental model. The zebrafish has proven to be a tractable model organism for studying both development and aging at the molecular genetic level. Zebrafish show an array of senescence symptoms resembling those in humans, which can be targeted to specific aging pathways conserved in vertebrates. However, no zebrafish models bearing human premature senescence currently exist. PRINCIPAL FINDINGS: We describe the induction of embryonic senescence and laminopathies in zebrafish harboring disturbed expressions of the lamin A gene (LMNA. Impairments in these fish arise in the skin, muscle and adipose tissue, and sometimes in the cartilage. Reduced function of lamin A/C by translational blocking of the LMNA gene induced apoptosis, cell-cycle arrest, and craniofacial abnormalities/cartilage defects. By contrast, induced cryptic splicing of LMNA, which generates the deletion of 8 amino acid residues lamin A (zlamin A-Δ8, showed embryonic senescence and S-phase accumulation/arrest. Interestingly, the abnormal muscle and lipodystrophic phenotypes were common in both cases. Hence, both decrease-of-function of lamin A/C and gain-of-function of aberrant lamin A protein induced laminopathies that are associated with mesenchymal cell lineages during zebrafish early development. Visualization of individual cells expressing zebrafish progerin (zProgerin/zlamin A-Δ37 fused to green fluorescent protein further revealed misshapen nuclear membrane. A farnesyltransferase inhibitor reduced these nuclear abnormalities and significantly prevented embryonic senescence and muscle fiber damage induced by zProgerin. Importantly, the adult

  2. Segmental patterning of the vertebrate embryonic axis.

    Science.gov (United States)

    Dequéant, Mary-Lee; Pourquié, Olivier

    2008-05-01

    The body axis of vertebrates is composed of a serial repetition of similar anatomical modules that are called segments or metameres. This particular mode of organization is especially conspicuous at the level of the periodic arrangement of vertebrae in the spine. The segmental pattern is established during embryogenesis when the somites--the embryonic segments of vertebrates--are rhythmically produced from the paraxial mesoderm. This process involves the segmentation clock, which is a travelling oscillator that interacts with a maturation wave called the wavefront to produce the periodic series of somites. Here, we review our current understanding of the segmentation process in vertebrates.

  3. Real-time cell analysis and heat shock protein gene expression in the TcA Tribolium castaneum cell line in response to environmental stress conditions.

    Science.gov (United States)

    García-Reina, Andrés; Rodríguez-García, María Juliana; Ramis, Guillermo; Galián, José

    2015-12-17

    The rust red flour beetle, Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae), is a pest of stored grain and one of the most studied insect model species. Some of the previous studies involved heat response studies in terms of survival and heat shock protein expression, which are regulated to protect other proteins against environmental stress conditions. In the present study, we characterize the impedance profile with the xCELLigence Real-Time Cell Analyzer and study the effect of increased temperature in cell growth and viability in the cell line BCIRL-TcA-CLG1 (TcA) of T. castaneum. This novel system measures cells behavior in real time and is applied for the first time to insect cells. Additionally, cells are exposed to heat shock, increased salinity, acidic pH and UV-A light with the aim of measuring the expression levels of Hsp27, Hsp68a and Hsp83 genes. Results show a high thermotolerance of TcA in terms of cell growth and viability. This result is likely related to gene expression results in which a significant up-regulation of all studied Hsp genes is observed after one hour of exposure to 40 ºC and UV light. All three genes show similar expression patterns, but Hsp27 seems to be the most affected. The results of this study validate the RTCA method and reveal the utility of insect cell lines, real-time analysis and gene expression studies to better understand the physiological response of insect cells, with potential applications in different fields of biology such as conservation biology and pest management. This article is protected by copyright. All rights reserved.

  4. Two major cuticular proteins are required for assembly of horizontal laminae and vertical pore canals in rigid cuticle of Tribolium castaneum.

    Science.gov (United States)

    Noh, Mi Young; Kramer, Karl J; Muthukrishnan, Subbaratnam; Kanost, Michael R; Beeman, Richard W; Arakane, Yasuyuki

    2014-10-01

    The insect exoskeleton is composed of cuticle primarily formed from structural cuticular proteins (CPs) and the polysaccharide chitin. Two CPs, TcCPR27 and TcCPR18, are major proteins present in the elytron (highly sclerotized and pigmented modified forewing) as well as the pronotum (dorsal sclerite of the prothorax) and ventral abdominal cuticle of the red flour beetle, Tribolium castaneum. Both CPs belong to the CPR family, which includes proteins that have an amino acid sequence motif known as the Rebers & Riddiford (R&R) consensus sequence. Injection of double-stranded RNA (dsRNA) for TcCPR27 and TcCPR18 resulted in insects with shorter, wrinkled, warped and less rigid elytra than those from control insects. To gain a more comprehensive understanding of the roles of CPs in cuticle assembly, we analyzed for the precise localization of TcCPR27 and the ultrastructural architecture of cuticle in TcCPR27- and TcCPR18-deficient elytra. Transmission electron microscopic analysis combined with immunodetection using gold-labeled secondary antibody revealed that TcCPR27 is present in dorsal elytral procuticle both in the horizontal laminae and in vertical pore canals. dsRNA-mediated RNA interference (RNAi) of TcCPR27 resulted in abnormal electron-lucent laminae and pore canals in elytra except for the boundary between these two structures in which electron-dense molecule(s) apparently accumulated. Insects subjected to RNAi for TcCPR18 also had disorganized laminae and pore canals in the procuticle of elytra. Similar ultrastructural defects were also observed in other body wall regions with rigid cuticle such as the thorax and legs of adult T. castaneum. TcCPR27 and TcCPR18 are required for proper formation of the horizontal chitinous laminae and vertical pore canals that are critical for formation and stabilization of rigid adult cuticle.

  5. Human embryonic stem cell derivation and directed differentiation.

    Science.gov (United States)

    Trounson, A

    2005-01-01

    Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-15

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

  7. Beneficial effects of melatonin on in vitro bovine embryonic development are mediated by melatonin receptor 1.

    Science.gov (United States)

    Wang, Feng; Tian, XiuZhi; Zhang, Lu; Gao, Chao; He, ChangJiu; Fu, Yao; Ji, PengYun; Li, Yu; Li, Ning; Liu, GuoShi

    2014-04-01

    In the current study, a fundamental question, that is, the mechanisms related to the beneficial effects of melatonin on mammalian embryonic development, was addressed. To examine the potential beneficial effects of melatonin on bovine embryonic development, different concentrations of melatonin (10(-11), 10(-9), 10(-7), 10(-5), 10(-3) M) were incubated with fertilized embryos. Melatonin in the range of 10(-11) to 10(-5) M significantly promoted embryonic development both in early culture medium (CR1aa +3 mg/mL BSA) and in later culture medium (CR1aa + 6%FBS). The most effective concentrations applied in the current studies were 10(-9) and 10(-7) M. Using quantitative real-time PCR with immunofluorescence and Western blot assays, the expression of melatonin receptor MT1 and MT2 genes was identified in bovine embryos. Further studies indicate that the beneficial effects of melatonin on bovine embryo development were mediated by the MT1 receptor. This is based on the facts that luzindole, a nonselective MT1 and MT2 antagonist, blocked the effect on melatonin-induced embryo development, while 4-P-PDOT, a selective MT2 antagonist, had little effect. Mechanistic explorations uncovered that melatonin application during bovine embryonic development significantly up-regulated the expression of antioxidative (Gpx4, SOD1, bcl-2) and developmentally important genes (SLC2A1, DNMT1A, and DSC2) while down-regulating expression of pro-apoptotic genes (P53, BAX, and Caspase-3). The results obtained from the current studies provide new information regarding the mechanisms by which melatonin promotes bovine embryonic development under both in vitro and in vivo conditions.

  8. Research on Isolation and Clone of Embryonic Stem Cell-Like in Bovine

    Institute of Scientific and Technical Information of China (English)

    AN Li-long; YANG Qi; XIAO Mei; FENG Xiu-Liang; YANG Chun-rong; LEI An-min; GAO Zhi-min; DOU Zhong-ying; QIU Huai

    2002-01-01

    Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary marine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS ,0.1μmol/L Na2SeO3, 0. 1mmol/L β-mercaptoethanol, 1 000ng/ml LIF,10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine), then, we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage marine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0. 125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification, karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.

  9. Mouse TEX14 Is Required for Embryonic Germ Cell Intercellular Bridges but Not Female Fertility1

    Science.gov (United States)

    Greenbaum, Michael P.; Iwamori, Naoki; Agno, Julio E.; Matzuk, Martin M.

    2008-01-01

    A conserved feature of germ cell cytokinesis is the formation of stable intercellular bridges between daughter cells. These intercellular bridges are seen in diverse species from Drosophila melanogaster to Homo sapiens and have been shown to have roles in communication of large numbers of germ cells. In testis expressed gene 14 (Tex14) knockout mice, intercellular bridges do not form during spermatogenesis, and male mice are sterile, demonstrating an essential role for intercellular bridges in postnatal spermatogenesis in mammals. Intercellular bridges also form between dividing germ cells in both male and female embryos. However, little is known about the formation or role of the embryonic intercellular bridges in mammals. In females, embryonic intercellular bridges have been proposed to have a role in development of the presumptive oocyte. Herein, we show that TEX14 is an essential component of male and female embryonic intercellular bridges. In addition, we demonstrate that mitotic kinesin-like protein 1 (MKLP1, official symbol KIF23), which we have discovered is a component of intercellular bridges during spermatogenesis, is also a component of male and female embryonic intercellular bridges. Germ cell intercellular bridges are readily identified by KIF23 immunofluorescence between the gonocytes and oogonia of control mice but are absent between germ cells of Tex14-null mice. Furthermore, by electron microscopy, intercellular bridges are present in all control newborn ovaries but are absent in the Tex14 knockout ovaries. Despite the absence of embryonic intercellular bridges in the Tex14-null mice, male mice initiate spermatogenesis, and female mice are fertile. Although fewer oocytes were present in Tex14-null neonatal ovaries, folliculogenesis was still active at 1 yr of age. Thus, while TEX14 and intercellular bridges have an essential role in postnatal spermatogenesis, they are not required in the embryo. PMID:19020301

  10. Effect of pregnancy and embryonic mortality on milk production in dromedary camels (Camelus dromedarius).

    Science.gov (United States)

    Nagy, P; Faigl, V; Reiczigel, J; Juhasz, J

    2015-02-01

    The main objective of the present study was to compare milk production in pregnant versus nonpregnant dromedary camels. In addition, we described the effect of embryonic mortality on lactation and measured serum progesterone levels until d 60 to 90 of gestation. Twenty-five multiparous camels were selected in midlactation for 2 studies in consecutive years. Camels were mated naturally when the size of the dominant follicle reached 1.2 to 1.5cm. Pregnancy was diagnosed by ultrasonography and progesterone determination. In the first experiment (Exp 1), 8 of 11 animals conceived at 284±21.5d postpartum. Three pregnant dromedaries were given PGF2α to induce luteolysis and pregnancy loss on d 62 and spontaneous embryonic loss was detected in 2 camels (on d 27 and 60). Animals were allotted to 3 groups retrospectively: nonpregnant camels (group 1, n=4), pregnant camels (group 2; n=3), and camels with embryonic loss after d 55 (group 3; n=4). In the second study (Exp 2), 14 dromedaries were mated during midlactation. Seven of them failed to conceive (group 1) and 7 became pregnant (group 2). No embryonic loss was detected in Exp 2. Turning points in milk production were identified by change point analysis. In nonpregnant dromedaries (group 1), milk decreased slowly over time without significant change point. In pregnant camels (group 2), a gradual decline until 4 wk after mating was followed by a sudden drop, and the change point model resulted in one breakpoint at d 28±7 and 35±3 of gestation in Exp 1 and Exp 2, respectively. In camels with embryonic mortality (group 3, Exp 1), milk yield started to decline similarly as in pregnant animals, but milk production increased gradually after embryonic loss and reached similar levels as in their nonpregnant herdmates. Change point analysis for group 3 resulted in 2 turning points at 30±4 and 48±4d after conception. Mean length of lactation was shorter by 230 (34.2%) and by 249d (37.6%) and mean total lactation production

  11. Dynamics and Mechanics of Zebrafish Embryonic Tissues.

    Science.gov (United States)

    Schoetz, Eva-Maria; Burdine, R. D.; Steinberg, M. S.; Heisenberg, C.-P.; Foty, R. A.; Julicher, F.

    2008-03-01

    In early zebrafish embryonic development, complex flows of cell populations occur, which ultimately lead to the spatial organization of the three germ layers: Ectoderm, mesoderm and endoderm. Here, we study the material properties of these germ layer tissues which are important for their dynamics and spatial organization in the embryo. In general, tissues can be classified as inherently active complex fluids. However, here we present examples of observed tissue behavior, which can be described satisfactorily in terms of passive visco-elastic fluids. We determined the material properties of the germ layer tissues quantitatively and found that differences in their properties influence tissue interaction. Specifically, quantitative differences in tissue surface tension result in tissue immiscibility and cell sorting behavior analogous to that of ordinary immiscible liquids. Surface tensions were measured with a tissue surface tensiometer. Furthermore, by tracking individual cells in the developing zebrafish embryo, we found differences in the migratory behavior of the different tissue types, which are, to some extent, governed by their mechanical properties. Finally, we generated a 3D velocity flow profile describing the tissue movements during zebrafish embryonic organizer development.

  12. Quantum dot imaging for embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Gambhir Sanjiv S

    2007-10-01

    Full Text Available Abstract Background Semiconductor quantum dots (QDs hold increasing potential for cellular imaging both in vitro and in vivo. In this report, we aimed to evaluate in vivo multiplex imaging of mouse embryonic stem (ES cells labeled with Qtracker delivered quantum dots (QDs. Results Murine embryonic stem (ES cells were labeled with six different QDs using Qtracker. ES cell viability, proliferation, and differentiation were not adversely affected by QDs compared with non-labeled control cells (P = NS. Afterward, labeled ES cells were injected subcutaneously onto the backs of athymic nude mice. These labeled ES cells could be imaged with good contrast with one single excitation wavelength. With the same excitation wavelength, the signal intensity, defined as (total signal-background/exposure time in millisecond was 11 ± 2 for cells labeled with QD 525, 12 ± 9 for QD 565, 176 ± 81 for QD 605, 176 ± 136 for QD 655, 167 ± 104 for QD 705, and 1,713 ± 482 for QD 800. Finally, we have shown that QD 800 offers greater fluorescent intensity than the other QDs tested. Conclusion In summary, this is the first demonstration of in vivo multiplex imaging of mouse ES cells labeled QDs. Upon further improvements, QDs will have a greater potential for tracking stem cells within deep tissues. These results provide a promising tool for imaging stem cell therapy non-invasively in vivo.

  13. Disseminated Cerebrospinal Embryonal Tumor in the Adult

    Science.gov (United States)

    Armocida, Daniele; Caporlingua, Federico; Lapadula, Gennaro; Elefante, Grazia Maria; Antonelli, Manila; Salvati, Maurizio

    2016-01-01

    Introduction. According to the 2016 World Health Organization classification of Tumors of the Central Nervous System, the term Primitive Neuroectodermal Tumor has been replaced by the term Embryonal Tumor (ET). We present a case of disseminated cerebrospinal ET presenting in an adult patient. Illustrative Case. A 49-year-old male presenting with low back pain, dysuria, and hypoesthesia of the lower extremities referred to our emergency department. Brain and whole spine contrast-enhanced MRI documented a diffusively disseminated heterogeneous neoplasm with intradural extra- and intramedullary involvement of the cervicothoracic tract and cauda equina. A primary biopsy of the lumbosacral localization was performed through L5 bilateral laminectomy. Histologic diagnosis was Embryonal Tumor Not Otherwise Specified. The patient underwent chemotherapy with postoperative adjuvant alternating Vincristine-Doxorubicin-Ifosfamide (VAI) and Ifosfamide-Etoposide (IE). Discussion. Spinal ETs are exceedingly rare especially when presenting in the adult patient. Neurosurgical and oncologic management is still unclear. When feasible, surgical removal should always be performed to obtain a histologic diagnosis. Postoperative adjuvant therapy might entail both chemo- and radiotherapy; however a consensus on this matter is still lacking. PMID:27818821

  14. Organotypic slice culture of embryonic brain tissue.

    Science.gov (United States)

    Daza, Ray A M; Englund, Chris; Hevner, Robert F

    2007-12-01

    INTRODUCTIONThis protocol describes how to dissect, assemble, and cultivate mouse embryonic (E) brain tissue from age E11.5 to E18.5 (days) for organotypic slice culture. These preparations can be used for a variety of assays and studies including coculture of different brain regions, cell migration assays, axon guidance assays, and DNA electroporation experiments. During electroporation, an electric current is applied to the surface of a specific target area of the brain slice in order to open holes in the plasma membrane and introduce a plasmid of coding DNA. The floating slice-on-membrane construct helps to preserve the structural integrity of the brain slices, while maintaining easy experimental access and optimal viability. Experiments can be monitored in living slices (e.g., with confocal imaging), and further studies can be completed using slices that have been fixed and cryosectioned at the end of the experiment. Any region of embryonic brain or spinal tissue can be used in this protocol.

  15. Embryonic stem cell differentiation: a chromatin perspective.

    Science.gov (United States)

    Rasmussen, Theodore P

    2003-11-13

    Embryonic stem (ES) cells hold immense promise for the treatment of human degenerative disease. Because ES cells are pluripotent, they can be directed to differentiate into a number of alternative cell-types with potential therapeutic value. Such attempts at "rationally-directed ES cell differentiation" constitute attempts to recapitulate aspects of normal development in vitro. All differentiated cells retain identical DNA content, yet gene expression varies widely from cell-type to cell-type. Therefore, a potent epigenetic system has evolved to coordinate and maintain tissue-specific patterns of gene expression. Recent advances show that mechanisms that govern epigenetic regulation of gene expression are rooted in the details of chromatin dynamics. As embryonic cells differentiate, certain genes are activated while others are silenced. These activation and silencing events are exquisitely coordinated with the allocation of cell lineages. Remodeling of the chromatin of developmentally-regulated genes occurs in conjunction with lineage commitment. Oocytes, early embryos, and ES cells contain potent chromatin-remodeling activities, an observation that suggests that chromatin dynamics may be especially important for early lineage decisions. Chromatin dynamics are also involved in the differentiation of adult stem cells, where the assembly of specialized chromatin upon tissue-specific genes has been studied in fine detail. The next few years will likely yield striking advances in the understanding of stem cell differentiation and developmental biology from the perspective of chromatin dynamics.

