Analysis of Imprinted Gene Expression in Normal Fertilized and Uniparental Preimplantation Porcine Embryos

In the present study quantitative real-time PCR was used to determine the expression status of eight imprinted genes (GRB10, H19, IGF2R, XIST, IGF2, NNAT, PEG1 and PEG10) during preimplantation development, in normal fertilized and uniparental porcine embryos. The results demonstrated that, in all observed embryo samples, a non imprinted gene expression pattern up to the 16-cell stage of development was common for most genes. This was true for all classes of embryo, regardless of parental-origins and the direction of imprint. However, several differentially expressed genes (H19, IGF2, XIST and PEG10) were detected amongst the classes at the blastocyst stage of development. Most interestingly and despite the fact that maternally and paternally expressed genes should not be expressed in androgenones and parthenogenones, respectively, both uniparental embryos expressed these genes when tested for in this study. In order to account for this phenomenon, we compared the expression patterns of eight imprinted genes along with the methylation status of the IGF2/H19 DMR3 in haploid and diploid parthenogenetic embryos. Our findings revealed that IGF2, NNAT and PEG10 were silenced in haploid but not diploid parthenogenetic blastocysts and differential methylation of the IGF2/H19 DMR3 was consistently observed between haploid and diploid parthenogenetic blastocysts. These results appear to suggest that there exists a process to adjust the expression status of imprinted genes in diploid parthenogenetic embryos and that this phenomenon may be associated with altered methylation at an imprinting control region. In addition we believe that imprinted expression occurs in at least four genes, namely H19, IGF2, XIST and PEG10 in porcine blastocyst stage embryos

The role of forensic anthropology in disaster victim identification (DVI):recent developments and future prospects

Forensic anthropological knowledge has been used in disaster victim identification (DVI) for over a century, but over the past decades, there have been a number of disaster events which have seen an increasing role for the forensic anthropologist. The experiences gained from some of the latest DVI operations have provided valuable lessons that have had an effect on the role and perceived value of the forensic anthropologist as part of the team managing the DVI process. This paper provides an overview of the ways in which forensic anthropologists may contribute to DVI with emphasis on how recent experiences and developments in forensic anthropology have augmented these contributions. Consequently, this paper reviews the value of forensic anthropological expertise at the disaster scene and in the mortuary, and discusses the way in which forensic anthropologists may use imaging in DVI efforts. Tissue-sampling strategies for DNA analysis, especially in the case of disasters with a large amount of fragmented remains, are also discussed. Additionally, consideration is given to the identification of survivors; the statistical basis of identification; the challenges related to some specific disaster scenarios; and education and training. Although forensic anthropologists can play a valuable role in different phases of a DVI operation, they never practice in isolation. The DVI process requires a multidisciplinary approach and, therefore, has a close collaboration with a range of forensic specialists

Transcriptome profiling of rabbit parthenogenetic blastocysts developed under in vivo conditions