  16. Embryonic stem cell differentiation: A chromatin perspective

    Directory of Open Access Journals (Sweden)

    Rasmussen Theodore P

    2003-11-01

    Full Text Available Abstract Embryonic stem (ES cells hold immense promise for the treatment of human degenerative disease. Because ES cells are pluripotent, they can be directed to differentiate into a number of alternative cell-types with potential therapeutic value. Such attempts at "rationally-directed ES cell differentiation" constitute attempts to recapitulate aspects of normal development in vitro. All differentiated cells retain identical DNA content, yet gene expression varies widely from cell-type to cell-type. Therefore, a potent epigenetic system has evolved to coordinate and maintain tissue-specific patterns of gene expression. Recent advances show that mechanisms that govern epigenetic regulation of gene expression are rooted in the details of chromatin dynamics. As embryonic cells differentiate, certain genes are activated while others are silenced. These activation and silencing events are exquisitely coordinated with the allocation of cell lineages. Remodeling of the chromatin of developmentally-regulated genes occurs in conjunction with lineage commitment. Oocytes, early embryos, and ES cells contain potent chromatin-remodeling activities, an observation that suggests that chromatin dynamics may be especially important for early lineage decisions. Chromatin dynamics are also involved in the differentiation of adult stem cells, where the assembly of specialized chromatin upon tissue-specific genes has been studied in fine detail. The next few years will likely yield striking advances in the understanding of stem cell differentiation and developmental biology from the perspective of chromatin dynamics.

  17. Chromosomal Aneuploidies and Early Embryonic Developmental Arrest

    Directory of Open Access Journals (Sweden)

    Maria Maurer

    2015-07-01

    Full Text Available Background: Selecting the best embryo for transfer, with the highest chance of achieving a vital pregnancy, is a major goal in current in vitro fertilization (IVF technology. The high rate of embryonic developmental arrest during IVF treatment is one of the limitations in achieving this goal. Chromosomal abnormalities are possibly linked with chromosomal arrest and selection against abnormal fertilization products. The objective of this study was to evaluate the frequency and type of chromosomal abnormalities in preimplantation embryos with developmental arrest. Materials and Methods: This cohort study included blastomeres of embryos with early developmental arrest that were biopsied and analyzed by fluorescence in-situ hybridization (FISH with probes for chromosomes 13, 16, 18, 21 and 22. Forty-five couples undergoing IVF treatment were included, and 119 arrested embryos were biopsied. All probes were obtained from the Kinderwunsch Zentrum, Linz, Austria, between August 2009 and August 2011. Results: Of these embryos, 31.6% were normal for all chromosomes tested, and 68.4% were abnormal. Eleven embryos were uniformly aneuploid, 20 were polyploid, 3 were haploid, 11 displayed mosaicism and 22 embryos exhibited chaotic chromosomal complement. Conclusion: Nearly 70% of arrested embryos exhibit chromosomal errors, making chromosomal abnormalities a major cause of embryonic arrest and may be a further explanation for the high developmental failure rates during culture of the embryos in the IVF setting.

  18. Embryonic stem cells: from markers to market.

    Science.gov (United States)

    Deb, Kaushik Dilip; Jayaprakash, Anitha Devi; Sharma, Vijay; Totey, Satish

    2008-02-01

    ABSTRACT Embryonic stem cells are considered the mother of all kinds of tissues and cells and it is envisioned as the holy grail of regenerative medicine. However, their use in cell replacement therapies (CRT) has so far been limited and their potentials are yet to be fully realized. The use of human embryonic stem cells (hESC) involves many safety issues pertaining to culture conditions and epigenetic changes. The role and importance of an epigenomic signature in derivation and maintenance of hESC are discussed. We provide a list of important epigenetic markers, which should be studied for evaluation of safety in hESC-based cell replacement therapies. These genes also need to be screened to determine an epigenetic signature for pluripotency in the hESCs. Finally a comprehensive list of all known stemness signature genes and the marker genes for different germ line lineages are presented. This review aims at summing up most of the intriguing molecules that can play a role in the maintenance of pluripotency and can help in determining hESC differentiation to various lineages. Extensive understanding of these markers will eventually help the researchers to transform the hESC research from bench to the bedside. The use of hESCs in CRTs is still in its infancy; much effort is warranted to turn them into the much dreamed about magic wand of regenerative medicine.

  19. Mechanical signaling coordinates the embryonic heart

    Science.gov (United States)

    Chiou, Kevin; Rocks, Jason; Prosser, Benjamin; Discher, Dennis; Liu, Andrea

    The heart is an active material which relies on robust signaling mechanisms between cells in order to produce well-timed, coordinated beats. Heart tissue is composed primarily of active heart muscle cells (cardiomyocytes) embedded in a passive extracellular matrix. During a heartbeat, cardiomyocyte contractions are coordinated across the heart to form a wavefront that propagates through the tissue to pump blood. In the adult heart, this contractile wave is coordinated via intercellular electrical signaling.Here we present theoretical and experimental evidence for mechanical coordination of embryonic heartbeats. We model cardiomyocytes as mechanically excitable Eshelby inclusions embedded in an overdamped elastic-fluid biphasic medium. For physiological parameters, this model replicates recent experimental measurements of the contractile wavefront which are not captured by electrical signaling models. We additionally challenge our model by pharmacologically blocking gap junctions, inhibiting electrical signaling between myocytes. We find that while adult hearts stop beating almost immediately after gap junctions are blocked, embryonic hearts continue beating even at significantly higher concentrations, providing strong support for a mechanical signaling mechanism.

  20. SpolvlgA is a DDX3/PL10-related DEAD-box RNA helicase expressed in blastomeres and embryonic cells in planarian embryonic development.

    Science.gov (United States)

    Solana, Jordi; Romero, Rafael

    2009-01-01

    Planarian flatworms have an impressive regenerative power. Although their embryonic development is still poorly studied and is highly derived it still displays some simple characteristics. We have identified SpolvlgA, a Schmidtea polychroa homolog of the DDX3/PL10 DEAD-box RNA helicase DjvlgA from the planarian species Dugesia japonica. This gene has been previously described as being expressed in planarian adult stem cells (neoblasts), as well as the germ line. Here we present the expression pattern of SpolvlgA in developing embryos of S. polychroa and show that it is expressed from the first cleavage rounds in blastomere cells and blastomere-derived embryonic cells. These cells are undifferentiated cells that engage in a massive wave of differentiation during stage 5 of development. SpolvlgA expression highlights this wave of differentiation, where nearly all previous structures are substituted by blastomere-derived embryonic cells. In late stages of development SpolvlgA is expressed in most proliferating and differentiating cells. Thus, SpolvlgA is a gene expressed in planarian embryos from the first stages of development and a good marker for the zygote-derived cell lineage in these embryos. Expression in adult worms is also monitored and is found in the planarian germ line, where it is showed to be expressed in spermatogonia, spermatocytes and differentiating spermatids.

  1. SpolvlgA is a DDX3/PL10-related DEAD-box RNA helicase expressed in blastomeres and embryonic cells in planarian embryonic development

    Directory of Open Access Journals (Sweden)

    Jordi Solana, Rafael Romero

    2009-01-01

    Full Text Available Planarian flatworms have an impressive regenerative power. Although their embryonic development is still poorly studied and is highly derived it still displays some simple characteristics. We have identified SpolvlgA, a Schmidtea polychroa homolog of the DDX3/PL10 DEAD-box RNA helicase DjvlgA from the planarian species Dugesia japonica. This gene has been previously described as being expressed in planarian adult stem cells (neoblasts, as well as the germ line. Here we present the expression pattern of SpolvlgA in developing embryos of S. polychroa and show that it is expressed from the first cleavage rounds in blastomere cells and blastomere-derived embryonic cells. These cells are undifferentiated cells that engage in a massive wave of differentiation during stage 5 of development. SpolvlgA expression highlights this wave of differentiation, where nearly all previous structures are substituted by blastomere-derived embryonic cells. In late stages of development SpolvlgA is expressed in most proliferating and differentiating cells. Thus, SpolvlgA is a gene expressed in planarian embryos from the first stages of development and a good marker for the zygote-derived cell lineage in these embryos. Expression in adult worms is also monitored and is found in the planarian germ line, where it is showed to be expressed in spermatogonia, spermatocytes and differentiating spermatids.

  2. Large-scale identification of microRNA targets in murine Dgcr8-deficient embryonic stem cell lines.

    Directory of Open Access Journals (Sweden)

    Matthew P A Davis

    Full Text Available Small RNAs such as microRNAs play important roles in embryonic stem cell maintenance and differentiation. A broad range of microRNAs is expressed in embryonic stem cells while only a fraction of their targets have been identified. We have performed large-scale identification of embryonic stem cell microRNA targets using a murine embryonic stem cell line deficient in the expression of Dgcr8. These cells are heavily depleted for microRNAs, allowing us to reintroduce specific microRNA duplexes and identify refined target sets. We used deep sequencing of small RNAs, mRNA expression profiling and bioinformatics analysis of microRNA seed matches in 3' UTRs to identify target transcripts. Consequently, we have identified a network of microRNAs that converge on the regulation of several important cellular pathways. Additionally, our experiments have revealed a novel candidate for Dgcr8-independent microRNA genesis and highlighted the challenges currently facing miRNA annotation.

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  17. Embryonic stem cell-like features of testicular carcinoma in situ revealed by genome-wide gene expression profiling

    DEFF Research Database (Denmark)

    Almstrup, Kristian; Hoei-Hansen, Christina E; Wirkner, Ute;

    2004-01-01

    in their stoichiometry on progression into embryonic carcinoma. We compared the CIS expression profile with patterns reported in embryonic stem cells (ESCs), which revealed a substantial overlap that may be as high as 50%. We also demonstrated an over-representation of expressed genes in regions of 17q and 12, reported......Carcinoma in situ (CIS) is the common precursor of histologically heterogeneous testicular germ cell tumors (TGCTs), which in recent decades have markedly increased and now are the most common malignancy of young men. Using genome-wide gene expression profiling, we identified >200 genes highly...

  18. Spontaneous cyclic embryonic movements in humans and guinea pigs

    NARCIS (Netherlands)

    Felt, Renee H. M.; Mulder, Eduard J. H.; Luchinger, Annemarie B.; van Kan, Colette M.; Taverne, Marcel A. M.; de Vries, J. I. P.

    2012-01-01

    Motility assessment before birth can be used to evaluate the integrity of the nervous system. Sideways bending (SB) of head and/or rump, the earliest embryonic motility in both humans and guinea pigs, can be visualized sonographically. We know from other species that early embryonic motility is cycl

  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 indefin

  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...

  1. Sox2 in Embryonic Stem Cells and Lung Development

    NARCIS (Netherlands)

    C.G. Pardo (Cristina Gontan)

    2009-01-01

    markdownabstract__Abstract__ Sox2 is a fascinating transcription factor with multiple roles during embryonic development. In early embryonic development, Sox2 is one of the key transcription factors in the maintenance of the pluripotent status of the cells of the inner cell mass (ICM). Sox2 is also

  2. The mechanical consequences of mineralization in embryonic bone.

    NARCIS (Netherlands)

    Tanck, E.J.M.; Donkelaar, C.C. van; Jepsen, K.J.; Goldstein, S.A.; Weinans, H.; Burger, E.H.; Huiskes, R.

    2004-01-01

    The purpose of this study was to examine the effect of mineralization on the mechanical properties of embryonic bone rudiments. For this purpose, four-point bending experiments were performed on unmineralized and mineralized embryonic mouse ribs at 16 and 17 days of gestational age. Young's modulus

  3. Meeting embryonic requirements of broilers throughout incubation: a review

    NARCIS (Netherlands)

    Molenaar, R.; Reijrink, I.A.M.; Meijerhof, R.; Brand, van den H.

    2010-01-01

    During incubation of chicken embryos, environmental conditions, such as temperature, relative humidity, and CO2 concentration, must be controlled to meet embryonic requirements that change during the different phases of embryonic development. In the current review, the effects of embryo temperature,

  4. 4D embryonic cardiography using gated optical coherence tomography

    Science.gov (United States)

    Jenkins, M. W.; Rothenberg, F.; Roy, D.; Nikolski, V. P.; Hu, Z.; Watanabe, M.; Wilson, D. L.; Efimov, I. R.; Rollins, A. M.

    2006-01-01

    Simultaneous imaging of very early embryonic heart structure and function has technical limitations of spatial and temporal resolution. We have developed a gated technique using optical coherence tomography (OCT) that can rapidly image beating embryonic hearts in four-dimensions (4D), at high spatial resolution (10-15 μm), and with a depth penetration of 1.5 - 2.0 mm that is suitable for the study of early embryonic hearts. We acquired data from paced, excised, embryonic chicken and mouse hearts using gated sampling and employed image processing techniques to visualize the hearts in 4D and measure physiologic parameters such as cardiac volume, ejection fraction, and wall thickness. This technique is being developed to longitudinally investigate the physiology of intact embryonic hearts and events that lead to congenital heart defects.

  5. Human embryonic stem cell lines derived from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhen Fu FANG; Fan JIN; Hui GAI; Ying CHEN; Li WU; Ai Lian LIU; Bin CHEN; Hui Zhen SHENG

    2005-01-01

    Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF,Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

  6. Dimethadione embryotoxicity in the rat is neither correlated with maternal systemic drug concentrations nor embryonic tissue levels.

    Science.gov (United States)

    Ozolinš, Terence R S; Weston, Andrea D; Perretta, Anthony; Thomson, Jason J; Brown, Nigel A

    2015-11-15

    Pregnant rats treated with dimethadione (DMO), the N-demethylated metabolite of the anticonvulsant trimethadione, produce offspring having a 74% incidence of congenital heart defects (CHD); however, the incidence of CHD has high inter-litter variability (40-100%) that presents a challenge when studying the initiating events prior to the presentation of an abnormal phenotype. We hypothesized that the variability in CHD incidence was the result of differences in maternal systemic concentrations or embryonic tissue concentrations of DMO. To test this hypothesis, dams were administered 300 mg/kg DMO every 12h from the evening of gestational day (GD) 8 until the morning of GD 11 (six total doses). Maternal serum levels of DMO were assessed on GD 11, 12, 13, 14, 15, 18 and 21. Embryonic tissue concentrations of DMO were assessed on GD 11, 12, 13 and 14. In a separate cohort of GD 12 embryos, DMO concentrations and parameters of growth and development were assessed to determine if tissue levels of DMO were correlated with these endpoints. Embryos were exposed directly to different concentrations of DMO with whole embryo culture (WEC) and their growth and development assessed. Key findings were that neither maternal systemic concentrations nor tissue concentrations of DMO identified embryos that were sensitive or resistant to DMO in vivo. Direct exposure of embryos to DMO via WEC also failed to show correlations between embryonic concentrations of DMO with developmental outcomes in vitro. We conclude that neither maternal serum nor embryonic tissue concentrations of DMO predict embryonic outcome.

  7. Pathway Analysis and Modeling of the Differentiation of Human Embryonic Stem Cells into Hepatocyte-like Cells

    Science.gov (United States)

    Daskalaki, Andriani; Jozefczuk, Justyna; Lehrach, Hans; Adjaye, James; Wierling, Christoph

    2011-06-01

    A more detailed understanding of the differentiation of human embryonic and induced pluripotent stem cells into hepatocyte-like cells can help to improve therapies for liver diseases, like steatohepatitis. In this work we used microarray-based expression data to analyze the in vitro differentiation of human embryonic stem cells into hepatocytes. Pathway analysis has been carried out on gene expression data of different stages of the differentiation process from embryonic stem cells into hepatocyte-like cells via definitive endoderm and hepatic endoderm. Based on pathway analysis we identified signaling pathways, like the GPCR signaling pathway as well as FOXA2 regulatory networks. Based on these highly enriched pathways we constructed a model prototype to better understand and study the differentiation of stem cells into hepatocytes.

  8. Identification of side population cells in chicken embryonic gonads.

    Science.gov (United States)

    Bachelard, Elodie; Raucci, Franca; Montillet, Guillaume; Pain, Bertrand

    2015-02-01

    The side population (SP) phenotype, defined by the ability of a cell to efflux fluorescent dyes such as Hoechst, is common to several stem/progenitor cell types. In avian species, SP phenotype has been identified in pubertal and adult testes, but nothing is known about its expression during prenatal development of a male gonad. In this study, we characterized the Hoechst SP phenotype via the cytofluorimetric analysis of disaggregated testes on different days of chicken embryonic development. Male prenatal gonads contained a fraction of SP cells at each stage analyzed. At least two main SP fractions, named P3 and P4, were identified. The percentage of P3 fraction decreased as development proceeds, whereas P4 cell number was not affected by gonad growth. Functional inhibition of BCRP1 channel membrane using Verapamil and/or Ko143 showed that P3, but not P4 phenotype, was dependent on BCRP1 activity. Molecular analysis of both P3- and P4-sorted fractions revealed a differential RNA expression pattern, indicating that P3 cells mainly contained germinal stem cell markers, whereas P4 was preferentially composed of both Sertoli and Leydig cell progenitor markers. Finally, these findings provided evidence that the SP phenotype is a common feature of both germ and somatic cells detected in chicken developing testis.

  9. Autocrine glutamatergic transmission for the regulation of embryonal carcinoma stem cells.

    Science.gov (United States)

    Teng, Lin; Lei, Hui-Min; Sun, Fan; An, Shi-Min; Tang, Ya-Bin; Meng, Shuang; Wang, Cong-Hui; Shen, Ying; Chen, Hong-Zhuan; Zhu, Liang

    2016-08-02

    Glutamate behaves as the principal excitatory neurotransmitter in the vertebrate central nervous system and recently demonstrates intercellular signaling activities in periphery cancer cells. How the glutamatergic transmission is organized and operated in cancer stem cells remains undefined. We have identified a glutamatergic transmission circuit in embryonal carcinoma stem cells. The circuit is organized and operated in an autocrine mechanism and suppresses the cell proliferation and motility. Biological analyses determined a repertoire of glutamatergic transmission components, glutaminase, vesicular glutamate transporter, glutamate NMDA receptor, and cell membrane excitatory amino-acid transporter, for glutamate biosynthesis, package for secretion, reaction, and reuptake in mouse and human embryonal carcinoma stem cells. The glutamatergic components were also identified in mouse transplanted teratocarcinoma and in human primary teratocarcinoma tissues. Released glutamate acting as the signal was directly quantified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Genetic and pharmacological abolishment of the endogenously released glutamate-induced tonic activation of the NMDA receptors increased the cell proliferation and motility. The finding suggests that embryonal carcinoma stem cells can be actively regulated by establishing a glutamatergic autocrine/paracrine niche via releasing and responding to the transmitter.

  10. Inference of Transcriptional Network for Pluripotency in Mouse Embryonic Stem Cells

    Science.gov (United States)

    Aburatani, S.