Parthenogenetic embryos are one attractive alternative as a source of embryonic stem cells, although many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. The present work was conducted to investigate the gene expression profile of rabbit parthenote embryos cultured under in vivo conditions using microarray analysis. Transcriptomic profiles indicate 2541 differentially expressed genes between parthenotes and normal in vivo fertilised blastocysts, of which 76 genes were upregulated and 16 genes downregulated in in vivo cultured parthenote blastocyst, using 3 fold-changes as a cut-off. While differentially upregulated expressed genes are related to transport and protein metabolic process, downregulated expressed genes are related to DNA and RNA binding. Using microarray data, 6 imprinted genes were identified as conserved among rabbits, humans and mice: GRB10, ATP10A, ZNF215, NDN, IMPACT and SFMBT2. We also found that 26 putative genes have at least one member of that gene family imprinted in other species. These data strengthen the view that a large fraction of genes is differentially expressed between parthenogenetic and normal embryos cultured under the same conditions and offer a new approach to the identification of imprinted genes in rabbit. © 2012 Naturil-Alfonso et al.This work was supported by Generalitat Valenciana research programme (Prometeo 2009/125). Carmen Naturil was supported by Generalitat Valenciana research programme (Prometeo 2009/125). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Naturil Alfonso, C.; Saenz De Juano Ribes, MDLD.; Peñaranda, D.; Vicente Antón, JS.; Marco Jiménez, F. (2012). Transcriptome profiling of rabbit parthenogenetic blastocysts developed under in vivo conditions. PLoS ONE. 7(12):1-11. https://doi.org/10.1371/journal.pone.0051271S111712Harness, J. V., Turovets, N. A., Seiler, M. J., Nistor, G., Altun, G., Agapova, L. S., … Keirstead, H. S. (2011). Equivalence of Conventionally-Derived and Parthenote-Derived Human Embryonic Stem Cells. PLoS ONE, 6(1), e14499. doi:10.1371/journal.pone.0014499Lu, Z., Zhu, W., Yu, Y., Jin, D., Guan, Y., Yao, R., … Zhou, Q. (2010). Derivation and long-term culture of human parthenogenetic embryonic stem cells using human foreskin feeders. Journal of Assisted Reproduction and Genetics, 27(6), 285-291. doi:10.1007/s10815-010-9408-5Koh, C. J., Delo, D. M., Lee, J. W., Siddiqui, M. M., Lanza, R. P., Soker, S., … Atala, A. (2009). Parthenogenesis-derived multipotent stem cells adapted for tissue engineering applications. Methods, 47(2), 90-97. doi:10.1016/j.ymeth.2008.08.002Vrana, K. E., Hipp, J. D., Goss, A. M., McCool, B. A., Riddle, D. R., Walker, S. J., … Cibelli, J. B. (2003). Nonhuman primate parthenogenetic stem cells. Proceedings of the National Academy of Sciences, 100(Supplement 1), 11911-11916. doi:10.1073/pnas.2034195100Chen, Z., Liu, Z., Huang, J., Amano, T., Li, C., Cao, S., … Liu, L. (2009). Birth of Parthenote Mice Directly from Parthenogenetic Embryonic Stem Cells. Stem Cells, 27(9), 2136-2145. doi:10.1002/stem.158Sritanaudomchai, H., Ma, H., Clepper, L., Gokhale, S., Bogan, R., Hennebold, J., … Mitalipov, S. (2010). Discovery of a novel imprinted gene by transcriptional analysis of parthenogenetic embryonic stem cells. Human Reproduction, 25(8), 1927-1941. doi:10.1093/humrep/deq144Fang, Z. F., Gai, H., Huang, Y. Z., Li, S. G., Chen, X. J., Shi, J. J., … Sheng, H. Z. (2006). Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos. Experimental Cell Research, 312(18), 3669-3682. doi:10.1016/j.yexcr.2006.08.013Wang, S., Tang, X., Niu, Y., Chen, H., Li, B., Li, T., … Ji, W. (2007). Generation and Characterization of Rabbit Embryonic Stem Cells. Stem Cells, 25(2), 481-489. doi:10.1634/stemcells.2006-0226Piedrahita, J. A., Anderson, G. B., & BonDurant, R. H. (1990). On the isolation of embryonic stem cells: Comparative behavior of murine, porcine and ovine embryos. Theriogenology, 34(5), 879-901. doi:10.1016/0093-691x(90)90559-cNaturil-Alfonso, C., Saenz-de-Juano, M. D., Peñaranda, D. S., Vicente, J. S., & Marco-Jiménez, F. (2011). Parthenogenic blastocysts cultured under in vivo conditions exhibit proliferation and differentiation expression genes similar to those of normal embryos. Animal Reproduction Science, 127(3-4), 222-228. doi:10.1016/j.anireprosci.2011.08.005Besenfelder, U., Strouhal, C., & Brem, G. (1998). A Method for Endoscopic Embryo Collection and Transfer in the Rabbit. Journal of Veterinary Medicine Series A, 45(1-10), 577-579. doi:10.1111/j.1439-0442.1998.tb00861.xMehaisen, G. M. K., Viudes-de-Castro, M. P., Vicente, J. S., & Lavara, R. (2006). In vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatment in rabbit does. Theriogenology, 65(7), 1279-1291. doi:10.1016/j.theriogenology.2005.08.007Bilodeau-Goeseels, S., & Schultz, G. A. (1997). Changes in Ribosomal Ribonucleic Acid Content Within in Vitro-produced Bovine Embryos1. Biology of Reproduction, 56(5), 1323-1329. doi:10.1095/biolreprod56.5.1323Conesa, A., Gotz, S., Garcia-Gomez, J. M., Terol, J., Talon, M., & Robles, M. (2005). Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics, 21(18), 3674-3676. doi:10.1093/bioinformatics/bti610Edgar, R. (2002). Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Research, 30(1), 207-210. doi:10.1093/nar/30.1.207Weltzien, F.-A., Pasqualini, C., Vernier, P., & Dufour, S. (2005). A quantitative real-time RT-PCR assay for European eel tyrosine hydroxylase. General and Comparative Endocrinology, 142(1-2), 134-142. doi:10.1016/j.ygcen.2004.12.019Llobat, L., Marco-Jiménez, F., Peñaranda, D., Saenz-de-Juano, M., & Vicente, J. (2011). Effect of Embryonic Genotype on Reference Gene Selection for RT-qPCR Normalization. Reproduction in Domestic Animals, 47(4), 629-634. doi:10.1111/j.1439-0531.2011.01934.xLiu, N., Enkemann, S. A., Liang, P., Hersmus, R., Zanazzi, C., Huang, J., … Liu, L. (2010). Genome-wide Gene Expression Profiling Reveals Aberrant MAPK and Wnt Signaling Pathways Associated with Early Parthenogenesis. Journal of Molecular Cell Biology, 2(6), 333-344. doi:10.1093/jmcb/mjq029Abdoon, A. S., Ghanem, N., Kandil, O. M., Gad, A., Schellander, K., & Tesfaye, D. (2012). cDNA microarray analysis of gene expression in parthenotes and in vitro produced buffalo embryos. Theriogenology, 77(6), 1240-1251. doi:10.1016/j.theriogenology.2011.11.004Labrecque, R., & Sirard, M.-A. (2011). Gene expression analysis of bovine blastocysts produced by parthenogenic activation or fertilisation. Reproduction, Fertility and Development, 23(4), 591. doi:10.1071/rd10243Rizos, D., Clemente, M., Bermejo-Alvarez, P., de La Fuente, J., Lonergan, P., & Gutiérrez-Adán, A. (2008). Consequences ofIn VitroCulture Conditions on Embryo Development and Quality. Reproduction in Domestic Animals, 43, 44-50. doi:10.1111/j.1439-0531.2008.01230.xLonergan, P., Rizos, D., Kanka, J., Nemcova, L., Mbaye, A., Kingston, M., … Boland, M. (2003). Temporal sensitivity of bovine embryos to culture environment after fertilization and the implications for blastocyst quality. Reproduction, 337-346. doi:10.1530/rep.0.1260337Memili, E., & First, N. L. (2000). Zygotic and embryonic gene expression in cow: a review of timing and mechanisms of early gene expression as compared with other species. Zygote, 8(1), 87-96. doi:10.1017/s0967199400000861Latham, K. E. (2001). Embryonic genome activation. Frontiers in Bioscience, 6(3), d748-759. doi:10.2741/a639Niemann, H., & Wrenzycki, C. (2000). Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: Implications for subsequent development. Theriogenology, 53(1), 21-34. doi:10.1016/s0093-691x(99)00237-xCorcoran, D., Fair, T., Park, S., Rizos, D., Patel, O. V., Smith, G. W., … Lonergan, P. (2006). Suppressed expression of genes involved in transcription and translation in in vitro compared with in vivo cultured bovine embryos. Reproduction, 131(4), 651-660. doi:10.1530/rep.1.01015Morison, I. M., Ramsay, J. P., & Spencer, H. G. (2005). A census of mammalian imprinting. Trends in Genetics, 21(8), 457-465. doi:10.1016/j.tig.2005.06.008Bischoff, S. R., Tsai, S., Hardison, N., Motsinger-Reif, A. A., Freking, B. A., Nonneman, D., … Piedrahita, J. A. (2009). Characterization of Conserved and Nonconserved Imprinted Genes in Swine1. Biology of Reproduction, 81(5), 906-920. doi:10.1095/biolreprod.109.078139Cruz-Correa, M., Zhao, R., Oveido, M., Bernabe, R. D., Lacourt, M., Cardona, A., … Giardiello, F. M. (2009). Temporal stability and age-related prevalence of loss of imprinting of the insulin-like growth factor-2 gene. Epigenetics, 4(2), 114-118. doi:10.4161/epi.4.2.7954Park, C.-H., Uh, K.-J., Mulligan, B. P., Jeung, E.-B., Hyun, S.-H., Shin, T., … Lee, C.-K. (2011). Analysis of Imprinted Gene Expression in Normal Fertilized and Uniparental Preimplantation Porcine Embryos. PLoS ONE, 6(7), e22216. doi:10.1371/journal.pone.0022216Thurston, A., Taylor, J., Gardner, J., Sinclair, K. D., & Young, L. E. (2007). Monoallelic expression of nine imprinted genes in the sheep embryo occurs after the blastocyst stage. Reproduction, 135(1), 29-40. doi:10.1530/rep-07-0211Li, Y., & Sasaki, H. (2011). Genomic imprinting in mammals: its life cycle, molecular mechanisms and reprogramming. Cell Research, 21(3), 466-473. doi:10.1038/cr.2011.15Mamo, S., Gal, A., Polgar, Z., & Dinnyes, A. (2008). Expression profiles of the pluripotency marker gene POU5F1 and validation of reference genes in rabbit oocytes and preimplantation stage embryos. BMC Molecular Biology, 9(1), 67. doi:10.1186/1471-2199-9-67Navarrete Santos, A., Tonack, S., Kirstein, M., Pantaleon, M., Kaye, P., & Fischer, B. (2004). Insulin acts via mitogen-activated protein kinase phosphorylation in rabbit blastocysts. Reproduction, 128(5), 517-526. doi:10.1530/rep.1.0020