    2015-01-01

    In embryonic stem cells, various transcription factors (TFs) maintain pluripotency. To gain insights into the regulatory system controlling pluripotency, I inferred the regulatory relationships between the TFs expressed in ES cells. In this study, I applied a method based on structural equation modeling (SEM), combined with factor analysis, to 649 expression profiles of 19 TF genes measured in mouse Embryonic Stem Cells (ESCs). The factor analysis identified 19 TF genes that were regulated by several unmeasured factors. Since the known cell reprogramming TF genes (Pou5f1, Sox2 and Nanog) are regulated by different factors, each estimated factor is considered to be an input for signal transduction to control pluripotency in mouse ESCs. In the inferred network model, TF proteins were also arranged as unmeasured factors that control other TFs. The interpretation of the inferred network model revealed the regulatory mechanism for controlling pluripotency in ES cells.

  11. Compatibility of embryonic stem cells with biomaterials.

    Science.gov (United States)

    Handschel, Jörg; Berr, Karin; Depprich, Rita; Naujoks, Christian; Kübler, Norbert R; Meyer, Ulrich; Ommerborn, Michelle; Lammers, Lydia

    2009-05-01

    Periodontal bone defects and atrophy of the jaws in an aging population are of special concern. Tissue engineering using embryonic stem cells (ESCs) and biomaterials may offer new therapeutic options. The purpose of this study is to evaluate the compatibility of ESCs with biomaterials and the influence of biomaterials on the osteogenic gene expression profile.Therefore, ESCs are cultured with various biomaterials. The cytocompatibility of murine ESCs is measured regarding the proliferation of the cells on the materials by CyQUANT assay, the morphology by scanning electron microscopy, and the influence on the gene expression by real time PCR.The results show that insoluble collagenous bone matrix, followed by beta-tricalciumphosphate, is most suitable for bone tissue engineering regarding cell proliferation, and phenotype. The gene expression analysis indicates that biomaterials do influence the gene expression of ESCs.Our results provide new insight into the cytocompatibility of ESCs on different scaffolds.

  12. Human embryonic stem cells: preclinical perspectives

    Directory of Open Access Journals (Sweden)

    Sarda Kanchan

    2008-01-01

    Full Text Available Abstract Human embryonic stem cells (hESCs have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic.

  13. Human embryonic stem cells for neuronal repair.

    Science.gov (United States)

    Ben-Hur, Tamir

    2006-02-01

    Human embryonic stem cells may serve as a potentially endeless source of transplantable cells to treat various neurologic disorders. Accumulating data have shown the therapeutic value of various neural precursor cell types in experimental models of neurologic diseases. Tailoring cell therapy for specific disorders requires the generation of cells that are committed to specific neural lineages. To this end, protocols were recently developed for the derivation of dopaminergic neurons, spinal motor neurons and oligodendrocytes from hESC. These protocols recapitulate normal development in culture conditions. However, a novel concept emerging from these studies is that the beneficial effect of transplanted stem cells is not only via cell replacement in damaged host tissue, but also by trophic and protective effects, as well as by an immunomodulatory effect that down-regulates detrimental brain inflammation.

  14. Current progress with primate embryonic stem cells.

    Science.gov (United States)

    Byrne, James A; Mitalipov, Shoukhrat M; Wolf, Don P

    2006-05-01

    Embryonic stem cells (ESCs) can proliferate indefinitely, maintain an undifferentiated pluripotent state and differentiate into any cell type. Differentiation of ESCs into various specific cell-types may be able to cure or alleviate the symptoms of various degenerative diseases. Unresolved issues regarding maintaining function, possible apoptosis and tumor formation in vivo mean a prudent approach should be taken towards advancing ESCs into human clinical trials. Rhesus macaques provide the ideal model organism for testing the feasibility, efficacy and safety of ESC based therapies and significant numbers of primate ESC lines are now available. In this review, we will summarize progress in evaluating the genetic and epigenetic integrity of primate ESCs, examine their current use in pre-clinical trials and discuss the potential of producing ESC-derived cell populations that are genetically identical (isogenic) to the host by somatic cell nuclear transfer.

  15. Functional analysis of Scr during embryonic and post-embryonic development in the cockroach, Periplaneta americana.

    Science.gov (United States)

    Hrycaj, Steven; Chesebro, John; Popadić, Aleksandar

    2010-05-01

    The cockroach, Periplaneta americana represents a basal insect lineage that undergoes the ancestral hemimetabolous mode of development. Here, we examine the embryonic and post-embryonic functions of the hox gene Scr in Periplaneta as a way of better understanding the roles of this gene in the evolution of insect body plans. During embryogenesis, Scr function is strictly limited to the head with no role in the prothorax. This indicates that the ancestral embryonic function of Scr was likely restricted to the head, and that the posterior expansion of expression in the T1 legs may have preceded any apparent gain of function during evolution. In addition, Scr plays a pivotal role in the formation of the dorsal ridge, a structure that separates the head and thorax in all insects. This is evidenced by the presence of a supernumerary segment that occurs between the labial and T1 segments of RNAiScr first nymphs and is attributed to an alteration in engrailed (en) expression. The fact that similar Scr phenotypes are observed in Tribolium but not in Drosophila or Oncopeltus reveals the presence of lineage-specific variation in the genetic architecture that controls the formation of the dorsal ridge. In direct contrast to the embryonic roles, Scr has no function in the head region during post-embryogenesis in Periplaneta, and instead, strictly acts to provide identity to the T1 segment. Furthermore, the strongest Periplaneta RNAiScr phenotypes develop ectopic wing-like tissue that originates from the posterior region of the prothoracic segment. This finding provides a novel insight into the current debate on the morphological origin of insect wings.

  16. Metabolic properties of chicken embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Cellular energy metabolism correlates with cell fate,but the metabolic properties of chicken embryonic stem (chES) cells are poorly understood.Using a previously established chES cell model and electron microscopy (EM),we found that undifferentiated chES cells stored glycogen.Additionally,undifferentiated chES cells expressed lower levels of glucose transporter 1 (GLUT1) and phosphofructokinase (PFK) mRNAs but higher levels of hexokinase 1 (HK1) and glycogen synthase (GYS) mRNAs compared with control primary chicken embryonic fibroblast (CEF) cells,suggesting that chES cells direct glucose flux towards the glycogenic pathway.Moreover,we demonstrated that undifferentiated chES cells block gluconeogenic outflow and impede the accumulation of glucose-6-phosphate (G6P) from this pathway,as evidenced by the barely detectable levels of pyruvate carboxylase (PCX) and mitochondrial phosphoenolpyruvate carboxykinase (PCK2) mRNAs.Additionally,cell death occurred in undifferentiated chES cells as shown by Hoechst 33342 and propidium iodide (PI) double staining,but it could be rescued by exogenous G6P.However,we found that differentiated chES cells decreased the glycogen reserve through the use of PAS staining.Moreover,differentiated chES cells expressed higher levels of GLUT1,HK1 and PFK mRNAs,while the level of GYS mRNA remained similar in control CEF cells.These data indicate that undifferentiated chES cells continue to synthesize glycogen from glucose at the expense of G6P,while differentiated chES cells have a decreased glycogen reserve,which suggests that the amount of glycogen is indicative of the chES cell state.

  17. 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

  18. 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.

  19. Integrative analysis of the mouse embryonic transcriptome.

    Science.gov (United States)

    Singh, Amar V; Knudsen, Kenneth B; Knudsen, Thomas B

    2007-04-10

    Monitoring global gene expression provides insight into how genes and regulatory signals work together to guide embryo development. The fields of developmental biology and teratology are now confronted with the need for automated access to a reference library of gene-expression signatures that benchmark programmed (genetic) and adaptive (environmental) regulation of the embryonic transcriptome. Such a library must be constructed from highly-distributed microarray data. Birth Defects Systems Manager (BDSM), an open access knowledge management system, provides custom software to mine public microarray data focused on developmental health and disease. The present study describes tools for seamless data integration in the BDSM library (MetaSample, MetaChip, CIAeasy) using the QueryBDSM module. A field test of the prototype was run using published microarray data series derived from a variety of laboratories, experiments, microarray platforms, organ systems, and developmental stages. The datasets focused on several developing systems in the mouse embryo, including preimplantation stages, heart and nerve development, testis and ovary development, and craniofacial development. Using BDSM data integration tools, a gene-expression signature for 346 genes was resolved that accurately classified samples by organ system and developmental sequence. The module builds a potential for the BDSM approach to decipher a large number developmental processes through comparative bioinformatics analysis of embryological systems at-risk for specific defects, using multiple scenarios to define the range of probabilities leading from molecular phenotype to clinical phenotype. We conclude that an integrative analysis of global gene-expression of the developing embryo can form the foundation for constructing a reference library of signaling pathways and networks for normal and abnormal regulation of the embryonic transcriptome. These tools are available free of charge from the web-site http

  20. Use of murine embryonic stem cells in embryotoxicity assays: the embryonic stem cell test.

    Science.gov (United States)

    Seiler, Andrea E M; Buesen, Roland; Visan, Anke; Spielmann, Horst

    2006-01-01

    The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body outgrowths compared to cytotoxic effects on murine ES cells and differentiated 3T3 fibroblasts. Through a number of prevalidation and validation studies, the EST has been demonstrated to be a reliable alternative method for embryotoxicity testing based on the most important mechanisms in embryotoxicity-cytotoxicity and differentiation--as well as on differences in sensitivity between differentiated and embryonic tissues. Improvements of the EST protocol using flow cytometry analysis showed that differential expression of sarcomeric myosin heavy chain and alpha-actinin proteins quantified under the influence of a test compound is a useful marker for detecting potential teratogenicity. The in vitro embryotoxicity test described in this chapter is rapid, simple, and sensitive and can be usefully employed as a component of the risk/hazard assessment process.

  1. Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells.

    Science.gov (United States)

    Morange, M; Diu, A; Bensaude, O; Babinet, C

    1984-04-01

    In a previous paper, we have shown that in the absence of stress, mouse embryonal carcinoma cells, like mouse early embryo multipotent cells, synthesize high levels of 89- and 70-kilodalton heat shock proteins (HSP)(O. Bensaude and M. Morange, EMBO J. 2:173-177, 1983). We report here the pattern of proteins synthesized after a short period of hyperthermia in various mouse embryonal carcinoma cell lines and early mouse embryo cells. Among the various cell lines tested, two of them, PCC4-Aza R1 and PCC7-S-1009, showed an unusual response in that stimulation of HSP synthesis was not observed in these cells after hyperthermia. However, inducibility of 68- and 105-kilodalton HSP can be restored in PCC7-S-1009 cells after in vitro differentiation triggered by retinoic acid. Similarly, in the early mouse embryo, hyperthermia does not induce the synthesis of nonconstitutive HSP at the eight-cell stage, but induction of the 68-kilodalton HSP does occur at the blastocyst stage. Such a transition in the expression of HSP has already been described for Drosophila melanogaster and sea urchin embryos and recently for mouse embryos. It may be a general property of early embryonic cells.

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

    Directory of Open Access Journals (Sweden)

    Ruixin Hao

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

  3. The single fgf receptor gene in the beetle Tribolium castaneum codes for two isoforms that integrate FGF8- and Branchless-dependent signals.

    Science.gov (United States)

    Sharma, Rahul; Beer, Katharina; Iwanov, Katharina; Schmöhl, Felix; Beckmann, Paula Indigo; Schröder, Reinhard

    2015-06-15

    The precise regulation of cell-cell communication by numerous signal-transduction pathways is fundamental for many different processes during embryonic development. One important signalling pathway is the evolutionary conserved fibroblast-growth-factor (FGF)-pathway that controls processes like cell migration, axis specification and mesoderm formation in vertebrate and invertebrate animals. In the model insect Drosophila, the FGF ligand / receptor combinations of FGF8 (Pyramus and Thisbe) / Heartless (Htl) and Branchless (Bnl) / Breathless (Btl) are required for the migration of mesodermal cells and for the formation of the tracheal network respectively with both the receptors functioning independently of each other. However, only a single fgf-receptor gene (Tc-fgfr) has been identified in the genome of the beetle Tribolium. We therefore asked whether both the ligands Fgf8 and Bnl could transduce their signal through a common FGF-receptor in Tribolium. Indeed, we found that the function of the single Tc-fgfr gene is essential for mesoderm differentiation as well as for the formation of the tracheal network during early development. Ligand specific RNAi for Tc-fgf8 and Tc-bnl resulted in two distinct non-overlapping phenotypes of impaired mesoderm differentiation and abnormal formation of the tracheal network in Tc-fgf8- and Tc-bnl(RNAi) embryos respectively. We further show that the single Tc-fgfr gene encodes at least two different receptor isoforms that are generated through alternative splicing. We in addition demonstrate through exon-specific RNAi their distinct tissue-specific functions. Finally, we discuss the structure of the fgf-receptor gene from an evolutionary perspective.

  4. Interspecific Competition between Tribolium castaneum and Cryptolestes ferrugineus%赤拟谷盗Tribolium castaneum与锈赤扁谷盗Cryptolestes ferrugineus种间竞争的研究

    Institute of Scientific and Technical Information of China (English)

    周蕊; 邓永学; 王进军; 崔晋波; 李平

    2012-01-01

    Interspecific competition between Tribolium castaneum and Cryptolestes femigineus was studied at 30℃ ,75 % RH and Votka-Volt-erra equation was used to simulate the competition. The results showed that population numbers of T. castaneum and C. ferrugineus under single rearing condition were more than that under mixture rearing condition. The interspecific competition( resource competition type) between the two species was remarkable. Population number of T. castaneum under single rearing and mixture rearing condition was greater than that of C. femigineus. The two species could coexist steadily under mixture rearing condition and T. castaneum was dominant species in the competition system.%在30℃、75% RH条件下研究赤拟谷盗Tribolium castaneum与锈赤扁谷盗Crptolestes ferrugineus的种间竞争,并采用Votka-Volterra种间竞争模型进行拟合.结果表明,赤拟谷盗与锈赤腐谷盗混合饲养时的种群数量均小于单独饲养时的种群数量,两物种之间存在着显著的种间竞争关系.属于资源利用型竞争;单独饲养和混合饲养条件下赤拟谷盗的种群数量均大于锈赤扁谷盗的种群数量;混合饲养条件下两个物种可以共存,并形成稳定的平衡状态,在这个竞争系统中,赤拟谷盗为优势种.

  5. 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.

  6. 七种糕点对赤拟谷盗(鞘翅目:拟步甲科)感染的抗性%Resistance of Seven Biscuit Types to Infestation by Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

    Institute of Scientific and Technical Information of China (English)

    Olusola O. ODEYEMI; Bernice M. OYEDARE; Michael O. ASHAMO

    2005-01-01

    Seven biscuit types namely, Okin, Digestive, Cabin, Peanut, Cream crackers, Hobnobs and Glucose and wheat flour were screened for their resistance to Tribolium castaneum (Herbst) at ambient temperature of 28±2 ℃ and 78±2% relative humidity in the laboratory. 50 g of each biscuit sample were infested with four female and two male adult beetles and left for 15 days then remove for a resistant experiment and 70 days for a mortality experiment. Each treatment and the control without beetles were replicated three times. Results showed that there was significant difference (P0.05) in the weight loss of biscuit types. The highest adult mortality of beetles (100%) was obtained from Peanut biscuits at 28 days after infestation while there was only 5.5% adult mortality in wheat flour at 70 days after infestation. The susceptibility index was 0 for all the biscuit types since there was no adult emergence indicating that they were resistant to T. castaneum infestation. Resistance in the biscuit types could be due to chemical additives (e.g. sodium chloride and sodium bicarbonate) used in the production of biscuits, which may have inhibitory effect on the development of T. castaneum and also the low moisture content of the biscuits. Packages that can easily be perforated or damaged to allow absorption of moisture from the environment should not be used in packing biscuits.

  7. A genetic screen for components of the mammalian RNA interference pathway in Bloom-deficient mouse embryonic stem cells

    OpenAIRE

    2009-01-01

    Genetic screens performed in model organisms have helped identify key components of the RNA interference (RNAi) pathway. Recessive genetic screens have recently become feasible through the use of mouse embryonic stem (ES) cells that are Bloom's syndrome protein (Blm) deficient. Here, we developed and performed a recessive genetic screen to identify components of the mammalian RNAi pathway in Blm-deficient ES cells. Genome-wide mutagenesis using a retroviral gene trap strategy resulted in the ...

  8. Screening of nanoparticle embryotoxicity using embryonic stem cells.

    Science.gov (United States)

    Campagnolo, Luisa; Fenoglio, Ivana; Massimiani, Micol; Magrini, Andrea; Pietroiusti, Antonio

    2013-01-01

    Due to the increasing use of engineered nanoparticles in many consumer products, rapid and economic tests for evaluating possible adverse effects on human health are urgently needed. In the present chapter the use of mouse embryonic stem cells as a valuable tool to in vitro screen nanoparticle toxicity on embryonic tissues is described. This in vitro method is a modification of the embryonic stem cell test, which has been widely used to screen soluble chemical compounds for their embryotoxic potential. The test offers an alternative to animal experimentation, reducing experimental costs and ethical issues.

  9. 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

  10. Stochastic Cell Fate Progression in Embryonic Stem Cells

    Science.gov (United States)

    Zou, Ling-Nan; Doyle, Adele; Jang, Sumin; Ramanathan, Sharad

    2013-03-01

    Studies on the directed differentiation of embryonic stem (ES) cells suggest that some early developmental decisions may be stochastic in nature. To identify the sources of this stochasticity, we analyzed the heterogeneous expression of key transcription factors in single ES cells as they adopt distinct germ layer fates. We find that under sufficiently stringent signaling conditions, the choice of lineage is unambiguous. ES cells flow into differentiated fates via diverging paths, defined by sequences of transitional states that exhibit characteristic co-expression of multiple transcription factors. These transitional states have distinct responses to morphogenic stimuli; by sequential exposure to multiple signaling conditions, ES cells are steered towards specific fates. However, the rate at which cells travel down a developmental path is stochastic: cells exposed to the same signaling condition for the same amount of time can populate different states along the same path. The heterogeneity of cell states seen in our experiments therefore does not reflect the stochastic selection of germ layer fates, but the stochastic rate of progression along a chosen developmental path. Supported in part by the Jane Coffin Childs Fund

  11. A novel marker for undifferentiated human embryonic stem cells.

    Science.gov (United States)

    Higashi, Kiyoshi; Yagi, Masaki; Arakawa, Tatsuhiko; Asano, Kouji; Kobayashi, Kumiko; Tachibana, Taro; Saito, Koichi

    2015-02-01

    Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs. The antigen recognized by MAb2 is expressed on the cell surface of undifferentiated hESCs; three diffused bands with molecular mass between 30 and 60 kDa in the lysates of hESCs were diminished during hESC differentiation into neural cells. The expression of MAb2 antigen was also observed on the plasma membrane of lung cancer cells, and MAb2 detected 55, 50, and 35 kDa protein bands in the cell lysates. Immunoprecipitation followed by proteomics analyses identified CD147/basigin as a MAb2 antigen. Finally, the positive expression of CD147/basigin protein in undifferentiated hESCs was confirmed. These results suggested that CD147/basigin could be another undifferentiated hESC marker.