Mouse Ribosomal RNA Genes Contain Multiple Differentially Regulated Variants

Previous cytogenetic studies suggest that various rDNA chromosomal loci are not equally active in different cell types. Consistent with this variability, rDNA polymorphism is well documented in human and mouse. However, attempts to identify molecularly rDNA variant types, which are regulated individually (i.e., independent of other rDNA variants) and tissue-specifically, have not been successful. We report here the molecular cloning and characterization of seven mouse rDNA variants (v-rDNA). The identification of these v-rDNAs was based on restriction fragment length polymorphisms (RFLPs), which are conserved among individuals and mouse strains. The total copy number of the identified variants is less than 100 and the copy number of each individual variant ranges from 4 to 15. Sequence analysis of the cloned v-rDNA identified variant-specific single nucleotide polymorphisms (SNPs) in the transcribed region. These SNPs were used to develop a set of variant-specific PCR assays, which permitted analysis of the v-rDNAs' expression profiles in various tissues. These profiles show that three v-rDNAs are expressed in all tissues (constitutively active), two are expressed in some tissues (selectively active), and two are not expressed (silent). These expression profiles were observed in six individuals from three mouse strains, suggesting the pattern is not randomly determined. Thus, the mouse rDNA array likely consists of genetically distinct variants, and some are regulated tissue-specifically. Our results provide the first molecular evidence for cell-type-specific regulation of a subset of rDNA

Key Labeling Technologies to Tackle Sizeable Problems in RNA Structural Biology

The ability to adopt complex three-dimensional (3D) structures that can rapidly interconvert between multiple functional states (folding and dynamics) is vital for the proper functioning of RNAs. Consequently, RNA structure and dynamics necessarily determine their biological function. In the post-genomic era, it is clear that RNAs comprise a larger proportion (>50%) of the transcribed genome compared to proteins (≤2%). Yet the determination of the 3D structures of RNAs lags considerably behind those of proteins and to date there are even fewer investigations of dynamics in RNAs compared to proteins. Site specific incorporation of various structural and dynamic probes into nucleic acids would likely transform RNA structural biology. Therefore, various methods for introducing probes for structural, functional, and biotechnological applications are critically assessed here. These probes include stable isotopes such as 2H, 13C, 15N, and 19F. Incorporation of these probes using improved RNA ligation strategies promises to change the landscape of structural biology of supramacromolecules probed by biophysical tools such as nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and Raman spectroscopy. Finally, some of the structural and dynamic problems that can be addressed using these technological advances are outlined

A search for the decay modes B+/- to h+/- tau l

We present a search for the lepton flavor violating decay modes B+/- to h+/- tau l (h= K,pi; l= e,mu) using the BaBar data sample, which corresponds to 472 million BBbar pairs. The search uses events where one B meson is fully reconstructed in one of several hadronic final states. Using the momenta of the reconstructed B, h, and l candidates, we are able to fully determine the tau four-momentum. The resulting tau candidate mass is our main discriminant against combinatorial background. We see no evidence for B+/- to h+/- tau l decays and set a 90% confidence level upper limit on each branching fraction at the level of a few times 10^-5.Comment: 15 pages, 7 figures, submitted to Phys. Rev.

Observation and study of baryonic B decays: B -> D(*) p pbar, D(*) p pbar pi, and D(*) p pbar pi pi

We present a study of ten B-meson decays to a D(*), a proton-antiproton pair, and a system of up to two pions using BaBar's data set of 455x10^6 BBbar pairs. Four of the modes (B0bar -> D0 p anti-p, B0bar -> D*0 p anti-p, B0bar -> D+ p anti-p pi-, B0bar -> D*+ p anti-p pi-) are studied with improved statistics compared to previous measurements; six of the modes (B- -> D0 p anti-p pi-, B- -> D*0 p anti-p pi-, B0bar -> D0 p anti-p pi- pi+, B0bar -> D*0 p anti-p pi- pi+, B- -> D+ p anti-p pi- pi-, B- -> D*+ p anti-p pi- pi-) are first observations. The branching fractions for 3- and 5-body decays are suppressed compared to 4-body decays. Kinematic distributions for 3-body decays show non-overlapping threshold enhancements in m(p anti-p) and m(D(*)0 p) in the Dalitz plots. For 4-body decays, m(p pi-) mass projections show a narrow peak with mass and full width of (1497.4 +- 3.0 +- 0.9) MeV/c2, and (47 +- 12 +- 4) MeV/c2, respectively, where the first (second) errors are statistical (systematic). For 5-body decays, mass projections are similar to phase space expectations. All results are preliminary.Comment: 28 pages, 90 postscript figures, submitted to LP0

Evidence for the h_b(1P) meson in the decay Upsilon(3S) --> pi0 h_b(1P)

Using a sample of 122 million Upsilon(3S) events recorded with the BaBar detector at the PEP-II asymmetric-energy e+e- collider at SLAC, we search for the $h_b(1P)$ spin-singlet partner of the P-wave chi_{bJ}(1P) states in the sequential decay Upsilon(3S) --> pi0 h_b(1P), h_b(1P) --> gamma eta_b(1S). We observe an excess of events above background in the distribution of the recoil mass against the pi0 at mass 9902 +/- 4(stat.) +/- 2(syst.) MeV/c^2. The width of the observed signal is consistent with experimental resolution, and its significance is 3.1sigma, including systematic uncertainties. We obtain the value (4.3 +/- 1.1(stat.) +/- 0.9(syst.)) x 10^{-4} for the product branching fraction BF(Upsilon(3S)-->pi0 h_b) x BF(h_b-->gamma eta_b).Comment: 8 pages, 4 postscript figures, submitted to Phys. Rev. D (Rapid Communications

Search for rare quark-annihilation decays, B --> Ds(*) Phi

We report on searches for B- --> Ds- Phi and B- --> Ds*- Phi. In the context of the Standard Model, these decays are expected to be highly suppressed since they proceed through annihilation of the b and u-bar quarks in the B- meson. Our results are based on 234 million Upsilon(4S) --> B Bbar decays collected with the BABAR detector at SLAC. We find no evidence for these decays, and we set Bayesian 90% confidence level upper limits on the branching fractions BF(B- --> Ds- Phi) Ds*- Phi)<1.2x10^(-5). These results are consistent with Standard Model expectations.Comment: 8 pages, 3 postscript figues, submitted to Phys. Rev. D (Rapid Communications