  12. Embryonic development of endoderm in chicken (Gallus gallus domesticus).

    Science.gov (United States)

    Alcântara, Dayane; Rodrigues, Marcio N; Franciolli, André L R; Da Fonseca, Erika T; Silva, Fernanda M O; Carvalho, Rafael C; Fratini, Paula; Sarmento, Carlos Alberto P; Ferreira, Antonio José P; Miglino, Maria Angelica

    2013-08-01

    The poultry industry is a sector of agribusiness which represents an important role in the country's agricultural exports. Therefore, the study about embryogenesis of the domestic chicken (Gallus gallus domesticus) has a great economic importance. The aim of this study was to evaluate embryonic development of the endoderm in chicken (Gallus gallus domesticus). Forty fertilized eggs of domestic chickens, starting from the 1st day of gestation and so on until the 19 days of the incubation were collected from the Granja São José (Amparo, SP, Brazil). Embryos and fetus were fixed in 10% formaldehyde solution, identified, weighed, measured, and subjected to light and scanning electron microscopy. The endoderm originates the internal lining epithelium of the digestive, immune, respiratory systems, and the organs can be visualized from the second day (48 h) when the liver is formed. The formation of the digestive system was complete in the 12th day. Respiratory system organs begin at the fourth day as a disorganized tissue and undifferentiated. Their complete differentiation was observed at the 10 days of incubation, however, until the 19 days the syrinx was not observed. The formation of immune system at 10th day was observed with observation of the spleen, thymus, and cloacal bursa. The study of the organogenesis of the chicken based on germ layers is very complex and underexplored, and the study of chicken embryology is very important due the economic importance and growth of the use of this animal model studies such as genetic studies.

  13. Embryonic stem cell biology: insights from molecular imaging.

    Science.gov (United States)

    Sallam, Karim; Wu, Joseph C

    2010-01-01

    Embryonic stem (ES) cells have therapeutic potential in disorders of cellular loss such as myocardial infarction, type I diabetes and neurodegenerative disorders. ES cell biology in living subjects was largely poorly understood until incorporation of molecular imaging into the field. Reporter gene imaging works by integrating a reporter gene into ES cells and using a reporter probe to induce a signal detectable by normal imaging modalities. Reporter gene imaging allows for longitudinal tracking of ES cells within the same host for a prolonged period of time. This has advantages over postmortem immunohistochemistry and traditional imaging modalities. The advantages include expression of reporter gene is limited to viable cells, expression is conserved between generations of dividing cells, and expression can be linked to a specific population of cells. These advantages were especially useful in studying a dynamic cell population such as ES cells and proved useful in elucidating the biology of ES cells. Reporter gene imaging identified poor integration of differentiated ES cells transplanted into host tissue as well as delayed donor cell death as reasons for poor long-term survival in vivo. This imaging technology also confirmed that ES cells indeed have immunogenic properties that factor into cell survival and differentiation. Finally, reporter gene imaging improved our understanding of the neoplastic risk of undifferentiated ES cells in forming teratomas. Despite such advances, much remains to be understood about ES cell biology to translate this technology to the bedside, and reporter gene imaging will certainly play a key role in formulating this understanding.

  14. Transcriptional profiling of rhesus monkey embryonic stem cells.

    Science.gov (United States)

    Byrne, James A; Mitalipov, Shoukhrat M; Clepper, Lisa; Wolf, Don P

    2006-12-01

    Embryonic stem cells (ESCs) may be able to cure or alleviate the symptoms of various degenerative diseases. However, unresolved issues regarding survival, functionality, and tumor formation mean a prudent approach should be adopted towards advancing ESCs into human clinical trials. The rhesus monkey provides an ideal model organism for developing strategies to prevent immune rejection and test the feasibility, safety, and efficacy of ESC-based medical treatments. Transcriptional profiling of rhesus monkey ESCs provides a foundation for pre-clinical ESC research in this species. In the present study, we used microarray technology, immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) to characterize and transcriptionally profile rhesus monkey ESCs. We identified 367 stemness gene candidates that were highly (>85%) conserved across five different ESC lines. Rhesus monkey ESC lines maintained a pluripotent undifferentiated state over a wide range of POU5F1 (also known as OCT4) expression levels, and comparisons between rhesus monkey, mouse, and human stemness genes revealed five mammalian stemness genes: CCNB1, GDF3, LEFTB, POU5F1, and NANOG. These five mammalian genes are strongly expressed in rhesus monkey, mouse, and human ESCs, albeit only in the undifferentiated state, and represent the core key mammalian stemness factors.

  15. DNA methylation programming and reprogramming in primate embryonic stem cells.

    Science.gov (United States)

    Cohen, Netta Mendelson; Dighe, Vikas; Landan, Gilad; Reynisdóttir, Sigrún; Palsson, Arnar; Mitalipov, Shoukhrat; Tanay, Amos

    2009-12-01

    DNA methylation is an important epigenetic mechanism, affecting normal development and playing a key role in reprogramming epigenomes during stem cell derivation. Here we report on DNA methylation patterns in native monkey embryonic stem cells (ESCs), fibroblasts, and ESCs generated through somatic cell nuclear transfer (SCNT), identifying and comparing epigenome programming and reprogramming. We characterize hundreds of regions that are hyper- or hypomethylated in fibroblasts compared to native ESCs and show that these are conserved in human cells and tissues. Remarkably, the vast majority of these regions are reprogrammed in SCNT ESCs, leading to almost perfect correlation between the epigenomic profiles of the native and reprogrammed lines. At least 58% of these changes are correlated in cis to transcription changes, Polycomb Repressive Complex-2 occupancy, or binding by the CTCF insulator. We also show that while epigenomic reprogramming is extensive and globally accurate, the efficiency of adding and stripping DNA methylation during reprogramming is regionally variable. In several cases, this variability results in regions that remain methylated in a fibroblast-like pattern even after reprogramming.

  16. Recapitulation of the embryonic cardiovascular progenitor cell niche.

    Science.gov (United States)

    Schenke-Layland, Katja; Nsair, Ali; Van Handel, Ben; Angelis, Ekaterini; Gluck, Jessica M; Votteler, Miriam; Goldhaber, Joshua I; Mikkola, Hanna K; Kahn, Michael; Maclellan, William R

    2011-04-01

    Stem or progenitor cell populations are often established in unique niche microenvironments that regulate cell fate decisions. Although niches have been shown to be critical for the normal development of several tissues, their role in the cardiovascular system is poorly understood. In this study, we characterized the cardiovascular progenitor cell (CPC) niche in developing human and mouse hearts, identifying signaling pathways and extracellular matrix (ECM) proteins that are crucial for CPC maintenance and expansion. We demonstrate that collagen IV (ColIV) and β-catenin-dependent signaling are essential for maintaining and expanding undifferentiated CPCs. Since niches are three-dimensional (3D) structures, we investigated the impact of a 3D microenvironment that mimics the in vivo niche ECM. Employing electrospinning technologies, 3D in vitro niche substrates were bioengineered to serve as culture inserts. The three-dimensionality of these structures increased mouse embryonic stem cell differentiation into CPCs when compared to 2D control cultures, which was further enhanced by incorporation of ColIV into the substrates. Inhibiting p300-dependent β-catenin signals with the small molecule IQ1 facilitated further expansion of CPCs. Our study represents an innovative approach to bioengineer cardiac niches that can serve as unique 3D in vitro systems to facilitate CPC expansion and study CPC biology.

  17. Toxic and Repellent effecto of Harmal (Peganum harmala L. Acetonic Extract on Several Aphids and Tribolium castaneum (Herbst Efecto Tóxico y Repelente del Extracto Acetónico de Harmal (Peganum harmala L. sobre varias especies de Áfidos y Tribolium castaneum (Herbst

    Directory of Open Access Journals (Sweden)

    Elham Salari

    2012-03-01

    Full Text Available To reduce the dependence on the sometimes unwise use of synthetic pesticides in fruit and vegetable plantations, the toxicity and repellence of Peganum harmala L. (Zygophyllaceae acetonic seed extract was assayed against several insect pests. For contact toxicity, 3- to 4-d-old individuals of Aphis fabae Scopoli, A. gossypii Glover, A. nerii Boyer de Fonscolombe, and Myzus persicae (Sulzer were included, as well as 1- to 7-d-old adult Tribolium castaneum (Herbst. Repellent effect experiments were conducted on adult, 1- to 2- and 3- to 4-d old M.persicae individuals. At 60 mg mL4, the topical bioassay mortality percentage was significantly higher in A. gossypii than in A. fabae and A. nerii after 12-72 h. Mortality of the treatments on M.persicae was 87.1% and 90.0% after 24 and 48 h, respectively, and significantly higher than A. fabae and A. nerii during this period. At 60 mg mL-1, the mortality of T. castaneum was much lower than that of the aphid species. The highest repellent index (over 72% was observed on 1- to 2-d-old M. persicae individuals.Para reducir la dependencia de los pesticidas sintéticos en plantaciones frutales y hortalizas, se realizó un ensayo para medir la toxicidad y repelencia de un extracto acetónico obtenido a partir de semillas de Peganum harmala L. (Zygophyllaceae contra diferentes especies de plagas. Para evaluar la toxicidad del extracto al contacto con los insectos, se incluyeron individuos de 3-4 d de edad de Aphis fabae Scopoli, Aphis gossypii Glover, Aphis nerii Boyer de Fonscolombe, y Myzus persicae (Sulzer, así como adultos 1-7 d de edad de Tribolium castaneum (Herbst. Experimentos para medir el efecto repelente se llevaron a cabo con individuos de 1-2 y 3-4 d de edad de M. persicae. En los resultados de los bioensayos tópicos el porcentaje de mortalidad fue significativamente mayor en la especie A. gossypii que en A. fabae y A. nerii, después de 12-72 h con una concentración de 60 mg mL-1. La mortalidad

  18. The production and directed differentiation of human embryonic stem cells.

    Science.gov (United States)

    Trounson, Alan

    2006-04-01

    Human embryonic stem cells (hESCs) are being rapidly produced from chromosomally euploid, aneuploid, and mutant human embryos that are available from in vitro fertilization clinics treating patients for infertility or preimplantation genetic diagnosis. These hESC lines are an important resource for functional genomics, drug screening, and, perhaps eventually, cell and gene therapy. The methods for deriving hESCs are well established and repeatable and are relatively successful with a ratio of 1:10 to 1:2 new hESC lines produced from 4- to 8-d-old morula and blastocysts and from isolated inner cell mass cell clusters of human blastocysts. The hESCs can be formed and maintained on human somatic cells in humanized serum-free culture conditions and for several passages in cell-free culture systems. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in vitro while maintaining their original karyotype and epigenetic status, but this needs to be confirmed from time to time in long-term cultures. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating flat attachment cultures and unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes, and characteristic morphology, and the cells thereafter enriched for progenitor types and further culture to more mature cell types. Directed differentiation systems are well developed for ectodermal pathways that result in neural and glial cells and the mesendodermal pathway for cardiac muscle cells and many other cell types including hematopoietic progenitors and endothelial cells. Directed differentiation into endoderm has been more difficult to achieve, perhaps because of the lack of markers of

  19. Possible Effect of 30K Proteins in Embryonic Development of Silkworm Bombyx mori

    Institute of Scientific and Technical Information of China (English)

    Bo-Xiong ZHONG; Jian-Ke LI; Jian-Rong LIN; Jian-She LIANG; Song-Kun SU; Hai-Sheng XU; Hai-Yan YAN; Ping-Bo ZHANG; Hiroshi FUJII

    2005-01-01

    The silkworm Bombyx mori possesses a 30K protein family of 3×104 Da, the biological functions of which have not been fully identified. The relationship between the 30K protein family and the embryonic development of temperature sensitive sex-linked mutant strain of silkworm was investigated by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Matrix assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS). The results show that protein spots 1-5 of the 30K protein family, mainly existing in normal strain, are possibly related to embryonic development. The early consumption of a 30K protein named 6G1-30K-1 and the accumulation of 30K proteins named 6G1-30K-3and 6G 1-30K-4 are likely caused by the destruction of physiological balance in normal embryonic development,which may lead to lower hatchability of the temperature sensitive strain. The results suggest that reasonable metabolism of 30K proteins is a prerequisite for the embryo's normal development.

  20. Identification and characterization of secondary neural tube-derived embryonic neural stem cells in vitro.

    Science.gov (United States)

    Shaker, Mohammed R; Kim, Joo Yeon; Kim, Hyun; Sun, Woong

    2015-05-15

    Secondary neurulation is an embryonic progress that gives rise to the secondary neural tube, the precursor of the lower spinal cord region. The secondary neural tube is derived from aggregated Sox2-expressing neural cells at the dorsal region of the tail bud, which eventually forms rosette or tube-like structures to give rise to neural tissues in the tail bud. We addressed whether the embryonic tail contains neural stem cells (NSCs), namely secondary NSCs (sNSCs), with the potential for self-renewal in vitro. Using in vitro neurosphere assays, neurospheres readily formed at the rosette and neural-tube levels, but less frequently at the tail bud tip level. Furthermore, we identified that sNSC-generated neurospheres were significantly smaller in size compared with cortical neurospheres. Interestingly, various cell cycle analyses revealed that this difference was not due to a reduction in the proliferation rate of NSCs, but rather the neuronal commitment of sNSCs, as sNSC-derived neurospheres contain more committed neuronal progenitor cells, even in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). These results suggest that the higher tendency for sNSCs to spontaneously differentiate into progenitor cells may explain the limited expansion of the secondary neural tube during embryonic development.

  1. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    Science.gov (United States)

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-08-10

    Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z.

  2. Control Effectiveness of Insect Growth Regulators on Tribolium castaneum%两种昆虫生长调节剂对赤拟谷盗的控制效果

    Institute of Scientific and Technical Information of China (English)

    陈雅群; 谢令德; 张建珍; 贺艳萍; 何君; 陈春武

    2012-01-01

    选取氟啶脲( Chlorfluazuron)和灭幼宝(Pyriproxyfen)两种昆虫生长调节剂对储藏物害虫赤拟谷盗进行了控制效果的研究.结果表明,在40、20、10 mg/kg剂量下,氟啶脲对赤拟谷盗幼虫有较高的校正死亡率,依次为94.44%、88.89%、83.33%,幼虫均不能化蛹,表明该药剂可能作用于几丁质合成酶,抑制了几丁质合成酶的活性.在相同浓度下,灭幼宝对赤拟谷盗幼虫没有明显的致死效果,但其能延长赤拟谷盗幼虫的发育历期,使其不能正常化蛹,最终导致畸形死亡,表明灭幼宝有类似保幼激素的作用.%The paper study the control effectiveness of two IGRs, Chlorfluazuron and Pyriproxyfen to Tribolium castaneum, a main insect pest occurring in stored product. The result showed that Chlorfluazuron had a high mortality rate on Tribolium castaneum larva, which is 94.44% ,,88. 89% ,,83. 33% corresponding to the concentration of 40,20, 10 mg/kg. ,and make them pupate hardly,so Chlorfluazuron had significant death effect on Tribolium castaneum larva , and it acted on chitin synthase, inhibiting the activity of chitin synthase. Pyriproxyfen didn't have obvious death effect on Tribolium castaneum at the same concentration as Chlorfluazuron, but Pyriproxyfen can delay the development of Tribolium castaneum larva, affect their normal pupa, and eventually cause their deformity death. The result preliminary shows that Pyriproxyfen has similar function as juvenile hormones.

  3. Derivation of multipotent mesenchymal precursors from human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    2005-06-01

    Full Text Available BACKGROUND: Human embryonic stem cells provide access to the earliest stages of human development and may serve as a source of specialized cells for regenerative medicine. Thus, it becomes crucial to develop protocols for the directed differentiation of embryonic stem cells into tissue-restricted precursors. METHODS AND FINDINGS: Here, we present culture conditions for the derivation of unlimited numbers of pure mesenchymal precursors from human embryonic stem cells and demonstrate multilineage differentiation into fat, cartilage, bone, and skeletal muscle cells. CONCLUSION: Our findings will help to elucidate the mechanism of mesoderm specification during embryonic stem cell differentiation and provide a platform to efficiently generate specialized human mesenchymal cell types for future clinical applications.

  4. 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.

  5. 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.

  6. Distribution of microglial cells in the cerebral hemispheres of embryonic and neonatal chicks

    Directory of Open Access Journals (Sweden)

    A.R. Ignácio

    2005-11-01

    Full Text Available The distribution, morphology and morphometry of microglial cells in the chick cerebral hemispheres from embryonic day 4 (E4 to the first neonatal day (P1 were studied by histochemical labeling with a tomato (Lycopersicon esculentum lectin. The histochemical analysis revealed lectin-reactive cells in the nervous parenchyma on day E4. Between E4 (5.7 ± 1.35 mm length and E17 (8.25 ± 1.2 mm length, the lectin-reactive cells were identified as ameboid microglia and observed starting from the subventricular layer, distributed throughout the mantle layer and in the proximity of the blood vessels. After day E13, the lectin-reactive cells exhibited elongated forms with small branched processes, and were considered primitive ramified microglia. Later, between E18 (5.85 ± 1.5 mm cell body length and P1 (3.25 ± 0.6 mm cell body length, cells with more elongated branched processes were observed, constituting the ramified microglia. Our findings provide additional information on the migration and differentiation of microglial cells, whose ramified form is observed at the end of embryonic development. The present paper focused on the arrangement of microglial cells in developing cerebral hemispheres of embryonic and neonatal chicks, which are little studied in the literature. Details of morphology, morphometry and spatial distribution of microglial cells contributed to the understanding of bird and mammal central nervous system ontogeny. Furthermore, the identification and localization of microglial cells during the normal development could be used as a morphological guide for embryonic brain injury researches.

  7. Genetic signatures shared in embryonic liver development and liver cancer define prognostically relevant subgroups in HCC

    Directory of Open Access Journals (Sweden)

    Becker Diana

    2012-08-01

    Full Text Available Abstract Multiple activations of individual genes during embryonic liver and HCC development have repeatedly prompted speculations about conserved embryonic signatures driving cancer development. Recently, the emerging discussion on cancer stem cells and the appreciation that generally tumors may develop from progenitor cells of diverse stages of cellular differentiation has shed increasing light on the overlapping genetic signatures between embryonic liver development and HCC. However there is still a lack of systematic studies investigating this area. We therefore performed a comprehensive analysis of differentially regulated genetic signaling pathways in embryonic and liver cancer development and investigated their biological relevance. Genetic signaling pathways were investigated on several publically available genome wide microarray experiments on liver development and HCC. Differentially expressed genes were investigated for pathway enrichment or underrepresentation compared to KEGG annotated pathways by Fisher exact evaluation. The comparative analysis of enrichment and under representation of differentially regulated genes in liver development and HCC demonstrated a significant overlap between multiple pathways. Most strikingly we demonstrated a significant overlap not only in pathways expected to be relevant to both conditions such as cell cycle or apoptosis but also metabolic pathways associated with carbohydrate and lipid metabolism. Furthermore, we demonstrated the clinical significance of these findings as unsupervised clustering of HCC patients on the basis of these metabolic pathways displayed significant differences in survival. These results indicate that liver development and liver cancer share similar alterations in multiple genetic signaling pathways. Several pathways with markedly similar patterns of enrichment or underrepresentation of various regulated genes between liver development and HCC are of prognostic relevance in

  8. 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.

  9. The liberation of embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kathryn Blair

    2011-04-01

    Full Text Available Mouse embryonic stem (ES cells are defined by their capacity to self-renew and their ability to differentiate into all adult tissues including the germ line. Along with efficient clonal propagation, these properties have made them an unparalleled tool for manipulation of the mouse genome. Traditionally, mouse ES (mES cells have been isolated and cultured in complex, poorly defined conditions that only permit efficient derivation from the 129 mouse strain; genuine ES cells have not been isolated from another species in these conditions. Recently, use of small molecule inhibitors of glycogen synthase kinase 3 (Gsk3 and the Fgf-MAPK signaling cascade has permitted efficient derivation of ES cells from all tested mouse strains. Subsequently, the first verified ES cells were established from a non-mouse species, Rattus norvegicus. Here, we summarize the advances in our understanding of the signaling pathways regulating mES cell self-renewal that led to the first derivation of rat ES cells and highlight the new opportunities presented for transgenic modeling on diverse genetic backgrounds. We also comment on the implications of this work for our understanding of pluripotent stem cells across mammalian species.

  10. Biological impact of human embryonic stem cells.

    Science.gov (United States)

    Martín, Miguel; Menéndez, Pablo

    2012-01-01

    Research on human embryonic stem cells (hESCs) and induced pluripotent (iPS) stem cells is currently a field of great potential in biomedicine. These cells represent a highly valuable tool for developmental biology studies, disease models, and drug screening and toxicity. The ultimate goal of hESCs and iPS cell research is the treatment of diseases or disorders for which there is currently no treatment or existing therapies are only partially effective. Despite the disproportionate short-term hopes generated, which are putting too much pressure on scientists, the international scientific community is making rapid progress in understanding hESCs and iPS cells. Nonetheless, great efforts have to be made to provide an answer to still quite basic questions concerning their biology. Moreover, translation to clinical applications in cell replacement therapy requires prior solution to ethical barriers. The recent development of iPS cells has provided a strong alternative to overcome ethical issues concerning hESCs. However, an in-depth characterization of their genetic and epigenetic features, as well as their differentiation potential still remains to be undertaken. This chapter will describe, precisely, what the critical issues are, where scientific and ethical barriers stand, and how we are to overcome them. Only then, we shall finally discover whether hESCs and iPS cells will allow building reproducible disease models, and whether they really are a safe tool, with great potential for regenerative medicine.

  11. Genetic Manipulation of Human Embryonic Stem Cells.

    Science.gov (United States)

    Eiges, Rachel

    2016-01-01

    One of the great advantages of embryonic stem (ES) cells over other cell types is their accessibility to genetic manipulation. They can easily undergo genetic modifications while remaining pluripotent, and can be selectively propagated, allowing the clonal expansion of genetically altered cells in culture. Since the first isolation of ES cells in mice, many effective techniques have been developed for gene delivery and manipulation of ES cells. These include transfection, electroporation, and infection protocols, as well as different approaches for inserting, deleting, or changing the expression of genes. These methods proved to be extremely useful in mouse ES cells, for monitoring and directing differentiation, discovering unknown genes, and studying their function, and are now being extensively implemented in human ES cells (HESCs). This chapter describes the different approaches and methodologies that have been applied for the genetic manipulation of HESCs and their applications. Detailed protocols for generating clones of genetically modified HESCs by transfection, electroporation, and infection will be described, with special emphasis on the important technical details that are required for this purpose. All protocols are equally effective in human-induced pluripotent stem (iPS) cells.

  12. Ontogeny and embryonic description of Betta splendens, Perciformes (Regan, 1910)

    OpenAIRE

    Shaytner Campos DUARTE; Vasconcellos,Breno de Faria e; Manuel Vazquez VIDAL JÚNIOR; Ferreira,Andre Veloso; Mattos,Douglas da Cruz; Branco,Allex Trindade

    2012-01-01

    Ontogeny process comprises the embryo development from the moment of fecundation, through embryonic development phase, until hatching or later phases. The study of embryogenesis is an important tool for growth study of the species within their natural environment. The present work observed and registered the main stages of Betta splendens embryonic development, one of the most promising ornamental species mostly because of the increasing commercial interest, to describe the initial ontogeny a...

  13. Human embryonic stem cell research: ethical and legal issues.

    Science.gov (United States)

    Robertson, J A

    2001-01-01

    The use of human embryonic stem cells to replace damaged cells and tissues promises future hope for the treatment of many diseases. However, many countries now face complex ethical and legal questions as a result of the research needed to develop these cell-replacement therapies. The challenge that must be met is how to permit research on human embryonic tissue to occur while maintaining respect for human life generally.

  14. Infection of Tribolium castaneum with Bacillus thuringiensis: Quantification of Bacterial Replication within Cadavers, Transmission via Cannibalism, and Inhibition of Spore Germination

    Science.gov (United States)

    Milutinović, Barbara; Höfling, Christina; Futo, Momir; Scharsack, Jörn P.

    2015-01-01

    Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen. PMID:26386058

  15. Infection of Tribolium castaneum with Bacillus thuringiensis: quantification of bacterial replication within cadavers, transmission via cannibalism, and inhibition of spore germination.

    Science.gov (United States)

    Milutinović, Barbara; Höfling, Christina; Futo, Momir; Scharsack, Jörn P; Kurtz, Joachim

    2015-12-01

    Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen.

  16. Toxicity of phosphine on the developmental stages of rust-red flour beetle, Tribolium castaneum Herbst over a range of concentrations and exposures.

    Science.gov (United States)

    Manivannan, S

    2015-10-01

    The susceptibility of the developmental stages of rust-red flour beetle, Tribolium castaneum to a range of concentrations of phosphine over varying durations from 24 to 168 h was reconnoitered in the laboratory at 25 ± 2 °C. Responses of the life stages exposed to phosphine were compared with those of un-treated controls over 24, 48, 72, 96, 120, 144 and 168 h exposures and mortality was assessed after 14 days. Among the life stages tested, pupae were more tolerant to phosphine followed by the egg and the larval instars. At 24 h, the maximum LC50 value was observed in case of egg; 1.571 mgL(-1); followed by the pupae, 6(th) instar, 4(th) instar and 2(nd) instar larvae with LC50 values of 1.184, 0.336, 0.212 and 0.081 mgL(-1) respectively. However, continued exposure of the developmental stages to phosphine, recorded maximum LC values in the pupae followed by egg and the larval instars. A linear increase in the mortality response was witnessed in all the insect stages when the exposure periods were extended from 24 to 168 h with increasing concentrations of phosphine, conversely significant increase in mortality was greatly apparent during the initial treatment periods.

  17. Insecticidal effects of extracts of seven plant species on larval development, alpha-amylase activity and offspring production of Tribolium castaneum (Herbst) (Insecta: Coleoptera: Tenebrionidae).

    Science.gov (United States)

    Jbilou, R; Amri, H; Bouayad, N; Ghailani, N; Ennabili, A; Sayah, F

    2008-03-01

    Bioinsecticidal effects of methanol extracts from seven plant species on Tribolium castaneum were investigated. Centaurium erythraea, Peganum harmala, Ajuga iva, Aristolochia baetica, Pteridium aquilinum and Raphanus raphanistrum extracts inhibit growth of larvae. C. erythraea was the most toxic with 63% mortality 10 days after treatment, followed by P. harmala with 58%. C. erythraea and P. aquilinum reduce the emergence rate respectively of 66% and 19%. The duration of larval period was shortened by Launaea arborescens, P. aquilinum and A. iva extracts, whereas R. raphanistrum and P. harmala extracts extend the larval period when compared to the control. Extracts of C. erythraea, P. harmala, A. iva and A. baetica inhibited F1 progeny production. Larvae possess three alpha-amylase isoforms as determined by SDS-PAGE. Larvae fed on treated diet had lower alpha-amylase activity than larvae feed on untreated diet. C. erythraea and P. harmala are the most potent extracts. These plant extracts could be useful to reduce seed damage caused by this pest species.

  18. Response of Tribolium castaneum to elevated copper concentrations is influenced by history of metal exposure, sex-specific defences, and infection by the parasite Steinernema feltiae.

    Science.gov (United States)

    Kramarz, Paulina E; Mordarska, Anna; Mroczka, Magdalena

    2014-07-01

    We studied how copper toxicity in the red flour beetle, Tribolium castaneum changed as a result of infection by the entomopathogenic nematode Steinernema feltiae. Measured traits were: respiration, growth and survival, as well as the concentrations of copper within beetle tissues and in its diet. By comparing F1 and F5 generation we were able to answer how long-term metal exposure changed the responses to both copper and the parasite. The beetles did accumulate copper; however, the results indicated that copper concentrations in beetle tissues were affected by nematode infection, the sex of the experimental animals, and the number of generations of exposure. Five generations of exposure to copper resulted in the highest dry body mass of infected beetles of both sexes; additionally, this group also had the lowest copper concentrations in their tissues. The only factor that had a significant effect on respiration was infection by nematodes: infected beetles of both sexes in both generational groups had significantly decreased respiration rates. Survival was lowest in nematode-infected animals of both sexes from both generations, regardless of exposure to copper. Our results confirm that an organism's response to metal pollution is dependent on its health status and sex. We also found that the history of exposure to metal was equally important-we found enhanced resistance to copper intoxication after only five generations of exposure.

  19. Genome-wide analysis of tandem repeats in Tribolium castaneum genome reveals abundant and highly dynamic tandem repeat families with satellite DNA features in euchromatic chromosomal arms.

    Science.gov (United States)

    Pavlek, Martina; Gelfand, Yevgeniy; Plohl, Miroslav; Meštrović, Nevenka

    2015-12-01

    Although satellite DNAs are well-explored components of heterochromatin and centromeres, little is known about emergence, dispersal and possible impact of comparably structured tandem repeats (TRs) on the genome-wide scale. Our bioinformatics analysis of assembled Tribolium castaneum genome disclosed significant contribution of TRs in euchromatic chromosomal arms and clear predominance of satellite DNA-typical 170 bp monomers in arrays of ≥5 repeats. By applying different experimental approaches, we revealed that the nine most prominent TR families Cast1-Cast9 extracted from the assembly comprise ∼4.3% of the entire genome and reside almost exclusively in euchromatic regions. Among them, seven families that build ∼3.9% of the genome are based on ∼170 and ∼340 bp long monomers. Results of phylogenetic analyses of 2500 monomers originating from these families show high-sequence dynamics, evident by extensive exchanges between arrays on non-homologous chromosomes. In addition, our analysis shows that concerted evolution acts more efficiently on longer than on shorter arrays. Efficient genome-wide distribution of nine TR families implies the role of transposition only in expansion of the most dispersed family, and involvement of other mechanisms is anticipated. Despite similarities in sequence features, FISH experiments indicate high-level compartmentalization of centromeric and euchromatic tandem repeats.

  20. The negative effect of starvation and the positive effect of mild thermal stress on thermal tolerance of the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Scharf, Inon; Wexler, Yonatan; MacMillan, Heath Andrew; Presman, Shira; Simson, Eddie; Rosenstein, Shai

    2016-04-01

    The thermal tolerance of a terrestrial insect species can vary as a result of differences in population origin, developmental stage, age, and sex, as well as via phenotypic plasticity induced in response to changes in the abiotic environment. Here, we studied the effects of both starvation and mild cold and heat shocks on the thermal tolerance of the red flour beetle, Tribolium castaneum. Starvation led to impaired cold tolerance, measured as chill coma recovery time, and this effect, which was stronger in males than females, persisted for longer than 2 days but less than 7 days. Heat tolerance, measured as heat knockdown time, was not affected by starvation. Our results highlight the difficulty faced by insects when encountering multiple stressors simultaneously and indicate physiological trade-offs. Both mild cold and heat shocks led to improved heat tolerance in both sexes. It could be that both mild shocks lead to the expression of heat shock proteins, enhancing heat tolerance in the short run. Cold tolerance was not affected by previous mild cold shock, suggesting that such a cold shock, as a single event, causes little stress and hence elicits only weak physiological reaction. However, previous mild heat stress led to improved cold tolerance but only in males. Our results point to both hardening and cross-tolerance between cold and heat shocks.

  1. LETHAL EFFECT OF MICROWAVE TREATMENT ON Tribolium castaneum(Herbst)%微波处理对赤拟谷盗各虫态的致死效果

    Institute of Scientific and Technical Information of China (English)

    王殿轩; 刘炎; 曹阳; 李淑荣

    2010-01-01

    分别采用微波功率160、320、480、640和800 W处理裸露的和混合于小麦粉中的各虫态赤拟谷盗Tribolium castaneum(Herbst)的卵、幼虫、蛹和成虫.结果表明:对于裸露的赤拟谷盗用上述功率各处理5、10、15、20、25和30 s时,800 W、25 s可完全致死卵,320 W、30 s可使幼虫和成虫完全致死;800 W、20s可完全致死幼虫、蛹和成虫.对混合于小麦粉中的各虫态赤拟谷盗,当用上述功率各处理20 s时,均不能完全致死各虫态;单独使用800 W分别处理5、10、15、20和25 s时,仅800 W、25 s可完全致死各虫态.微波处理对赤拟谷盗杀虫效果显著.

  2. The negative effect of starvation and the positive effect of mild thermal stress on thermal tolerance of the red flour beetle, Tribolium castaneum

    Science.gov (United States)

    Scharf, Inon; Wexler, Yonatan; MacMillan, Heath Andrew; Presman, Shira; Simson, Eddie; Rosenstein, Shai

    2016-04-01

    The thermal tolerance of a terrestrial insect species can vary as a result of differences in population origin, developmental stage, age, and sex, as well as via phenotypic plasticity induced in response to changes in the abiotic environment. Here, we studied the effects of both starvation and mild cold and heat shocks on the thermal tolerance of the red flour beetle, Tribolium castaneum. Starvation led to impaired cold tolerance, measured as chill coma recovery time, and this effect, which was stronger in males than females, persisted for longer than 2 days but less than 7 days. Heat tolerance, measured as heat knockdown time, was not affected by starvation. Our results highlight the difficulty faced by insects when encountering multiple stressors simultaneously and indicate physiological trade-offs. Both mild cold and heat shocks led to improved heat tolerance in both sexes. It could be that both mild shocks lead to the expression of heat shock proteins, enhancing heat tolerance in the short run. Cold tolerance was not affected by previous mild cold shock, suggesting that such a cold shock, as a single event, causes little stress and hence elicits only weak physiological reaction. However, previous mild heat stress led to improved cold tolerance but only in males. Our results point to both hardening and cross-tolerance between cold and heat shocks.

  3. Residual efficacy of methoprene for control of Tribolium castaneum (Coleoptera: Tenebrionidae) larvae at different temperatures on varnished wood, concrete, and wheat.

    Science.gov (United States)

    Wijayaratne, L K Wolly; Fields, Paul G; Arthur, Frank H

    2012-04-01

    The residual efficacy of the juvenile hormone analog methoprene (Diacon II) was evaluated in bioassays using larvae of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) exposed on unsealed concrete or varnished wood treated with a liquid formulation and held at different temperatures. When these two types of surfaces were stored at 20, 30 or 35 degrees C for 0-24 wk, the percentage of adult emergence on concrete increased with time. In contrast, there was no adult emergence from larvae exposed to varnished wood at 24 wk after treatment at any of these temperatures. The presence of flour reduced residual efficacy of methoprene on concrete, but not on varnished wood, with no differences between cleaning frequencies. Methoprene was also stable for 48 h on concrete held at 65 degrees C and wheat, Triticum aestivum L., held at 46 degrees C. Results show that methoprene is stable at a range of temperatures commonly encountered in indoor food storage facilities and at high temperatures attained during insecticidal heat treatments of structures. The residual persistence of methoprene applied to different surface substrates may be affected more by the substrate than by temperature.

  4. Efficacy of chlorfenapyr against adult Tribofium castaneum exposed on concrete: effects of exposure interval, concentration and the presence of a food source after exposure

    Institute of Scientific and Technical Information of China (English)

    Frank H. Arthur

    2009-01-01

    Chlorfenapyr, an insecticidal pyrrole, was applied to concrete arenas at concentrations of 1.1, 0.825, 0.55, and 0.275 g of active ingredient [AI]/m2. Adult Tribolium castaneum (Herbst), the red flour beetle, were exposed for 2, 4, or 8 h at each concentration, then removed and held either with or without food (wheat flour) for 7 days. Survival was assessed when the beetles were removed from the exposure arenas and daily during the post-exposure period. In the presence of food, survival was high regardless of concentration and the day on which post-treatment survival was assessed, but survival did decrease as the exposure period increased from 4 to 8 h. When the beetles were not given food after exposure, survival at each concentration and exposure period declined during the 1-week post-exposure assessments. This pattern of decrease could be described by linear and non-linear equations. Results show the presence of food material greatly compromised effectiveness of the insecticide, and emphasize the importance of cleaning and sanitation in conjunction with insecticide treatments.

  5. Potential of embryonic and adult stem cells in vitro.

    Science.gov (United States)

    Czyz, Jaroslaw; Wiese, Cornelia; Rolletschek, Alexandra; Blyszczuk, Przemyslaw; Cross, Michael; Wobus, Anna M

    2003-01-01

    Recent developments in the field of stem cell research indicate their enormous potential as a source of tissue for regenerative therapies. The success of such applications will depend on the precise properties and potentials of stem cells isolated either from embryonic, fetal or adult tissues. Embryonic stem cells established from the inner cell mass of early mouse embryos are characterized by nearly unlimited proliferation, and the capacity to differentiate into derivatives of essentially all lineages. The recent isolation and culture of human embryonic stem cell lines presents new opportunities for reconstructive medicine. However, important problems remain; first, the derivation of human embryonic stem cells from in vitro fertilized blastocysts creates ethical problems, and second, the current techniques for the directed differentiation into somatic cell populations yield impure products with tumorigenic potential. Recent studies have also suggested an unexpectedly wide developmental potential of adult tissue-specific stem cells. Here too, many questions remain concerning the nature and status of adult stem cells both in vivo and in vitro and their proliferation and differentiation/transdifferentiation capacity. This review focuses on those issues of embryonic and adult stem cell biology most relevant to their in vitro propagation and differentiation. Questions and problems related to the use of human embryonic and adult stem cells in tissue regeneration and transplantation are discussed.

  6. Regulation of post-embryonic neuroblasts by Drosophila Grainyhead.

    Science.gov (United States)

    Almeida, Mara S; Bray, Sarah J

    2005-12-01

    The Drosophila post-embryonic neuroblasts (pNBs) are neural stem cells that persist in the larval nervous system where they proliferate to produce neurons for the adult CNS. These pNBs provide a good model to investigate mechanisms regulating the maintenance and proliferation of stem cells. The transcription factor Grainyhead (Grh), which is required for morphogenesis of epidermal and tracheal cells, is also expressed in all pNBs. Here, we show that grh is essential for pNBs to adopt the stem cell programme appropriate to their position within the CNS. In grh mutants the abdominal pNBs produced more progeny while the thoracic pNBs, in contrast, divided less and produced fewer progeny than wild type. We investigated three candidates; the Neuroblast identify gene Castor, the signalling molecule Notch and the adhesion protein E-Cadherin, to determine whether they could mediate these effects. Neither Castor nor Notch fulfilled the criteria for intermediaries, and in particular Notch activity was found to be dispensable for the normal proliferation and survival of the pNBs. In contrast E-Cadherin, which has been shown to regulate pNB proliferation, was present at greatly reduced levels in the grh mutant pNBs. Furthermore, ectopic expression of Grh was sufficient to promote ectopic E-Cadherin and two conserved Grh-binding sites were identified in the E-Cadherin/shotgun flanking sequences, arguing that this gene is a downstream target. Thus one way Grh could regulate pNBs is through expression of E-cadherin, a protein that is thought to mediate interactions with the glial niche.

  7. Nucleosome Organization in Human Embryonic Stem Cells.

    Science.gov (United States)

    Yazdi, Puya G; Pedersen, Brian A; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Chen, Yumay; Jacobsen, Steven E; Wang, Ping H

    2015-01-01

    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 analysis of nucleosome

  8. 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

  9. Stable isotope labelling with amino acids in cell culture for human embryonic stem cell proteomic analysis

    DEFF Research Database (Denmark)

    Harkness, Linda; Prokhorova, Tatyana A; Kassem, Moustapha

    2012-01-01

    The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass...... spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we...

  10. Differentiation of embryonic stem cells transfected by ibeB gene

    Institute of Scientific and Technical Information of China (English)

    SHANG Deshu; FANG Wengang; CHEN Yuhua

    2005-01-01

    We have previously identified an E. coli deter- minant, ibeB gene locus contributing to invasion of human brain microvascular endothelial cells. In the present study, we established embryonic stem (ES) cell lines overexpressing IbeB and found that exogenic ibeB gene could start-up expression of a neural stem cell specific marker, nestin, and give rise to polar changes. In analysis of IbeB location, it was found that GFP-IbeB fusion protein targeted at the ES cell nucleus. These data suggests that ibeB gene may play an important role in the regulation of nestin expression.

  11. Cannabinoid Receptor-2 Regulates Embryonic Hematopoietic Stem Cell Development via Prostaglandin E2 and P-Selectin Activity.

    Science.gov (United States)

    Esain, Virginie; Kwan, Wanda; Carroll, Kelli J; Cortes, Mauricio; Liu, Sarah Y; Frechette, Gregory M; Sheward, Lea M V; Nissim, Sahar; Goessling, Wolfram; North, Trista E

    2015-08-01

    Cannabinoids (CB) modulate adult hematopoietic stem and progenitor cell (HSPCs) function, however, impact on the production, expansion, or migration of embryonic HSCs is currently uncharacterized. Here, using chemical and genetic approaches targeting CB-signaling in zebrafish, we show that CB receptor (CNR) 2, but not CNR1, regulates embryonic HSC development. During HSC specification in the aorta-gonad-mesonephros (AGM) region, CNR2 stimulation by AM1241 increased runx1;cmyb(+) HSPCs, through heightened proliferation, whereas CNR2 antagonism decreased HSPC number; FACS analysis and absolute HSC counts confirmed and quantified these effects. Epistatic investigations showed AM1241 significantly upregulated PGE2 synthesis in a Ptgs2-dependent manner to increase AGM HSCs. During the phases of HSC production and colonization of secondary niches, AM1241 accelerated migration to the caudal hematopoietic tissue (CHT), the site of embryonic HSC expansion, and the thymus; however these effects occurred independently of PGE2. Using a candidate approach for HSC migration and retention factors, P-selectin was identified as the functional target of CNR2 regulation. Epistatic analyses confirmed migration of HSCs into the CHT and thymus was dependent on CNR2-regulated P-selectin activity. Together, these data suggest CNR2-signaling optimizes the production, expansion, and migration of embryonic HSCs by modulating multiple downstream signaling pathways.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-09-01

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

  13. Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells

    DEFF Research Database (Denmark)

    Melo-Braga, Marcella Nunes; Schulz, Melanie; Liu, Qiuyue;

    2014-01-01

    Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important......ESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well...... in which 78% of phosphopeptides were identified with ≥99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development...

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

    Science.gov (United States)

    Brenneis, Georg; Scholtz, Gerhard

    2014-01-01

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

  15. 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

  16. Evolution of the mammalian embryonic pluripotency gene regulatory network

    Science.gov (United States)

    Fernandez-Tresguerres, Beatriz; Cañon, Susana; Rayon, Teresa; Pernaute, Barbara; Crespo, Miguel; Torroja, Carlos; Manzanares, Miguel

    2010-01-01

    Embryonic pluripotency in the mouse is established and maintained by a gene-regulatory network under the control of a core set of transcription factors that include octamer-binding protein 4 (Oct4; official name POU domain, class 5, transcription factor 1, Pou5f1), sex-determining region Y (SRY)-box containing gene 2 (Sox2), and homeobox protein Nanog. Although this network is largely conserved in eutherian mammals, very little information is available regarding its evolutionary conservation in other vertebrates. We have compared the embryonic pluripotency networks in mouse and chick by means of expression analysis in the pregastrulation chicken embryo, genomic comparisons, and functional assays of pluripotency-related regulatory elements in ES cells and blastocysts. We find that multiple components of the network are either novel to mammals or have acquired novel expression domains in early developmental stages of the mouse. We also find that the downstream action of the mouse core pluripotency factors is mediated largely by genomic sequence elements nonconserved with chick. In the case of Sox2 and Fgf4, we find that elements driving expression in embryonic pluripotent cells have evolved by a small number of nucleotide changes that create novel binding sites for core factors. Our results show that the network in charge of embryonic pluripotency is an evolutionary novelty of mammals that is related to the comparatively extended period during which mammalian embryonic cells need to be maintained in an undetermined state before engaging in early differentiation events. PMID:21048080

  17. Embryonic development of Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida, Paludicola).

    Science.gov (United States)

    Vara, D C; Leal-Zanchet, A M; Lizardo-Daudt, H m

    2008-11-01

    The embryonic development of freshwater triclads is mainly known from studies of species of Dendrocoelum, Planaria, Polycelis, and, more recently, Schmidtea. The present study characterizes the development of Girardia tigrina (Girard, 1850) by means of optical microcopy using glycol methacrylate semi-thin sections. 94 cocoons were collected in the period from laying to hatching, with intervals of up to twenty-four hours. The sequence of morphological changes occurring in the embryo permitted the identification of nine embryonic stages. At the time of cocoon laying, numerous embryos were dispersed among many yolk cells, with a rigid capsule covering the entire cocoon. In the first stage (approx. up to 6 hours after cocoon laying), yolk cells and embryonic cells showed random distribution. Stage II (between 12 and 24 hours after cocoon laying) is characterized by aggregates of blastomeres, which later aggregate forming an enteroblastula. Approximately 2 days after cocoon laying (stage III), formation of the embryonic epidermis and embryonic digestive system took place, the latter degenerating during the subsequent stage. Stage V (until the fourth day) is characterized by the formation of the definitive epidermis. Between 4 and 6 days after laying, organogenesis of the definitive inner organs starts (stage VI). Approximately 14 days after laying (stage IX), formation of the nervous system is completed. At this stage, the embryo shows similar characteristics to those of newly hatched juveniles. The hatching of Girardia tigrina occurs in the period between twelve to twenty-two days after cocoon laying.

  18. Population growth rate and genetic variability of small and large populations of Red flour beetle (Tribolium castaneum) following multigenerational exposure to copper.

    Science.gov (United States)

    Laskowski, Ryszard; Radwan, Jacek; Kuduk, Katarzyna; Mendrok, Magdalena; Kramarz, Paulina

    2015-07-01

    We reared large (1000 individuals) and small (20 individuals) populations of Tribolium castaneum on diet contaminated with copper in order to determine if the size of a population affects its ability to adapt to adverse environmental conditions. After 10 generations, we used microsatellite markers to estimate and subsequently compare the genetic variability of the copper-treated populations with that of the control populations, which were reared on uncontaminated medium. Additionally, we conducted a full cross-factorial experiment which evaluated the effects of 10 generations of "pre-exposure" to copper on a population's fitness in control and copper-contaminated environments. In order to distinguish results potentially arising from genetic adaptation from those due to non-genetic effects associated to parental exposure to copper, we subjected also F11 generation, originating from parents not exposed to copper, to the same cross-factorial experiment. The effects of long-term exposure to copper depended on population size: the growth rates of small populations that were pre-exposed to copper were inhibited compared to those of small populations reared in uncontaminated environments. Large Cu-exposed populations had a higher growth rate in the F10 generation compared to the control groups, while the growth rate of the F11 generation was unaffected by copper exposure history. The only factor that had a significant effect on genetic variability was population size, but this was to be expected given the large difference in the number of individuals between large and small populations. Neither copper contamination nor its interaction with population size affected the number of microsatellite alleles retained in the F10 generation.

  19. Metamorphic labral axis patterning in the beetle Tribolium castaneum requires multiple upstream, but few downstream, genes in the appendage patterning network.

    Science.gov (United States)

    Smith, Frank W; Angelini, David R; Gaudio, Matthew S; Jockusch, Elizabeth L

    2014-03-01

    The arthropod labrum is an anterior appendage-like structure that forms the dorsal side of the preoral cavity. Conflicting interpretations of fossil, nervous system, and developmental data have led to a proliferation of scenarios for labral evolution. The best supported hypothesis is that the labrum is a novel structure that shares development with appendages as a result of co-option. Here, we use RNA interference in the red flour beetle Tribolium castaneum to compare metamorphic patterning of the labrum to previously published data on ventral appendage patterning. As expected under the co-option hypothesis, depletion of several genes resulted in similar defects in the labrum and ventral appendages. These include proximal deletions and proximal-to-distal transformations resulting from depletion of the leg gap genes homothorax and extradenticle, large-scale deletions resulting from depletion of the leg gap gene Distal-less, and smaller distal deletions resulting from knockdown of the EGF ligand Keren. However, depletion of dachshund and many of the genes that function downstream of the leg gap genes in the ventral appendages had either subtle or no effects on labral axis patterning. This pattern of partial similarity suggests that upstream genes act through different downstream targets in the labrum. We also discovered that many appendage axis patterning genes have roles in patterning the epipharyngeal sensillum array, suggesting that they have become integrated into a novel regulatory network. These genes include Notch, Delta, and decapentaplegic, and the transcription factors abrupt, bric à brac, homothorax, extradenticle and the paralogs apterous a and apterous b.

  20. Unexpected functional diversity in the fatty acid desaturases of the flour beetle Tribolium castaneum and identification of key residues determining activity.

    Science.gov (United States)

    Haritos, Victoria S; Horne, Irene; Damcevski, Katherine; Glover, Karen; Gibb, Nerida

    2014-08-01

    Desaturases catalyse modifications to fatty acids which are essential to homeostasis and for pheromone and defensive chemical production. All desaturases of the flour beetle Tribolium castaneum were investigated via query of the sequenced genome which yielded 15 putative acyl-Coenzyme A genes. Eleven desaturase mRNA were obtained in full length and functionally expressed in yeast. Phylogenetic analysis separated the desaturases into 4 distinct clades; one clade contained conserved beetle Δ9 desaturases, second clade was Tribolium-specific having diverse activities including Δ5, Δ9 and Δ12 desaturation and the other 2 clades had mixed insect representatives. Three members of this clade contained unusual inserted sequences of ∼20 residues in the C-terminal region and were related to desaturases that all contained similar inserts. Deletion of the entirety of the insert in the flour beetle Δ12 desaturase abolished its activity but this was partially restored by the reintroduction of two histidine residues, suggesting the histidine(s) are required for activity but the full length insert is not. Five new desaturase activities were discovered: Δ9 desaturation of C12:0-C16:0 substrates; two unprecedented Δ5 enzymes acting on C18:0 and C16:0; Δ9 activity exclusively on C16:0 and a further stearate Δ9 desaturase. qPCR analysis ruled out a role in sex pheromone synthesis for the Δ5 and Δ9/C16:0 desaturases. The flour beetle genome has underpinned an examination of all transcribed desaturases in the organism and revealed a diversity of novel and unusual activities, an improved understanding of the evolutionary relationships among insect desaturases and sequence determinants of activity.

  1. Germ-cell cluster formation in the telotrophic meroistic ovary of Tribolium castaneum (Coleoptera, Polyphaga, Tenebrionidae) and its implication on insect phylogeny.

    Science.gov (United States)

    Trauner, Jochen; Büning, Jürgen

    2007-01-01

    Tribolium castaneum has telotrophic meroistic ovarioles of the Polyphaga type. During larval stages, germ cells multiply in a first mitotic cycle forming many small, irregularly branched germ-cell clusters which colonize between the anterior and posterior somatic tissues in each ovariole. Because germ-cell multiplication is accompanied by cluster splitting, we assume a very low number of germ cells per ovariole at the beginning of ovariole development. In the late larval and early pupal stages, we found programmed cell death of germ-cell clusters that are located in anterior and middle regions of the ovarioles. Only those clusters survive that rest on posterior somatic tissue. The germ cells that are in direct contact with posterior somatic cells transform into morphologically distinct pro-oocytes. Intercellular bridges interconnecting pro-oocytes are located posteriorly and are filled with fusomes that regularly fuse to form polyfusomes. Intercellular bridges connecting pro-oocytes to pro-nurse cells are always positioned anteriorly and contain small fusomal plugs. During pupal stages, a second wave of metasynchronous mitoses is initiated by the pro-oocytes, leading to linear subclusters with few bifurcations. We assume that the pro-oocytes together with posterior somatic cells build the center of determination and differentiation of germ cells throughout the larval, pupal, and adult stages. The early developmental pattern of germ-cell multiplication is highly similar to the events known from the telotrophic ovary of the Sialis type. We conclude that among the common ancestors of Neuropterida and Coleoptera, a telotrophic meroistic ovary of the Sialis type evolved, which still exists in Sialidae, Raphidioptera, and a myxophagan Coleoptera family, the Hydroscaphidae. Consequently, the telotrophic ovary of the Polyphaga type evolved from the Sialis type.

  2. Characterizing cancer cells with cancer stem cell-like features in 293T human embryonic kidney cells

    OpenAIRE

    Buchholz Thomas A; Lacerda Lara; Xu Wei; Robertson Fredika; Ueno Naoto T; Lucci Anthony; Landis Melissa D; Rodriguez Angel A; Li Li; Cohen Evan; Gao Hui; Krishnamurthy Savitri; Zhang Xiaomei; Debeb Bisrat G; Cristofanilli Massimo

    2010-01-01

    Abstract Background Since the first suggestion of prospectively identifiable cancer stem cells in solid tumors, efforts have been made to characterize reported cancer stem cell surrogates in existing cancer cell lines, and cell lines rich with these surrogates have been used to screen for cancer stem cell targeted agents. Although 293T cells were derived from human embryonic kidney, transplantation of these cells into the mammary fat pad yields aggressive tumors that self-renew as evidenced b...

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

    Directory of Open Access Journals (Sweden)

    Paola A Bignone

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

  4. Human embryonic stem cell technologies and drug discovery.

    Science.gov (United States)

    Jensen, Janne; Hyllner, Johan; Björquist, Petter

    2009-06-01

    Development of new drugs is costly and takes huge resources into consideration. The big pharmaceutical companies are currently facing increasing developmental costs and a lower success-rate of bringing new compounds to the market. Therefore, it is now of outmost importance that the drug-hunting companies minimize late attritions due to sub-optimal pharmacokinetic properties or unexpected toxicity when entering the clinical programs. To achieve this, a strong need to test new candidate drugs in assays of high human relevance in vitro as early as possible has been identified. The traditionally used cell systems are however remarkably limited in this sense, and new improved technologies are of greatest importance. The human embryonic stem cells (hESC) is one of the most powerful cell types known. They have not only the possibility to divide indefinitely; these cells can also differentiate into all mature cell types of the human body. This makes them potentially very valuable for pharmaceutical development, spanning from use as tools in early target studies, DMPK or safety assessment, as screening models to find new chemical entities modulating adult stem cell fate, or as the direct use in cell therapies. This review illustrates the use of hESC in the drug discovery process, today, as well as in a future perspective. This will specifically be exemplified with the most important cell type for pharmaceutical development-the hepatocyte. We discuss how hESC-derived hepatocyte-like cells could improve this process, and how these cells should be cultured if optimized functionality and usefulness should be achieved. J. Cell. Physiol. 219: 513-519, 2009. (c) 2009 Wiley-Liss, Inc.

  5. Cryopreservation of human embryonic stem cells by vitrification

    Institute of Scientific and Technical Information of China (English)

    周灿权; 麦庆云; 李涛; 庄广伦

    2004-01-01

    Background The efficiency of traditional cryopreservation of human embryonic stem (ES) cells is low, and there have been few attempts to prove new cryopreservation methods effective. This study was designed to evaluate the efficiency of cryopreservation of human ES cells using vitrification method.Methods Human ES cells clumped from an identical cell line were randomly allocated to be cryopreserved by vitrification or by slow freezing. The recovery rates, the growth and differentiation potential of thawed human ES cells were compared between these two groups. The pluripotency of human ES cells after thawing was identified.Results Eighty-one point nine percent (59/72) of human ES cell clumps were recovered after vitrification, while only 22.8% (16/70) were recovered after slow freezing (P<0.01). The colonies after vitrification manifested have not only faster growth but also a lower level of differentiation when compared to colonies subjected to the slow freezing protocol. However, the rates of growth and differentiation in undifferentiated colonies from both groups were identical to the rates in those of non-cryopreserved stem cells after a prolonged culture period. Passage 6 of vitrified human ES cells retained the properties of pluripotent cells, a normal karyotype and expressed the transcription factor OCT-4, stage specific expressed antigen-4 (SSEA-4) and SSEA-3. Teratoma growth of these cells demonstrated the ability to develop into all three germ layers.Conclusions Vitrification is effective in cryopreserving human ES cells. During a prolonged culture, human ES cells retain their pluripotency after cryopreservation.

  6. Insecticide activity of essential oils of Mentha longifolia, Pulicaria gnaphalodes and Achillea wilhelmsii against two stored product pests, the flour beetle, Tribolium castaneum, and the cowpea weevil, Callosobruchus maculatus.

    Science.gov (United States)

    Khani, Abbas; Asghari, Javad

    2012-01-01

    Essential oils extracted from the foliage of Mentha longifolia (L.) (Lamiales: Lamiaceae) and Pulicaria gnaphalodes Ventenat (Asterales: Asteraceae), and flowers of Achillea wilhelmsii C. Koch (Asterales: Asteraceae) were tested in the laboratory for volatile toxicity against two storedproduct insects, the flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) and the cowpea weevil, Callosobruchus maculatus F. (Coleoptera: Bruchidae). The chemical composition of the isolated oils was examined by gas chromatography-mass spectrometry. InM longifolia, the major compounds were piperitenon (43.9%), tripal (14.3%), oxathiane (9.3%), piperiton oxide (5.9%), and d-limonene (4.3%). In P. gnaphalodes, the major compounds were chrysanthenyl acetate (22.38%), 2L -4L-dihydroxy eicosane (18.5%), verbenol (16.59%), dehydroaromadendrene (12.54%), β-pinen (6.43%), and 1,8 cineol (5.6%). In A. wilhelmsii, the major compounds were 1,8 cineole (13.03%), caranol (8.26%), alpha pinene (6%), farnesyl acetate (6%), and p-cymene (6%). C maculatus was more susceptible to the tested plant products than T castaneum. The oils of the three plants displayed the same insecticidal activity against C. maculatus based on LC(50) values (between 1.54µl/L air in P. gnaphalodes, and 2.65 µl/L air in A. wilhelmsii). While the oils of A. wilhelmsii and M. longifolia showed the same strong insecticidal activity against T. castaneum (LC(50) = 10.02 and 13.05 µl/L air, respectively), the oil of P. gnaphalodes revealed poor activity against the insect (LC(50) = 297.9 µl/L air). These results suggested that essential oils from the tested plants could be used as potential control agents for stored-product insects.

  7. New tools for the identification of developmentally regulated enhancer regions in embryonic and adult zebrafish.

    Science.gov (United States)

    Levesque, Mitchell P; Krauss, Jana; Koehler, Carla; Boden, Cindy; Harris, Matthew P

    2013-03-01

    We have conducted a screen to identify developmentally regulated enhancers that drive tissue-specific Gal4 expression in zebrafish. We obtained 63 stable transgenic lines with expression patterns in embryonic or adult zebrafish. The use of a newly identified minimal promoter from the medaka edar locus resulted in a relatively unbiased set of expression patterns representing many tissue types derived from all germ layers. Subsequent detailed characterization of selected lines showed strong and reproducible Gal4-driven GFP expression in diverse tissues, including neurons from the central and peripheral nervous systems, pigment cells, erythrocytes, and peridermal cells. By screening adults for GFP expression, we also isolated lines expressed in tissues of the adult zebrafish, including scales, fin rays, and joints. The new and efficient minimal promoter and large number of transactivating driver-lines we identified will provide the zebrafish community with a useful resource for further enhancer trap screening, as well as precise investigation of tissue-specific processes in vivo.

  8. A recessive genetic screen for components of the RNA interference pathway in mouse embryonic stem cells.

    Science.gov (United States)

    Trombly, Melanie I; Wang, Xiaozhong

    2010-01-01

    Several key components of the RNA interference (RNAi) pathway were identified in genetic screens performed in nonmammalian model organisms. To identify components of the mammalian RNAi pathway, we developed a recessive genetic screen in mouse embryonic stem (ES) cells. Recessive genetic screens are feasible in ES cells that are Bloom-syndrome protein (Blm-) deficient. Therefore, we constructed a reporter cell line in Blm-deficient ES cells to isolate RNAi mutants through a simple drug-selection scheme. This chapter describes how we used retroviral gene traps to mutagenize the reporter cell line and select for RNAi mutants. Putative RNAi mutants were confirmed using a separate functional assay. The location of the gene trap was then identified using molecular techniques such as Splinkerette PCR. Our screening strategy successfully isolated several mutant clones of Argonaute2, a vital component of the RNAi pathway.

  9. 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.

  10. Microfluidics for gametes, embryos, and embryonic stem cells.

    Science.gov (United States)

    Smith, G D; Swain, J E; Bormann, C L

    2011-01-01

    Microfluidics is a young but established field that holds significant potential for scientific discovery. The utility of microfluidics can improve our knowledge of basic biology as well as expand our understanding in specialized areas such as assisted reproduction and stem cell developmental biology. This review describes the technology of microfluidics and discusses applications within assisted reproduction technology and embryonic stem cell growth and directed differentiation. Development of an integrated microfluidic platform for assisted reproduction, which can manipulate gametes, embryos, embryonic stem cells, their culture environment, and incorporate biomarker analysis, could have a dramatic impact on the basic understanding of embryo/embryonic stem cell development, as well as provide significant improvements in current technologies used to treat infertility, preserve fertility, and derive therapeutic cells from stem cells.

  11. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

    Institute of Scientific and Technical Information of China (English)

    Qingyun Mai; Yang Yu; Tao Li; Liu Wang; Mei-jue Chen; Shu-zhen Huang; Canquan Zhou; Qi Zhou

    2007-01-01

    Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

  12. Efficiency of haplotype-based methods to fine-map QTLs and embryonic lethals variants affecting fertility: illustration with a deletion segregating in Nordic Red cattle Corresponding

    DEFF Research Database (Denmark)

    Kadri, Naveen Kumar; Sahana, Goutam; Guldbrandtsen, Bernt;

    2014-01-01

    -negligible fraction of the fertility decline. Therefore identification of such embryonic lethal variants is essential to improve fertility. We herein illustrate, with an example of a large recessive lethal deletion recently identified in Nordic Red cattle, that haplotype-based method are particularly efficient...

  13. PICKLE acts throughout the plant to repress expression of embryonic traits and may play a role in Giberellin-Dependent responses

    NARCIS (Netherlands)

    Henderson, J.; Li, H.C.; Mordhorst, A.P.; Romero-Severson, J.; Cheng, J.C.; Robey, J.; Sung, Z.R.; Vries, de S.C.; Ogas, J.

    2004-01-01

    A seed marks the transition between two developmental states; a plant is an embryo during seed formation, whereas it is a seedling after emergence from the seed. Two factors have been identified in Arabidopsis that play a role in establishment of repression of the embryonic state: PKL (PICKLE), whic

  14. Metabolomic Analysis of the Secretome of Human Embryonic Stem Cells Following Methyl Parathion and Methyl Paraoxon Exposure. Phase 3. LC-MS-MS Structural Confirmation

    Science.gov (United States)

    2014-01-01

    drug, valproate, has a dramatic toxic effect on embryonic development; leading to neural tube deficits, autism , and cognitive dysfunction. In their...TR-1178) down- selected the putatively identified metabolites for follow-on definitive structural confirmation using several criteria. That subset of

  15. Disruption of G-protein γ5 subtype causes embryonic lethality in mice.

    Directory of Open Access Journals (Sweden)

    Anne M Moon

    Full Text Available Heterotrimeric G-proteins modulate many processes essential for embryonic development including cellular proliferation, migration, differentiation, and survival. Although most research has focused on identifying the roles of the various αsubtypes, there is growing recognition that similarly divergent βγ dimers also regulate these processes. In this paper, we show that targeted disruption of the mouse Gng5 gene encoding the γ5 subtype produces embryonic lethality associated with severe head and heart defects. Collectively, these results add to a growing body of data that identify critical roles for the γ subunits in directing the assembly of functionally distinct G-αβγ trimers that are responsible for regulating diverse biological processes. Specifically, the finding that loss of the G-γ5 subtype is associated with a reduced number of cardiac precursor cells not only provides a causal basis for the mouse phenotype but also raises the possibility that G-βγ5 dependent signaling contributes to the pathogenesis of human congenital heart problems.

  16. Systematic identification of cis-regulatory sequences active in mouse and human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Marica Grskovic

    2007-08-01

    Full Text Available Understanding the transcriptional regulation of pluripotent cells is of fundamental interest and will greatly inform efforts aimed at directing differentiation of embryonic stem (ES cells or reprogramming somatic cells. We first analyzed the transcriptional profiles of mouse ES cells and primordial germ cells and identified genes upregulated in pluripotent cells both in vitro and in vivo. These genes are enriched for roles in transcription, chromatin remodeling, cell cycle, and DNA repair. We developed a novel computational algorithm, CompMoby, which combines analyses of sequences both aligned and non-aligned between different genomes with a probabilistic segmentation model to systematically predict short DNA motifs that regulate gene expression. CompMoby was used to identify conserved overrepresented motifs in genes upregulated in pluripotent cells. We show that the motifs are preferentially active in undifferentiated mouse ES and embryonic germ cells in a sequence-specific manner, and that they can act as enhancers in the context of an endogenous promoter. Importantly, the activity of the motifs is conserved in human ES cells. We further show that the transcription factor NF-Y specifically binds to one of the motifs, is differentially expressed during ES cell differentiation, and is required for ES cell proliferation. This study provides novel insights into the transcriptional regulatory networks of pluripotent cells. Our results suggest that this systematic approach can be broadly applied to understanding transcriptional networks in mammalian species.

  17. Cell cycle regulation and cytoskeletal remodelling are critical processes in the nutritional programming of embryonic development.

    Science.gov (United States)

    Swali, Angelina; McMullen, Sarah; Hayes, Helen; Gambling, Lorraine; McArdle, Harry J; Langley-Evans, Simon C

    2011-01-01

    Many mechanisms purport to explain how nutritional signals during early development are manifested as disease in the adult offspring. While these describe processes leading from nutritional insult to development of the actual pathology, the initial underlying cause of the programming effect remains elusive. To establish the primary drivers of programming, this study aimed to capture embryonic gene and protein changes in the whole embryo at the time of nutritional insult rather than downstream phenotypic effects. By using a cross-over design of two well established models of maternal protein and iron restriction we aimed to identify putative common "gatekeepers" which may drive nutritional programming.Both protein and iron deficiency in utero reduced the nephron complement in adult male Wistar and Rowett Hooded Lister rats (Pmolecular mechanisms which may initiate the sequelae of events involved in nutritional programming of embryonic development. We propose that despite differences in the individual genes and proteins affected in each strain and with each diet, the general response to nutrient deficiency in utero is perturbation of the cell cycle, at the level of interaction with the cytoskeleton and the mitotic checkpoints, thereby diminishing control over the integrity of DNA which is allowed to replicate. These findings offer novel insight into the primary causes and mechanisms leading to the pathologies which have been identified by previous programming studies.

  18. ISOLATION AND EXPANSION OF HUMAN EMBRYONIC NEURAL STEM/PROGENITOR CELLS IN VITRO

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective: To isolate, culture and identify human embryonic neural stem cells and to establish a practical passaging method. Method:The cerebral cortex cells were isolated from aborted embryos (11~13 weeks)by mechanical dissociation, and cultured in DMEM/F12 culture medium supplemented with N2 and growth factors for proliferation. Upon passaging, the neurospheres were pipetted gentlely to separate them into several cell masses and then grown in growth medium. The cells were grown in DMEM/F12 medium with serum (without growth factors) to induce differentiation. The stem cell, neuron, astrocyte and oligodendrocyte were identified by immunocytochemistry with antibodies to vimentin, MAP2, GFAP and GalC, respectively. Results:The primary cells grew together and formed neurospheres at 5th~7th day. They were all vimentin positive and could be passaged for at least 8passages. After passaging, the cell masses grew up and formed new neurospheres rapidly. These cells could differentiated into MAP2 ( + ), GFAP( + ) or GalC( + ) cells. Conclusion: The neural stem cells from human embryonic cerebral cortex have the capacity of proliferation and multi - differentiation in vitro. The passaging methods we used in this experiment were practical and convenient.

  19. Single-cell RNA sequencing identifies distinct mouse medial ganglionic eminence cell types

    Science.gov (United States)

    Chen, Ying-Jiun J.; Friedman, Brad A.; Ha, Connie; Durinck, Steffen; Liu, Jinfeng; Rubenstein, John L.; Seshagiri, Somasekar; Modrusan, Zora

    2017-01-01

    Many subtypes of cortical interneurons (CINs) are found in adult mouse cortices, but the mechanism generating their diversity remains elusive. We performed single-cell RNA sequencing on the mouse embryonic medial ganglionic eminence (MGE), the major birthplace for CINs, and on MGE-like cells differentiated from embryonic stem cells. Two distinct cell types were identified as proliferating neural progenitors and immature neurons, both of which comprised sub-populations. Although lineage development of MGE progenitors was reconstructed and immature neurons were characterized as GABAergic, cells that might correspond to precursors of different CINs were not identified. A few non-neuronal cell types were detected, including microglia. In vitro MGE-like cells resembled bona fide MGE cells but expressed lower levels of Foxg1 and Epha4. Together, our data provide detailed understanding of the embryonic MGE developmental program and suggest how CINs are specified. PMID:28361918

  20. Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells

    OpenAIRE

    2015-01-01

    Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4...

  1. Research Resource: The Dexamethasone Transcriptome in Hypothalamic Embryonic Neural Stem Cells.

    Science.gov (United States)

    Frahm, Krystle A; Peffer, Melanie E; Zhang, Janie Y; Luthra, Soumya; Chakka, Anish B; Couger, Matthew B; Chandran, Uma R; Monaghan, A Paula; DeFranco, Donald B

    2016-01-01

    Exposure to excess glucocorticoids during fetal development has long-lasting physiological and behavioral consequences, although the mechanisms are poorly understood. The impact of prenatal glucocorticoids exposure on stress responses in juvenile and adult offspring implicates the developing hypothalamus as a target of adverse prenatal glucocorticoid action. Therefore, primary cultures of hypothalamic neural-progenitor/stem cells (NPSCs) derived from mouse embryos (embryonic day 14.5) were used to identify the glucocorticoid transcriptome in both males and females. NPSCs were treated with vehicle or the synthetic glucocorticoid dexamethasone (dex; 100nM) for 4 hours and total RNA analyzed using RNA-Sequencing. Bioinformatic analysis demonstrated that primary hypothalamic NPSC cultures expressed relatively high levels of a number of genes regulating stem cell proliferation and hypothalamic progenitor function. Interesting, although these cells express glucocorticoid receptors (GRs), only low levels of sex-steroid receptors are expressed, which suggested that sex-specific differentially regulated genes identified are mediated by genetic and not hormonal influences. We also identified known or novel GR-target coding and noncoding genes that are either regulated equivalently in male and female NPSCs or differential responsiveness in one sex. Using gene ontology analysis, the top functional network identified was cell proliferation and using bromodeoxyuridine (BrdU) incorporation observed a reduction in proliferation of hypothalamic NPSCs after dexamethasone treatment. Our studies provide the first characterization and description of glucocorticoid-regulated pathways in male and female embryonically derived hypothalamic NPSCs and identified GR-target genes during hypothalamic development. These findings may provide insight into potential mechanisms responsible for the long-term consequences of fetal glucocorticoid exposure in adulthood.

  2. Cytoskeletal heart-enriched actin-associated protein (CHAP) is expressed in striated and smooth muscle cells in chick and mouse during embryonic and adult stages.

    Science.gov (United States)

    van Eldik, Willemijn; Beqqali, Abdelaziz; Monshouwer-Kloots, Jantine; Mummery, Christine; Passier, Robert

    2011-01-01

    We recently identified a new Z-disc protein, CHAP (Cytoskeletal Heart-enriched Actin-associated Protein), which is expressed in striated muscle and plays an important role during embryonic muscle development in mouse and zebrafish. Here, we confirm and further extend these findings by (i) the identification and characterization of the CHAP orthologue in chick and (ii) providing a detailed analysis of CHAP expression in mouse during embryonic and adult stages. Chick CHAP contains a PDZ domain and a nuclear localization signal, resembling the human and mouse CHAPa. CHAP is expressed in the developing heart and somites, as well as muscle precursors of the limb buds in mouse and chick embryos. CHAP expression in heart and skeletal muscle is maintained in adult mice, both in slow and fast muscle fibers. Moreover, besides expression in striated muscle, we demonstrate that CHAP is expressed in smooth muscle cells of aorta, carotid and coronary arteries in adult mice, but not during embryonic development.

  3. Preliminary study on human fibroblasts as feeder layer for human embryonic stem cells culture in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    To avoid the direct contact with mouse cells and possible heterogeneous pathogen in future application, we need to replace mouse embryonic fibroblastswith human fibroblasts as the feeder layer to maintain human embryonic stem cells growth in the undifferentiated state. We successfully use human fibroblasts derived from aborted fetus and adult prepuce as feeder layer to maintain human embryonic stem cells growth. During the passage and growth on this feeder layer, the human embryonic stem cells can keep their undifferentiated state.

  4. 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....

  5. Stage-dependent remodeling of the nuclear envelope and lamina during rabbit early embryonic development.

    Science.gov (United States)

    Popken, Jens; Schmid, Volker J; Strauss, Axel; Guengoer, Tuna; Wolf, Eckhard; Zakhartchenko, Valeri

    2016-04-22

    Utilizing 3D structured illumination microscopy, we investigated the quality and quantity of nuclear invaginations and the distribution of nuclear pores during rabbit early embryonic development and identified the exact time point of nucleoporin 153 (NUP153) association with chromatin during mitosis. Contrary to bovine early embryonic nuclei, featuring almost exclusively nuclear invaginations containing a small volume of cytoplasm, nuclei in rabbit early embryonic stages show additionally numerous invaginations containing a large volume of cytoplasm. Small-volume invaginations frequently emanated from large-volume nuclear invaginations but not vice versa, indicating a different underlying mechanism. Large- and small-volume nuclear envelope invaginations required the presence of chromatin, as they were restricted to chromatin-positive areas. The chromatin-free contact areas between nucleolar precursor bodies (NPBs) and large-volume invaginations were free of nuclear pores. Small-volume invaginations were not in contact with NPBs. The number of invaginations and isolated intranuclear vesicles per nucleus peaked at the 4-cell stage. At this stage, the nuclear surface showed highly concentrated clusters of nuclear pores surrounded by areas free of nuclear pores. Isolated intranuclear lamina vesicles were usually NUP153 negative. Cytoplasmic, randomly distributed NUP153-positive clusters were highly abundant at the zygote stage and decreased in number until they were almost absent at the 8-cell stage and later. These large NUP153 clusters may represent a maternally provided NUP153 deposit, but they were not visible as clusters during mitosis. Major genome activation at the 8- to 16-cell stage may mark the switch from a necessity for a deposit to on-demand production. NUP153 association with chromatin is initiated during metaphase before the initiation of the regeneration of the lamina. To our knowledge, the present study demonstrates for the first time major remodeling

  6. Ligand-induced fate of embryonic species in the shape-controlled synthesis of rhodium nanoparticles.

    Science.gov (United States)

    Biacchi, Adam J; Schaak, Raymond E

    2015-02-24

    The shapes of noble metal nanoparticles directly impact their properties and applications, including in catalysis and plasmonics, and it is therefore important to understand how multiple distinct morphologies can be controllably synthesized. Solution routes offer powerful capabilities for shape-controlled nanoparticle synthesis, but the earliest stages of the reaction are difficult to interrogate experimentally and much remains unknown about how metal nanoparticle morphologies emerge and evolve. Here, we use a well-established polyol process to synthesize uniform rhodium nanoparticle cubes, icosahedra, and triangular plates using bromide, trifluoroacetate, and chloride ligands, respectively. In all of these systems, we identified rhodium clusters with diameters of 1-2 nm that form early in the reactions. The colloidally stable metal cluster intermediates served as a stock solution of embryonic species that could be transformed predictably into each type of nanoparticle morphology. The anionic ligands that were added to the embryonic species determined their eventual fate, e.g., the morphologies into which they would ultimately evolve. Extensive high-resolution transmission electron microscopy experiments revealed that the growth pathway-monomer addition, coalescence, or a combination of the two-was different for each of the morphologies, and was likely controlled by the interactions of each specific anionic adsorbate with the embryonic species. Similar phenomena were observed for related palladium and platinum nanoparticle systems. These studies provide important insights into how noble metal nanoparticles nucleate, the pathways by which they grow into several distinct morphologies, and the imperative role of the anonic ligand in controlling which route predominates in a particular system.

  7. Sertad1 encodes a novel transcriptional co-activator of SMAD1 in mouse embryonic hearts

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Yin [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Zhao, Shaomin [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069 (China); Song, Langying [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Wang, Manyuan [School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069 (China); Jiao, Kai, E-mail: kjiao@uab.edu [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

    2013-11-29

    Highlights: •SERTAD1 interacts with SMAD1. •Sertad1 is expressed in mouse embryonic hearts. •SERTAD1 is localized in both cytoplasm and nucleus of cardiomyocytes. •SERTAD1 enhances expression of BMP target cardiogenic genes as a SMAD1 co-activator. -- Abstract: Despite considerable advances in surgical repairing procedures, congenital heart diseases (CHDs) remain the leading noninfectious cause of infant morbidity and mortality. Understanding the molecular/genetic mechanisms underlying normal cardiogenesis will provide essential information for the development of novel diagnostic and therapeutic strategies against CHDs. BMP signaling plays complex roles in multiple cardiogenic processes in mammals. SMAD1 is a canonical nuclear mediator of BMP signaling, the activity of which is critically regulated through its interaction partners. We screened a mouse embryonic heart yeast two-hybrid library using Smad1 as bait and identified SERTAD1 as a novel interaction partner of SMAD1. SERTAD1 contains multiple potential functional domains, including two partially overlapping transactivation domains at the C terminus. The SERTAD1-SMAD1 interaction in vitro and in mammalian cells was further confirmed through biochemical assays. The expression of Sertad1 in developing hearts was demonstrated using RT-PCR, western blotting and in situ hybridization analyses. We also showed that SERTAD1 was localized in both the cytoplasm and nucleus of immortalized cardiomyocytes and primary embryonic cardiomyocyte cultures. The overexpression of SERTAD1 in cardiomyocytes not only enhanced the activity of two BMP reporters in a dose-dependent manner but also increased the expression of several known BMP/SMAD regulatory targets. Therefore, these data suggest that SERTAD1 acts as a SMAD1 transcriptional co-activator to promote the expression of BMP target genes during mouse cardiogenesis.

  8. Bloomsbury report on mouse embryo phenotyping: recommendations from the IMPC workshop on embryonic lethal screening

    Directory of Open Access Journals (Sweden)

    David Adams

    2013-05-01

    Full Text Available Identifying genes that are important for embryo development is a crucial first step towards understanding their many functions in driving the ordered growth, differentiation and organogenesis of embryos. It can also shed light on the origins of developmental disease and congenital abnormalities. Current international efforts to examine gene function in the mouse provide a unique opportunity to pinpoint genes that are involved in embryogenesis, owing to the emergence of embryonic lethal knockout mutants. Through internationally coordinated efforts, the International Knockout Mouse Consortium (IKMC has generated a public resource of mouse knockout strains and, in April 2012, the International Mouse Phenotyping Consortium (IMPC, supported by the EU InfraCoMP programme, convened a workshop to discuss developing a phenotyping pipeline for the investigation of embryonic lethal knockout lines. This workshop brought together over 100 scientists, from 13 countries, who are working in the academic and commercial research sectors, including experts and opinion leaders in the fields of embryology, animal imaging, data capture, quality control and annotation, high-throughput mouse production, phenotyping, and reporter gene analysis. This article summarises the outcome of the workshop, including (1 the vital scientific importance of phenotyping embryonic lethal mouse strains for basic and translational research; (2 a common framework to harmonise international efforts within this context; (3 the types of phenotyping that are likely to be most appropriate for systematic use, with a focus on 3D embryo imaging; (4 the importance of centralising data in a standardised form to facilitate data mining; and (5 the development of online tools to allow open access to and dissemination of the phenotyping data.

  9. Zebrafish embryonic stromal trunk (ZEST) cells support hematopoietic stem and progenitor cell (HSPC) proliferation, survival, and differentiation.

    Science.gov (United States)

    Campbell, Clyde; Su, Tammy; Lau, Ryan P; Shah, Arpit; Laurie, Payton C; Avalos, Brenda; Aggio, Julian; Harris, Elena; Traver, David; Stachura, David L

    2015-12-01

    Forward genetic screens in zebrafish have been used to identify genes essential for the generation of primitive blood and the emergence of hematopoietic stem cells (HSCs), but have not elucidated the genes essential for hematopoietic stem and progenitor cell (HSPC) proliferation and differentiation because of the lack of methodologies to functionally assess these processes. We previously described techniques used to test the developmental potential of HSPCs by culturing them on zebrafish kidney stromal (ZKS) cells, derived from the main site of hematopoiesis in the adult teleost. Here we describe an additional primary stromal cell line we refer to as zebrafish embryonic stromal trunk (ZEST) cells, derived from tissue surrounding the embryonic dorsal aorta, the site of HSC emergence in developing fish. ZEST cells encouraged HSPC differentiation toward the myeloid, lymphoid, and erythroid pathways when assessed by morphologic and quantitative reverse transcription polymerase chain reaction analyses. Additionally, ZEST cells significantly expanded the number of cultured HSPCs in vitro, indicating that these stromal cells are supportive of both HSPC proliferation and multilineage differentiation. Examination of ZEST cells indicates that they express numerous cytokines and Notch ligands and possess endothelial characteristics. Further characterization of ZEST cells should prove to be invaluable in understanding the complex signaling cascades instigated by the embryonic hematopoietic niche required to expand and differentiate HSPCs. Elucidating these processes and identifying possibilities for the modulation of these molecular pathways should allow the in vitro expansion of HSPCs for a multitude of therapeutic uses.

  10. Post-embryonic nerve-associated precursors to adult pigment cells: genetic requirements and dynamics of morphogenesis and differentiation.

    Directory of Open Access Journals (Sweden)

    Erine H Budi

    2011-05-01

    Full Text Available The pigment cells of vertebrates serve a variety of functions and generate a stunning variety of patterns. These cells are also implicated in human pathologies including melanoma. Whereas the events of pigment cell development have been studied extensively in the embryo, much less is known about morphogenesis and differentiation of these cells during post-embryonic stages. Previous studies of zebrafish revealed genetically distinct populations of embryonic and adult melanophores, the ectotherm homologue of amniote melanocytes. Here, we use molecular markers, vital labeling, time-lapse imaging, mutational analyses, and transgenesis to identify peripheral nerves as a niche for precursors to adult melanophores that subsequently migrate to the skin to form the adult pigment pattern. We further identify genetic requirements for establishing, maintaining, and recruiting precursors to the adult melanophore lineage and demonstrate novel compensatory behaviors during pattern regulation in mutant backgrounds. Finally, we show that distinct populations of latent precursors having differential regenerative capabilities persist into the adult. These findings provide a foundation for future studies of post-embryonic pigment cell precursors in development, evolution, and neoplasia.

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

    Directory of Open Access Journals (Sweden)

    Shen-Hsi Yang

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

  12. Expression of the fibroblast growth factor 4 (FGF-4) gene is regulated by serum in Tera-2 embryonal carcinoma cells.

    Science.gov (United States)

    Lucas, J; Knobloch, T; Lang, J

    1996-02-01

    Expression of the fibroblast growth factor 4 (FGF-4) gene is tightly regulated during mammalian development. Dysregulation of FGF-4 gene expression results in cell transformation and tumorigenesis. It is therefore pertinent to investigate the regulatory mechanisms which control expression of FGF-4. In an initial attempt to identify exogenous factors other than retinoic acid which might control FGF-4 expression, we have investigated the response of endogenous FGF-4 to serum in a number of embryonal carcinoma and embryonic stem cell lines. We have identified a human embryonal carcinoma cell line (Tera-2) in which the FGF-4 gene can be induced by serum. In Tera-2 cells made quiescent by serum deprivation, expression of the FGF-4 gene is repressed. Subsequent addition of serum reactivates FGF-LC expression and further addition of cycloheximide results in superinduction of mRNA suggesting that FGF-4 may be classified as an early response gene. It is suggested that this observation may be explained, at least in part, by the stage of differentiation of the Tera-2 cells.

  13. 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...

  14. Adverse effects of advanced glycation end products on embryonal development

    Directory of Open Access Journals (Sweden)

    Hiramatsu,Yuji

    2008-04-01

    Full Text Available We studied the effects of advanced glycation end products (AGEs, which are known to accumulate in patients with diabetes, autoimmune diseases, or those who smoke, on embryonal development. Pronuclear (PN embryos were obtained by flushing the fallopian tubes of rats after superovulation and mating. The cleavage rate and blastocyst yield were evaluated at 24, 72, 96, and 120 h of culture. Glyoxal, an AGE-forming aldehyde, suppressed embryonal development at every stage from PN to blastocyst in a concentration-dependent manner. The cleavage rate of the embryo was also signifi cantly decreased by treatment with glyoxal at concentrations of 1 mM or higher. The blastocyst yield was significantly decreased by treatment with glyoxal at concentrations of 0.5 mM or higher. N-acetyl-L-cysteine (L-NAC at 1 mM significantly suppressed the glyoxal-induced embryonal toxicity. BSA-AGEs at 5 microg/ml or higher concentration signifi cantly reduced the cleavage rate and blastocyst yield compared to those for BSA-treated embryos. L-NAC at 1 mM significantly suppressed BSAAGE-induced embryonal toxicity. Because AGEs are embryo-toxic, AGE contamination may influence the pregnancy rate of in vitro fertilization and embryo transfer. AGEs, which are increased in women under pathological conditions, may also be involved in their infertility.

  15. 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...

  16. Formation of transcription factor complexes during embryonic erythroid development

    NARCIS (Netherlands)

    X. Yu (Xiao)

    2013-01-01

    textabstractHematopoiesis is a classic model for the study of embryonic and adult stem cell differentiation. Erythropoiesis is the process of generating erythrocytes from hematopoietic stem cells (HSC). In Chapter1, we introduce the process of erythropoiesis and discuss proteins and protein complexe

  17. Endothelial cells derived from human embryonic stem cells

    Science.gov (United States)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  18. Dihydroartemisinin promotes angiogenesis during the early embryonic development of zebrafish

    Institute of Scientific and Technical Information of China (English)

    Qian BA; Juan DUAN; Jia-qiang TIAN; Zi-liang WANG; Tao CHEN; Xiao-guang LI; Pei-zhan CHEN

    2013-01-01

    Aim:To investigate the embryotoxicity of dihydroartemisinin (DHA),the main active metabolite of artemisinin,in zebrafish,and explore the corresponding mechanisms.Methods:The embryos of wild type and TG (flk1:GFP) transgenic zebrafish were exposed to DHA.Developmental phenotypes of the embryos were observed.Development of blood vessels was directly observed in living embryos of TG (flk1:GFP) transgenic zebrafish under fluorescence microscope.The expression of angiogenesis marker genes vegfa,flk1,and flt1 in the embryos was detected using real-time PCR and RNA in situ hybridization assays.Results:Exposure to DHA (1-10 mg/L) dose-dependently caused abnormal zebrafish embryonic phenotypes in the early developmental stage.Furthermore,exposure to DHA (10 mg/L) resulted in more pronounced embryonic angiogenesis in TG (flk1:GFP)zebrafish line.Exposure to DHA (10 mg/L) significantly increased the mRNA expression of vegfa,flk1,and flt1 in the embryos.Knockdown of the ilk1 protein partially blocked the effects of DHA on embryogenesis.Conclusion:DHA causes abnormal embryonic phenotypes and promotes angiogenesis in zebrafish early embryonic development,demonstrating the potential embryotoxicity of DHA.

  19. Twenty years of embryonic stem cell research in farm animals

    Science.gov (United States)

    Notable distinctions between an embryonic stem cell (ESC) and somatic cell are that the ESC can maintain an undifferentiated state indefinitely, self renew, and is pluripotent, meaning that the ESC can potentially generate cells representing all the three primordial germ layers and contribute to the...

  20. 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.

  1. 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 different

  2. 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 requ

  3. 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 cardiomyocy

  4. Differentiation of neuroepithelia from human embryonic stem cells

    OpenAIRE

    2009-01-01

    We describe the method for in vitro differentiation of neuroepithelial cells from human embryonic stem cells under a chemically defined condition. The protocol is established following the fundamental principle of in vivo neuroectodermal specification. The primitive neuroepithelial cells generated by this protocol can be further induced into neuronal and glia cells with forebrain, mid/hind brain, and spinal cord identities.

  5. Origin and the embryonic transformations of vertebrate heart conducting system.

    Directory of Open Access Journals (Sweden)

    Tverdokhleb I.V.

    2007-01-01

    Full Text Available As the embryonic structure, the cardiac conduction system must continue to develop in a coordinated manner at all embryonic stages. This requires not only the formation of distinct components of the conduction system, but the integration of these components into a functioning whole. The development of the chambered heart and a conduction system requires the proper arrangement of a number of embryonic building blocks, comprising inflow tract, atria, atrioventricular canal, compact and trabecular ventricular myocardium, and outflow tract. In the mammalian heart, the sinoatrial and atrioventricular nodes will aggregate in the slow-conducting inflow tract and atrioventricular canal. The ventricular conduction system may develop in its entirety from the trabecular ventricular myocardium, the remodeling of which results in a gradual transition toward the compact myocardium. So the development of the conduction system does not require the invention of new building components but a remodeling of existing components. Many details in the fashioning of the embryonic blocks of the heart into the conduction system of the formed heart still need to be worked out. The factors that specify the cardiac building blocks and regulate their coordinated morphogenesis have remained largely unknown. Their identification will benefit from combined molecular, genetic, and morphological approaches.

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

    DEFF Research Database (Denmark)

    Van Hoof, Dennis; Muñoz, Javier; Braam, Stefan R

    2009-01-01

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

  7. 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

  8. Profiling post-translational modifications of histones in neural differentiation of embryonic stem cells using liquid chromatography-mass spectrometry.

    Science.gov (United States)

    Zheng, Shuzhen; Sun, Ming; Zhang, Kai; Gu, Junjie; Guo, Zhenchang; Tian, Shanshan; Zhai, Guijin; He, Xiwen; Jin, Ying; Zhang, Yukui

    2016-04-01

    The neural differentiation of embryonic stem cells (ESCs) is of great significance for understanding of the mechanism of diseases. Histone post-translational modifications (HPTMs) play a key role in the regulation of ESCs differentiation. Here, we combined the stable isotope chemical derivatization with nano-HPLC-mass spectrometry (MS) for comprehensive analysis and quantification of histone post-translational modifications (HPTMs) in mouse embryonic stem cells (mESCs) and neural progenitor cells (mNPCs) that was derived from ESCs. We identified 85 core HPTM sites in ESCs and 78HPTM sites in NPCs including some novel lysine modifications. Our quantitative analysis results further revealed the changes of HPTMs from ESCs to NPCs and suggested effect of combinational HPTMs in the differentiation. This study demonstrates that HPLC-MS-based quantitative proteomics has a considerable advantage on quantification of combinational PTMs and expands our understanding of HPTMs in the differentiation.

  9. Secreted proteoglycans directly mediate human embryonic stem cell-basic fibroblast growth factor 2 interactions critical for proliferation.

    Science.gov (United States)

    Levenstein, Mark E; Berggren, W Travis; Lee, Ji Eun; Conard, Kevin R; Llanas, Rachel A; Wagner, Ryan J; Smith, Lloyd M; Thomson, James A

    2008-12-01

    Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface, and their removal from CM impairs proliferation. Finally, we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography, immunoblotting, and mass spectrometry-based proteomic analysis, we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture.

  10. Publishing SNP genotypes of human embryonic stem cell lines: policy statement of the International Stem Cell Forum Ethics Working Party.

    Science.gov (United States)

    Knoppers, Bartha M; Isasi, Rosario; Benvenisty, Nissim; Kim, Ock-Joo; Lomax, Geoffrey; Morris, Clive; Murray, Thomas H; Lee, Eng Hin; Perry, Margery; Richardson, Genevra; Sipp, Douglas; Tanner, Klaus; Wahlström, Jan; de Wert, Guido; Zeng, Fanyi

    2011-09-01

    Novel methods and associated tools permitting individual identification in publicly accessible SNP databases have become a debatable issue. There is growing concern that current technical and ethical safeguards to protect the identities of donors could be insufficient. In the context of human embryonic stem cell research, there are no studies focusing on the probability that an hESC line donor could be identified by analyzing published SNP profiles and associated genotypic and phenotypic information. We present the International Stem Cell Forum (ISCF) Ethics Working Party's Policy Statement on "Publishing SNP Genotypes of Human Embryonic Stem Cell Lines (hESC)". The Statement prospectively addresses issues surrounding the publication of genotypic data and associated annotations of hESC lines in open access databases. It proposes a balanced approach between the goals of open science and data sharing with the respect for fundamental bioethical principles (autonomy, privacy, beneficence, justice and research merit and integrity).

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  14. File list: Pol.Emb.50.AllAg.Embryonic_heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: Pol.Emb.10.AllAg.Embryonic_brains [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  4. File list: DNS.Emb.50.AllAg.Embryonic_brains [Chip-atlas[Archive

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  8. File list: DNS.Emb.10.AllAg.Embryonic_brains [Chip-atlas[Archive

    Lifescience Database Archive (English)

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